Alberts Visualizing Cells2

Molecular Biology of
THE CELL Seventh Edition

Molecular Biology of
THE CELL Seventh Edition
Bruce Alberts
Rebecca Heald
Alexander Johnson
David Morgan
Martin Raff
Keith Roberts
Peter Walter
With problems by
John Wilson
Tim Hunt
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Copyright © 2022 by Bruce Alberts, Rebecca Heald, Alexander Johnson, David Morgan,
Martin Raff, Keith Roberts, Peter Walter, the Estate of Julian Lewis, John Wilson, and
Tim Hunt
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Library of Congress Cataloging-in-Publication Data
Names: Alberts, Bruce, author.

Title: Molecular biology of the cell / Bruce Alberts, Rebecca Heald,
Alexander Johnson, David Morgan, Martin Raff, Keith Roberts, Peter
Walter.
Description: Seventh edition. | New York : W. W. Norton & Company, [2022] |
Includes bibliographical references and index.
Identifiers: LCCN 2021049376 | ISBN 9780393884821 (hardcover) | ISBN
9780393884630 (epub)
Subjects: MESH: Cells | Molecular Biology
Classification: LCC QH581.2 | NLM QU 300 | DDC 572.8—dc23/eng/20211015
LC record available at https://lccn.loc.gov/2021049376
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xxiii
Brief Contents
PART I INTRODUCTION TO THE CELL

Chapter 1 Cells, Genomes, and the Diversity of Life 1
Chapter 2 Cell Chemistry and Bioenergetics 49
Chapter 3 Proteins 115
PART II BASIC GENETIC MECHANISMS
Chapter 4 DNA, Chromosomes, and Genomes 183
Chapter 5 DNA Replication, Repair, and Recombination 253
Chapter 6 How Cells Read the Genome: From DNA to Protein 321
Chapter 7 Control of Gene Expression 397
PART III WAYS OF WORKING WITH CELLS
Chapter 8 Analyzing Cells, Molecules, and Systems 475
Chapter 9 Visualizing Cells and Their Molecules 563
PART IV INTERNAL ORGANIZATION OF THE CELL
Chapter 10 Membrane Structure 603
Chapter 11 Small-Molecule Transport and Electrical Properties of Membranes 637
Chapter 12 Intracellular Organization and Protein Sorting 683
Chapter 13 Intracellular Membrane Traffic 749
Chapter 14 Energy Conversion and Metabolic Compartmentation: Mitochondria and Chloroplasts 811
Chapter 15 Cell Signaling 873
Chapter 16 The Cytoskeleton 949
Chapter 17 The Cell Cycle 1027
Chapter 18 Cell Death 1089
PART V CELLS IN THEIR SOCIAL CONTEXT
Chapter 19 Cell Junctions and the Extracellular Matrix 1105
Chapter 20 Cancer 1163
Chapter 21 Development of Multicellular Organisms 1217
Chapter 22 Stem Cells in Tissue Homeostasis and Regeneration 1279
Chapter 23 Pathogens and Infection 1313
Chapter 24 The Innate and Adaptive Immune Systems 1353
563
CHAPTER
Visualizing Cells and Their
Molecules 9
Understanding the structural organization of cells, and the macromolecules
that build and animate them, is essential for learning how they function. In this IN THIS CHAPTER
chapter, we briefly describe some of the principal light and electron microscopy
methods used to study cells and molecules. In the past decade or so, there have Looking at Cells and Molecules
been major technical developments in both methods that allow us to see biolog-
ical structures with increasing resolution and clarity. Optical microscopy will be
in the Light Microscope
our starting point because cell biology began with the light microscope, and it is
Looking at Cells and Molecules
still an indispensable tool. The development of methods for the specific labeling
and imaging of individual cellular constituents and the reconstruction of their in the Electron Microscope
three-dimensional architecture has meant that, far from falling into disuse, opti-
cal microscopy continues to increase in importance. One advantage of optical
microscopy is that light is relatively nondestructive. By tagging specific cell com-
ponents with fluorescent probes, such as intrinsically fluorescent proteins, we
can watch their movement, dynamics, and interactions in living cells.
Although conventional optical microscopy is limited in resolution by the
wavelength of visible light, new methods cleverly bypass this limitation and allow
the exact position of even single molecules to be mapped. By using a beam of
electrons instead of visible light, electron microscopy can image the interior of
cells, and their macromolecular components, at almost atomic resolution and in
three dimensions. But all imaging methods involve trade-offs; in this case, the
higher resolution means only small objects are imaged and only in fixed, dead
cells. There is now a bewildering variety of imaging technologies for the cell biol-
ogist to choose from, and when some of these are described later in the chapter,
it is worth considering why you might use one rather than another. Trade-offs
will always have to be made between thin and thick specimens, living and fixed
cells, high and low resolution, fast and slow imaging, signal and noise, or cells
and molecules.
This chapter is intended as a companion, rather than an introduction, to
the chapters that follow; readers may wish to refer back to it as applications of
microscopy to basic biological problems are encountered in other chapters of
the book.
LOOKING AT CELLS AND MOLECULES

IN THE LIGHT MICROSCOPE
A typical animal cell is 10–20 μm in diameter, which is just less than a tenth the
size of the smallest object that we can normally see with the naked eye. Only after
good light microscopes became available in the early part of the nineteenth cen-
tury did Matthias Schleiden and Theodor Schwann propose that all plant and
animal tissues were aggregates of individual cells. Their proposal in 1838, known
as the cell doctrine, marks the formal birth of cell biology.
Animal cells are not only tiny, but they are also colorless and translucent.
The discovery of their main internal features, therefore, depended on the devel-
opment, in the late nineteenth century, of a variety of stains that provided
sufficient color and contrast to make those features visible. Similarly, the far
564 Chapter 9: Visualizing Cells and Their Molecules
visible with
0.2 mm
(200 µm) unaided eye
×10
CELLS
20 µm
×10
20 mm 2 mm 0.2 mm
2 µm
ORGANELLES
×10
0.2 µm light
(200 nm) microscope
×10
superresolution
20 nm fluorescence
microscope
20 µm 2 µm 0.2 µm
MOLECULES
×10
2 nm
×10
electron
ATOMS
0.2 nm
microscope
20 nm 2 nm 0.2 nm
(A) (B)
more powerful electron microscope introduced in the early 1940s required the Figure 9–1 A sense of scale between
development of new techniques for preserving and staining cells before the full living cells and atoms. (A) Each diagram
shows an image magnified by a factor
complexities of their internal fine structure could begin to emerge. To this day,
of 10 in an imaginary progression from a
microscopy often relies as much on techniques for preparing the specimen as on thumb, through skin cells, to a ribosome,
the performance of the microscope itself. In the following discussions, we there- to a cluster of atoms forming part of one
fore consider both instruments and specimen preparation, beginning with the of the many protein molecules in the
light microscope. ribosome. Atomic details of biological
macromolecules, as shown in the last
The images in Figure 9–1A illustrate a stepwise progression from a thumb to a two panels, are just within the power of
cluster of atoms. Each successive image represents a tenfold increase in magnifi- the electron microscope. While color has
cation. The naked eye can see features in the first two panels, the light microscope been used here in all the panels, it is not a
allows us to see details corresponding to about the fifth panel, and the electron feature of objects much smaller than the
microscope takes us to about the eighth or ninth panel. Figure 9–1B shows the wavelength of light, so the last five panels
should really be in black and white.
sizes of various cellular and subcellular structures and the ranges of size that (B) Sizes of cells and their components
different types of microscopes can visualize. are shown on a logarithmic scale, indicating
the range of objects that can readily be
resolved by the naked eye and in the
The Conventional Light Microscope Can Resolve light and electron microscopes. Note
Details 0.2 μm Apart that new superresolution microscopy
techniques, discussed in detail later, allow
For well over 100 years, all microscopes were constrained by a fundamental lim- an improvement in resolution by an order
itation: that a given type of radiation cannot be used to probe structural details of magnitude compared with conventional
much smaller than its own wavelength. A limit to the resolution of a light micro- light microscopy.
scope was therefore set by the wavelength of visible light, which ranges from
about 0.4 μm (for violet) to 0.7 μm (for deep red). In practical terms, bacteria and
mitochondria, which are about 500 nm (0.5 μm) wide, are generally the small-
est objects whose shape we can clearly discern in a standard light microscope;
LOOKING AT CELLS AND MOLECULES IN THE LIGHT MICROSCOPE 565
(A) image on retina (B) Figure 9–2 A light microscope.

(A) Diagram showing the light path in an
eye upright compound microscope. Light is
focused on the specimen by lenses in the
condenser. A combination of objective
eyepiece lenses, tube lenses, and eyepiece lenses
is arranged to focus an image of the
illuminated specimen in the eye. (B) A
modern upright research light microscope.
(C) A modern inverted microscope,
particularly useful for looking at cells in
culture. Both microscopes are equipped for
tube lens fluorescence imaging (B and C, courtesy of
Carl Zeiss Microscopy, GmbH.)
(C)
objective
specimen
condenser
iris diaphragm
light
source
details smaller than this are obscured by effects resulting from the wave-like
The following units of length are
nature of light. Let us follow the behavior of a beam of light as it passes through commonly employed in microscopy:
the lenses of a microscope (Figure 9–2).
μm (micrometer) = 10–6 m
Because of its wave nature, light does not follow the idealized straight ray
nm (nanometer) = 10–9 m
paths that geometrical optics predicts. Instead, light waves travel through an
optical system by many slightly different routes, like ripples in water, so that they Å (angstrom) = 10–10 m
interfere with one another and cause optical diffraction effects. If two trains of
waves reaching the same point by different paths are precisely in phase, with crest
matching crest and trough matching trough, they will reinforce each other so as
to increase brightness. In contrast, if the trains of waves are out of phase, they will
interfere with each other in such a way as to cancel each other partly or entirely
(Figure 9–3). The interaction of light with an object changes the phase relation-
ships of the light waves in a way that produces complex interference effects. At
high magnification, for example, the shadow of an edge that is evenly illuminated
with light of uniform wavelength appears as a set of parallel lines (Figure 9–4A),
TWO WAVES IN PHASE TWO WAVES OUT OF PHASE
Figure 9–3 Interference between light

waves. When two light waves combine in
phase, the amplitude of the resultant wave
is larger, and the brightness is increased.
Two light waves that are out of phase
DIM
cancel each other partly and produce a
wave whose amplitude, and therefore
BRIGHT brightness, is decreased.
whereas the smallest focused image of a bright circular aperture appears as a set
of concentric rings (Figure 9–4B). For the same reason, a single point seen
through a microscope appears as a blurred disc, and two point objects close
together give overlapping images and may merge into one. Although no amount
of refinement of the lenses can overcome the diffraction limit imposed by the
wave-like nature of light, other ways of cleverly bypassing this limit have emerged,
creating so-called superresolution imaging techniques that can even detect the (A) (B)
position of single molecules. These are discussed later in the chapter.
The limiting separation at which two objects appear distinct—the so-called Figure 9–4 Images of an edge and of a
limit of resolution—depends on both the wavelength of the light and the point of light. (A) The interference effects,
numerical aperture of the lens system used. The numerical aperture affects the or fringes, seen at high magnification when
light of a specific wavelength passes the
light-gathering ability of the lens and is related both to the angle of the cone of edge of a solid object placed between
light that can enter it and to the refractive index of the medium the lens is operat- the light source and the observer. (B) The
ing in; the wider the microscope opens its eye, so to speak, the more sharply it can image of a point source of light. Diffraction
see (Figure 9–5). The refractive index is the ratio of the speed of light in a vacuum spreads this out into a complex, circular
pattern, whose width depends on the
to the speed of light in a particular transparent medium. For example, for water numerical aperture of the optical system:
this is 1.33, meaning that light travels 1.33 times slower in water than in a vacuum. the smaller the aperture, the bigger (more
Under the best conditions, with violet light (wavelength = 0.4 μm) and a numer- blurred) the diffracted image. Two point
ical aperture of 1.4, the basic light microscope can theoretically achieve a limit sources can be just resolved when the
of resolution of about 0.2 μm, or 200 nm. Some microscope makers at the end center of the image of one lies within the
first dark ring in the image of the other: this
of the nineteenth century achieved this resolution, but it is routinely matched in is used to define the limit of resolution.
contemporary, factory-produced microscopes. Although it is possible to enlarge
an image as much as we want—for example, by projecting it onto a screen—it is
not possible, in a conventional light microscope, to resolve two objects in the light
microscope that are separated by less than about 0.2 μm; they will always appear
as a single object. It is important, however, to distinguish between resolution and
detection. If a small object, below the resolution limit, itself emits light, then we
may still be able to see or detect it. Thus, we can see a single fluorescently labeled
microtubule even though it is about 10 times thinner than the resolution limit of
the light microscope. Diffraction effects, however, will cause it to appear blurred
and at least 0.2 μm thick (see Figure 9–14). In a similar way, we can see the stars
in the night sky, even though their diameters are far below the angular resolution
of our unaided eyes: they all appear as similar, slightly blurred points of light, dif-
fering only in their color and brightness.
LENSES RESOLUTION: the resolving power of the

microscope depends on the width of the
cone of illumination and therefore on both
the condenser and the objective lens. It is
IMAGE calculated using the formula
the objective lens 0.61 λ

resolution =
collects a cone of n sin θ
light rays to create where:
an image
specimen θ = half the angular width of the cone of
rays collected by the objective lens
2θ from a typical point in the central
region of the specimen (because the
maximum width is 180o, sin θ has a
the condenser lens
maximum value of 1)
focuses a cone of
n = the refractive index of the medium
light rays onto
(usually air or oil) separating the
each point of the
LIGHT specimen from the objective and
specimen
condenser lenses
λ = the wavelength of light used (for white
light a figure of 0.53 µm is commonly
assumed)
Figure 9–5 Basic principles of light
microscopy. The path of light rays
NUMERICAL APERTURE: n sin θ in the aperture, the greater the resolution and the passing through a transparent specimen
equation above is called the numerical aperture brighter the image (brightness is important in in a microscope illustrates the concept of
of the lens and is a function of its light- fluorescence microscopy). However, this advan-
collecting ability. For dry lenses this cannot be tage does necessitate very short working
numerical aperture and its relation to the
more than 1, but for oil-immersion lenses it can distances and a very small depth of field, just limit of resolution. The higher the numerical
be as high as 1.4. The higher the numerical as in a conventional camera. aperture of a lens, the brighter the image it
forms and the higher its resolution.
Photon Noise Creates Additional Limits to Resolution When Light

Levels Are Low
Any image, whether produced by an electron microscope or by an optical micro-
scope, is made by particles—electrons or photons—striking a detector of some
sort. But these particles are governed by quantum mechanics, so the numbers
reaching the detector are predictable only in a statistical sense. Finite samples,
collected by imaging for a limited period of time (that is, by taking a snapshot),
will show random variation: successive snapshots of the same scene will not be
exactly identical. Moreover, every detection method has some level of background
signal or noise, adding to the statistical uncertainty. With bright illumination,
corresponding to very large numbers of photons or electrons, the features of the
imaged specimen are accurately determined on the basis of the distribution of
these particles at the detector. However, with smaller numbers of particles, the
structural details of the specimen are obscured by the statistical fluctuations in
the numbers of particles detected in each region, which give the image a speckled
appearance and limit its precision. The term noise describes this random variabil-
ity. Because noise in the image is proportional to the square root of the number of
photons that are detected (or electrons in electron microscopy), then as the num- Figure 9–6 Contrast in light microscopy.
(A) The stained portion of the cell will
ber of photons or electrons recorded increases, the absolute noise also increases, absorb light of some wavelengths, which
but because of the square root relationship, the percentage of noise decreases, in depends on the stain, but will allow
other words the signal-to-noise ratio improves. A poor signal-to-noise ratio is an other wavelengths to pass through it.
important consideration when weak fluorescent light signals are recorded or low, A colored image of the cell is thereby
but less damaging, electron doses are required. obtained that is visible in the normal
bright-field light microscope. (B) In the
dark-field microscope, oblique rays of light
Living Cells Are Seen Clearly in a Phase-Contrast focused on the specimen do not enter the
or a Differential-Interference-Contrast Microscope objective lens, but light that is scattered
by components in the living cell can be
There are many ways in which contrast in a specimen can be generated collected to produce a bright image on a
(Figure 9–6). While fixing and staining a specimen can generate contrast dark background. (C) Light passing through
the unstained living cell experiences very
through color (Figure 9–6A), microscopists have always been challenged by the little change in amplitude, and the structural
possibility that some components of the cell may be lost or distorted during spec- details cannot be seen even if the image
imen preparation. The only certain way to avoid the problem is to examine cells is highly magnified. The phase of the light,
while they are alive, without fixing or freezing. For this purpose, light microscopes however, is altered by its passage through
with special optical systems are especially useful. either thicker or denser parts of the cell,
and small phase differences can be made
In the normal bright-field microscope, light passing through a cell in culture visible by exploiting interference effects
forms the image directly. Another system, dark-field microscopy, exploits the using a phase-contrast or a differential-
fact that light rays can be scattered in all directions by small objects in their path. interference-contrast microscope.
only scattered waves out of phase

light rays enter generate contrast
stained objective when combined
section
of cell
unstained
cell
waves in
phase
(A) incident light (B) oblique incident light (C) incident light
(white) (white)
(A) (B)
(C) (D)
50 µm
If oblique lighting from the condenser is used, which does not directly enter the Figure 9–7 Four types of light
objective, unstained objects in a living cell can scatter the rays, some of which microscopy. Four images are shown
of the same fibroblast cell in culture. All
then enter the objective to create a bright image against a black background
images can be obtained with most modern
(Figure 9–6B). microscopes by interchanging optical
When light passes through a living cell, the phase of the light wave is changed components. (A) Bright-field microscopy,
according to the cell’s refractive index: a relatively thick or dense part of the cell, in which light is transmitted straight
such as a nucleus, slows the light passing through it. The phase of the light, con- through the specimen. (B) Phase-contrast
microscopy, in which phase alterations
sequently, is shifted relative to light that has passed through an adjacent thinner of light transmitted through the specimen
region of the cytoplasm (Figure 9–6C). The phase-contrast microscope and, in a are translated into brightness changes.
more complex way, the differential-interference-contrast microscope increase (C) Differential-interference-contrast
these phase differences to produce amplitude differences, or contrast, when the sets microscopy, which highlights edges where
of waves recombine, thereby creating an image of the cell’s structure. Both types of there is a steep change of refractive index.
(D) Dark-field microscopy, in which the
light microscopy are widely used to look at living cells (see Movie 17.2). Figure 9–7 specimen is lit from the side and only the
compares images of the same cell obtained by four kinds of light microscopy. scattered light is seen.
Phase-contrast, differential-interference-contrast, and dark-field microscopy
make it possible to watch the movements involved in such processes as mitosis
and cell migration. Because many cellular motions are too slow to be seen in real
time, it is often helpful to make time-lapse videos in which the camera records
successive frames separated by a short time delay, so that when the resulting pic-
ture series is played at normal speed, events appear greatly speeded up.
Images Can Be Enhanced and Analyzed by Digital Techniques

Digital imaging systems, and the associated technology of image processing, have
had a major impact on light microscopy. Certain practical limitations of micro-
scopes relating to imperfections in their optical components have been largely
overcome. Digital imaging systems have also circumvented two fundamental lim-
itations of the human eye: the eye cannot see well in extremely dim light, and it
cannot perceive small differences in light intensity against a bright background.
To increase our ability to observe cells in these difficult conditions, we can attach
a sensitive digital camera to a microscope. These cameras detect light by means
of high-sensitivity complementary metal-oxide semiconductor (CMOS) sensors,
similar to those now found in digital cameras and smartphones. Such image sen-
sors can count individual photons and are many times more sensitive than the
human eye and can detect 100 times more intensity levels. It is therefore possible to
observe cells for long periods at very low light levels, thereby avoiding the damaging
effects of prolonged bright light (and heat). Such sensitive detectors are especially
important for viewing fluorescent molecules in living cells, as explained later.
Because images produced by digital cameras are in electronic form, they can
be processed in various ways to extract latent information. Such image processing
makes it possible to compensate for several aberrations in the lenses of micro- movement of
microtome arm
scopes. Moreover, by digital image processing, contrast can be greatly enhanced
to overcome the eye’s limitations in detecting small differences in light intensity, specimen embedded
in wax or resin
and background irregularities in the optical system can be digitally subtracted.
This procedure reveals small transparent objects that were previously impossible fixed blade
to distinguish from the background.
ribbon of
sections
Intact Tissues Are Usually Fixed and Sectioned Before Microscopy
Looking at individual living cells in culture is relatively easy, but most cells are
found in complex tissues and organs, and this forces another trade-off when we
want to look at them. Because most tissue samples are too thick for their individ- ribbon of sections on
glass slide, stained
ual cells to be examined directly at high resolution, they are often cut into very and mounted under
thin transparent slices, or sections. To preserve the cells within the tissue they a glass cover slip
must first be treated with a fixative. A common fixative is glutaraldehyde, which
Figure 9–8 Making tissue sections.
forms covalent bonds with the free amino groups of proteins, cross-linking them This illustration shows how an embedded
so they are stabilized and locked into position. tissue is sectioned with a microtome in
Because tissues are generally soft and fragile, even after fixation, they need to preparation for examination in the light
be either frozen or embedded in a supporting medium before they can be sec- microscope. Very rapidly frozen samples
can also be sectioned, and these better
tioned. The usual embedding media are waxes or resins. In liquid form, these preserve the structure of cells in their
media both permeate and surround the fixed tissue before being hardened (by native state.
cooling or by polymerization) to form a solid block, which is readily sectioned
with a microtome. This is a machine with a sharp blade, usually of steel or glass,
which operates like a meat slicer (Figure 9–8). The sections (typically 0.5–10 μm
thick) are then laid flat on the surface of a glass microscope slide.
There is little in the contents of most cells (which are 70% water by weight) to
impede the passage of light rays. Thus, most cells in their natural state, particu-
larly if fixed and sectioned, are almost invisible in an ordinary light microscope.
We have seen that cellular components can be made visible by techniques such
as phase-contrast and differential-interference-contrast microscopy, but these
methods tell us almost nothing about the underlying chemistry. There are three
main approaches to working with thin tissue sections that reveal differences in
the types of molecules that are present.
First, and traditionally, sections can be stained with organic dyes that have
some specific affinity for particular subcellular components. The dye hema-
toxylin, for example, has an affinity for negatively charged molecules and
therefore reveals the general distribution of DNA, RNA, and acidic proteins in a
cell (Figure 9–9). The chemical basis for the specificity of many dyes, however, is
not known, although they are used widely in hospital laboratories.
Figure 9–9 Staining of cell components.

(A) This section of cells in a salivary gland
was stained with hematoxylin and eosin,
two dyes commonly used in histology. The
central duct is made of closely packed cells
with nuclei stained purple and cytoplasm
stained red. The duct is surrounded by
groups of saliva-secreting cells. (B) This
section of a young plant root is stained
with two dyes, safranin and fast green.
Fast green stains the cellulosic cell walls,
while the safranin stains the lignified xylem
cell walls red. (A, from R.L. Sorenson and
T.C. Brelje, Atlas of Human Histology: A
Guide to Microscopic Structure of Cells,
Tissues and Organs, 3rd ed., 2014. With
permission from the authors; B, courtesy
(A) (B) of University of Wisconsin Plant Teaching
50 µm 100 µm Collection.)
Second, sectioned tissues can be used to visualize specific patterns of differ-

ential gene expression. A third and very sensitive approach, generally and widely
applicable for localizing proteins of interest, depends on the use of fluorescent
probes and markers, as we explain next.
Specific Molecules Can Be Located in Cells by Fluorescence

Microscopy
Fluorescent molecules absorb light at one wavelength and emit it at another,
longer wavelength (Figure 9–10A and B). If we illuminate such a molecule at
its absorbing wavelength and then view it through a filter that allows only light of
the emitted wavelength to pass, it will glow against a dark background. Because
the background is dark, even a minute amount of the glowing fluorescent dye can
be detected. In contrast, the same number of molecules of a nonfluorescent stain,
viewed conventionally, would be practically indiscernible because the absorp-
tion of light by molecules in the stain would result in only the faintest tinge of
color in the light transmitted through that part of the specimen.
The fluorescent dyes used for staining cells are visualized with a fluorescence
microscope. This microscope is similar to an ordinary upright or inverted light
microscope except that the illuminating light, from a very powerful source, is
passed through two sets of filters—one to filter the light before it reaches the spec-
imen, and one to filter the light obtained from the specimen. The first filter passes
only the wavelengths that excite the particular fluorescent dye, while the second
filter blocks out this light and passes only those wavelengths emitted when the
dye fluoresces (Figure 9–10C).
Fluorescence microscopy is most often used to detect specific proteins or
other molecules in cells and tissues. For example, when using fluorescent nucle-
otide probes, in situ hybridization, discussed earlier (see Figure 8–63), can reveal
EXCITED STATE
energy of orbital electron
in fluorophore
absorption emission of photon

of photon at longer wavelength eyepiece
GROUND STATE 3 3 second barrier filter: cuts out

unwanted fluorescent signals,
passing the specific green
(A) fluorescein emission between
520 and 560 nm
LIGHT
excitation emission
relative intensity
SOURCE
2
2 beam-splitting mirror: reflects
light below 510 nm but
1
transmits light above 510 nm
1 first barrier filter: lets through
only blue light with a wavelength
between 450 and 490 nm objective lens
400 450 500 550 600 650
(B) wavelength (nm) (C) object
Figure 9–10 Fluorescence and the fluorescence microscope. (A) An orbital electron of a fluorochrome molecule can be
raised to an excited state after the absorption of a photon. Fluorescence occurs when the electron returns to its ground
state and emits a photon of light at a longer wavelength. Too much exposure to light or too bright a light can destroy
the fluorochrome molecule in a process called photobleaching. (B) The excitation and emission spectra for the common
fluorescent dye fluorescein isothiocyanate (FITC). (C) In the fluorescence microscope, a filter set consists of two barrier filters
(1 and 3) and a dichroic (beam-splitting) mirror (2). This example shows the filter set for detection of the fluorescent molecule
fluorescein. High-numerical-aperture objective lenses are especially important in this type of microscopy because, for a given
magnification, the brightness of the fluorescent image is proportional to the fourth power of the numerical aperture (see also
Figure 9–5).
(A) (B)
100 µm 10 µm
Figure 9–11 RNA in situ hybridization. (A) As described in Chapter 8 (see Figure 8–63), it is
possible to visualize the distribution of different RNAs in tissues using in situ hybridization. Here,
the transcription pattern of five different genes involved in patterning the early fruit fly embryo is
revealed in a single embryo. Each RNA probe has been fluorescently labeled, and the resulting
images are displayed each in a different color (“false-colored”) and then combined to give an image
where different color combinations represent different sets of genes expressed. The genes whose
expression pattern is revealed here are wingless (yellow), engrailed (blue), short gastrulation (red),
intermediate neuroblasts defective (green), and muscle specific homeobox (purple). (B) Individual
RNA transcripts can be detected in a single cell. Each of these six yeast cells is expressing less
than 20 transcripts of a particular gene. Using multiple DNA oligonucleotide probes to that particular
gene, each labeled with many fluorescent Cy5 molecules, each individual RNA transcript can be
detected as a red spot. [A, from D. Kosman et al., Science 305:846, 2004. With permission from
AAAS; B, from G.M. Wadsworth, R.Y. Parikh, and H.D. Kim, Single-probe RNA FISH in yeast. Bio
Protoc. 8(11):e2868, 2018, doi 10.21769/BioProtoc.2868.]
the cellular distribution and abundance of specific expressed RNA molecules

in sectioned material or in whole mounts of small organisms, organs, or cells
(Figure 9–11).
A versatile and widely used technique is to couple fluorescent dyes to antibody
molecules, which then serve as highly specific and versatile staining reagents that
bind selectively to the particular macromolecules they recognize in cells or in the
extracellular matrix. Two fluorescent dyes that have been commonly used for this
DAPI
purpose are fluorescein, which emits an intense green fluorescence when excited
with blue light, and rhodamine, which emits a deep red fluorescence when excited
with green-yellow light (Figure 9–12). By coupling one antibody to fluorescein and
another to rhodamine, the distributions of different molecules can be compared 420 nm
in the same cell; the two molecules are visualized separately in the microscope CFP
by switching back and forth between two sets of filters, each specific for one dye.
As shown in Figure 9–13, multiple fluorescent dyes can be used in the same way 460 nm
to clearly distinguish several different types of molecules in the same cell. Many GFP
fluorescent dyes, such as Cy3, Cy5, and the Alexa dyes, have been specifically
FITC
developed for fluorescence microscopy, but, like many organic fluorochromes,
they fade fairly rapidly when continually illuminated. Later in the chapter, addi- YFP 500 nm
tional fluorescence microscopy methods will be discussed that can be used to
monitor changes in the concentration and location of specific molecules inside rhodamine
Cy3 540 nm
Figure 9–12 Fluorescent probes. The maximum excitation and emission wavelengths of several
Alexa 568
commonly used fluorescent probes are shown in relation to the corresponding colors of the
spectrum. The photon emitted by a fluorescent molecule is necessarily of lower energy (longer RFP 580 nm
wavelength) than the absorbed photon, and this accounts for the difference between the excitation
and emission peaks. CFP, GFP, YFP, and RFP are cyan, green, yellow, and red fluorescent proteins,
respectively. DAPI is widely used as a general fluorescent DNA probe, which absorbs ultraviolet
light and fluoresces bright blue. FITC is an abbreviation for fluorescein isothiocyanate—a widely
620 nm
used derivative of fluorescein—which fluoresces bright green. The other probes are all commonly
used to fluorescently label antibodies and other proteins. Note that although the true fluorescence
Cy5
emission colors are shown here, the actual color seen in the microscope will depend on the second
barrier filter used (see Figure 9–10), and these are usually optimized so as to allow as many different
non-overlapping colored probes to be seen in the same specimen. Thus although YFP emits in the 660 nm
green spectrum, it actually appears as a yellow-green in the microscope because of the filter used.
The use of fluorescent proteins will be discussed later in the chapter. EXCITATION EMISSION
Figure 9–13 Different fluorescent probes

can be visualized in the same cell. In this
composite micrograph of an epithelial cell
in culture, three different fluorescent probes
have been used to label three different
cellular components. The actin filaments
of the cytoskeleton are revealed with a
green fluorescent probe, the numerous
mitochondria with a red fluorescent dye
that accumulates inside the organelles,
and the nucleus with a blue fluorescent
dye that binds to DNA. (Courtesy of
Carl Zeiss Microscopy, GmbH.)
5 µm
living cells. As with all microscopy methods there are trade-offs to consider. In all
fluorescence microscopes, the only molecules that can be imaged are those that
are fluorescently labeled; all the other molecules in the cell remain hidden.
Antibodies Can Be Used to Detect Specific Proteins

Antibodies are proteins produced by the vertebrate immune system as a defense
against infection (discussed in Chapter 24). They are unique among proteins in
that they are made in billions of different forms, each with a different binding
site that recognizes a specific target molecule (or antigen). The precise antigen
specificity of antibodies makes them powerful tools for the cell biologist. When
chemically coupled to fluorescent dyes, antibodies are invaluable for locating
specific molecules in cells by fluorescence microscopy (Figure 9–14). When
labeled with electron-dense particles such as colloidal gold spheres, they are used
for similar purposes in the electron microscope (discussed later). The antibodies
employed in microscopy are commonly either purified from antiserum so as to
remove all nonspecific antibodies or they are specific monoclonal antibodies that
only recognize the target molecule.
When we use antibodies as probes to detect and assay specific molecules in
cells, we frequently use methods to amplify the fluorescent signal they produce.
For example, although a marker molecule such as a fluorescent dye can be linked
directly to an antibody—the primary antibody—a stronger signal is achieved by
Figure 9–14 Immunofluorescence.

(A) A transmission electron micrograph of
the periphery of a cultured epithelial cell
showing the distribution of microtubules
and other filaments. (B) The same area
stained with fluorescent antibodies
against tubulin, the protein that assembles
to form microtubules, using the technique
of indirect immunocytochemistry (see
Figure 9–15). Red arrows indicate
individual microtubules that are readily
recognizable in both images. Note
that, because of diffraction effects, the
microtubules in the light microscope
appear 0.2 μm wide rather than their true
width of 0.025 μm. (© 1978 M. Osborn
et al. Originally published in J. Cell Biol.
(A) (B) doi 10.1083/jcb.77.3.R27. With permission
10 µm from Rockefeller University Press.)
primary antibody: secondary antibodies: Figure 9–15 Indirect immunocytochemistry.

directed against marker-coupled antibodies This detection method is very sensitive
antigen A directed against primary because many molecules of the secondary
antibodies marker
antibody recognize each primary antibody.
The secondary antibody is covalently coupled
immobilized to a marker molecule that makes it readily
antigen A detectable. Commonly used marker
molecules include fluorescent dyes (for
fluorescence microscopy) and colloidal gold
spheres (for electron microscopy).
using an unlabeled primary antibody and then detecting it with a group of labeled
secondary antibodies that bind to it (Figure 9–15). This process is called indirect
immunocytochemistry.
Individual Proteins Can Be Fluorescently Tagged in Living Cells

and Organisms
Even the most stable cell structures must be assembled, disassembled, and
reorganized during the cell’s life cycle. Other structures, often enormous on the
molecular scale, rapidly change, move, and reorganize themselves as the cell
conducts its internal affairs and responds to its environment. Complex, highly
organized pieces of molecular machinery move components around the cell,
controlling traffic into and out of the nucleus, from one organelle to another, and
into and out of the cell itself.
Various techniques have been developed to visualize the specific components
involved in such dynamic phenomena, and many of these methods use fluores-
cent proteins. All of the fluorescent molecules discussed so far are made outside
the cell and then artificially introduced into it. But the use of genes encoding
protein molecules that are themselves inherently fluorescent also enables the
creation of organisms and cell lines that make their own visible tags and labels,
without the introduction of foreign molecules. These cellular exhibitionists dis-
play their inner workings in glowing fluorescent color. Figure 9–16 Green fluorescent protein
Foremost among the fluorescent proteins used for these purposes by cell biol- (GFP). (A) The structure of GFP, shown
here schematically, highlights the eleven
ogists is the green fluorescent protein (GFP), isolated from the jellyfish Aequorea β strands that form the staves of a
victoria. This protein is encoded by a single gene, which can be cloned and intro- barrel, buried within which is the active
duced into cells of other species. The freshly translated protein is not fluorescent, chromophore (dark green). (B) The
but within an hour or so (less for some alleles of the gene, more for others) some chromophore is formed post-translationally
of the amino acids undergo a self-catalyzed post-translational modification to from the protruding side chains of
two amino acid residues in a series of
generate an efficient fluorochrome, shielded within the interior of a barrel-like autocatalytic steps. (A, PDB code: 1EMA,
protein, which will now fluoresce green when illuminated appropriately with from M. Ormö et al., Science 273:1392–
blue light (Figure 9–16). Extensive site-directed mutagenesis performed on the 1395, 1996. With permission from AAAS.)
(A) (B)
N
O tyrosine O
H
N N
C
O NH N
protein OH
backbone
HN
serine OH
HN
autocatalytic OH mature GFP
OH
O steps fluorophore
(A) (B)
500 µm 500 µm
Figure 9–17 Fluorescent proteins as reporter molecules. (A) For this experiment, carried out
in the fruit fly, the GFP gene was joined (using recombinant DNA techniques) to a fly promoter
that is active only in a specialized set of neurons. This image of a live fly embryo was captured
by a fluorescence microscope and shows approximately 20 neurons, each with long projections
(axons and dendrites) that communicate with other (nonfluorescent) cells. These neurons are
located just under the surface of the animal and allow it to sense its immediate environment.
(B) In a variation of this method, three different fluorescent proteins, red, yellow, and cyan, can
be expressed at random in neurons of the live fly embryo. The genetic constructs have been
arranged such that a strong pulse of blue light will activate the expression of one or other of the
three fluorescent proteins at random in neuronal cells, where they are then targeted to the plasma
membrane. This noninvasive control of the timing of cell labeling allows the behavior of individual
cells to be followed subsequently over time. The fine detail of all the dendrites of individual sensory
neurons can be clearly seen. The lines of pale dots arise from the autofluorescence of the bands
of denticles in the cuticle that define the segments of the embryo (see Figure 21–24). (A, from W.B.
Grueber et al., Curr. Biol. 13:618–626, 2003. With permission from Elsevier; B, from M. Boulina
et al., Development 140:1605–1613, 2013, doi 10.1242/dev.088930. © 2013. Published by the
Company of Biologists Ltd.)
original gene sequence has resulted in multiple variants that can be used effec-
tively in organisms ranging from animals and plants to fungi and microbes. The
fluorescence efficiency has also been improved, and variants have been gener-
ated with altered absorption and emission spectra from the blue-green, like blue
fluorescent protein (BFP), to the far visible red. Other, related fluorescent proteins
have since been discovered (for example, in corals) that also extend the range into
the red region of the spectrum, like red fluorescent protein (RFP).
One of the simplest uses of GFP is as a reporter molecule, a fluorescent probe
to monitor gene expression. A transgenic organism can be made with the
GFP-coding sequence placed under the transcriptional control of the promoter
belonging to a gene of interest, giving a directly visible readout of the gene’s
expression pattern in the living organism (Figure 9–17). In another applica-
tion, a peptide location signal can be added to the GFP to direct it to a particular 2 µm
cell compartment, such as the endoplasmic reticulum or a mitochondrion (see
Figure 9–18 GFP-tagged proteins. This
Figure 9–25B), lighting up these organelles so they can be observed in the cultured mammalian cell is expressing EB3,
living state. a plus-end tracking protein that is fused
The GFP DNA-coding sequence can also be inserted at the beginning or end of to a GFP-derived blue fluorescent protein
the coding sequence for another protein, yielding a chimeric product consisting (BFP). These proteins associate with
of that protein with a new GFP domain attached. In many cases, this GFP fusion the plus ends of growing microtubules
(see Figure 16–49), and their dynamics
protein behaves in the same way as the original protein, directly revealing its loca- can be followed as they appear to zoom
tion and activities by means of its genetically encoded fluorescence (Figure 9–18). brightly around the cell. (Courtesy of
It is often possible to prove that the GFP fusion protein is functionally equivalent Carl Zeiss Microscopy, GmbH.)
to the untagged protein, for example by using it to rescue a mutant lacking that
protein. GFP tagging is the clearest and most unequivocal way of showing the dis-
tribution and dynamics of a protein in a living organism (see Movie 16.8).
Protein Dynamics Can Be Followed in Living Cells

Fluorescent proteins are now exploited not just to see where in a cell a particular
protein is located, but also to uncover its kinetic properties and to find out whether
it might interact with other molecules. We now describe techniques in which
fluorescent proteins are used in this way.
First, interactions between one protein and another can be monitored by
Förster resonance energy transfer, also called fluorescence resonance energy
transfer but both abbreviated to FRET. In this technique, two molecules of inter-
est are each labeled with a different fluorochrome, chosen so that the emission
spectrum of one fluorochrome, the donor, overlaps with the absorption spec-
trum of the other, the acceptor. If the two proteins interact in such a way as to
bring their fluorochromes into very close proximity (closer than about 5 nm),
one fluorochrome, when excited, can transfer energy from the absorbed light
directly (by resonance, nonradiatively) to the other. Thus, when the complex is
illuminated at the excitation wavelength of the first fluorochrome, fluorescent
light is produced at the emission wavelength of the second (Figure 9–19). This
method can be used with two different spectral variants of GFP as fluorochromes
to monitor processes such as the interaction of signaling molecules with their
receptors (see Figure 15–49) or proteins in macromolecular complexes at specific
locations inside living cells. The FRET can be measured by quantifying the reduc-
tion of the donor fluorescence in the presence of the acceptor. The efficiency of
FRET is inversely proportional to the sixth power of the distance between the
donor and acceptor molecules and so is extremely sensitive to small changes
in distance.
The genes encoding GFP and related fluorescent proteins can be engineered
to produce protein variants, usually with one or more amino acid changes, that
fluoresce only weakly under normal excitation conditions but can be induced to
fluoresce either more strongly or with a color shift (for example, from green to
red) by activating them with a strong pulse of light at a different wavelength in Figure 9–19 Fluorescence resonance
a process called photoactivation. In principle, the biologist can then follow the energy transfer (FRET). To determine
local in vivo behavior of any protein that can be expressed as a fusion with one of whether (and when) two proteins interact
inside a cell, the proteins are first produced
as fusion proteins attached to different
color variants of green fluorescent protein
blue fluorescent green fluorescent (GFP). (A) In this example, protein X is
protein blue protein green coupled to a blue fluorescent protein,
violet light blue light
light emission light which is excited by violet light and emits
emission
excitation excitation blue light; protein Y is coupled to a green
fluorescent protein, which is excited by blue
light and emits green light. (B) If protein
X and Y do not interact, illuminating the
sample with violet light yields fluorescence
only from the blue fluorescent protein.
protein X protein Y
(C) When protein X and protein Y interact,
(A) the resonance transfer of energy, FRET,
can now occur. Illuminating the sample
blue FRET green with violet light excites the blue fluorescent
violet violet
light light light light protein, which transfers its energy to the
IN OUT IN OUT green fluorescent protein, resulting in an
emission of green light. The fluorochromes
must be quite close together—within about
1–5 nm of one another—for FRET to occur.
Because not every molecule of protein X
and protein Y is bound at all times, some
blue light may still be detected. But as the
(B) NO PROTEIN INTERACTION (C) PROTEIN INTERACTION two proteins begin to interact, emission
NO EXCITATION OF GREEN FLUORESCENCE RESONANCE from the donor blue fluorescent protein
FLUORESCENT PROTEIN; ENERGY TRANSFER; falls as the emission from the acceptor
BLUE LIGHT DETECTED GREEN LIGHT DETECTED GFP rises.
(A) (C)
before photobleach after photobleach 90 sec pre-bleach
0.8
fluorescence intensity
recovery
0.6
(B)
area to be bleached plasma fluorescence 0.4 mobile
photobleached membrane recovery fraction
0.2
0
0 5 10 15 20 25 30
time (seconds)
bleach
Figure 9–20 Fluorescence recovery after photobleaching (FRAP). A strong focused pulse
of laser light will extinguish, or bleach, fluorescent proteins. By selectively photobleaching a set
of fluorescently tagged protein molecules within a defined region of a cell, the microscopist can
monitor recovery over time, as the remaining fluorescent molecules move into the bleached region
(see Movie 10.6). (A) This cultured mammalian cell is expressing an integral membrane protein
called CD86, which is fused with a fluorescent protein. CD86 is a co-stimulatory protein present
in the plasma membrane of antigen-presenting cells and is required for the activation of T cells
(see Figure 24–34). After a small region of the plasma membrane is selectively photobleached, the
remaining fluorescent molecules diffuse rapidly within the plane of the membrane and populate
the bleached region. This recovery can be followed as a function of time. (B) Schematic diagram of
the experiment shown in A. (C) Measurements of the fluorescence intensity in the bleached area as
a function of time can be plotted as a fluorescence recovery curve. From such graphs quantitative
data can be derived about the rate of recovery and the fraction of fluorescent protein molecules that
are either mobile or immobile. (A, from S. Dorsch et al., Nat. Methods 6:225–230, 2009.)
these GFP variants. These genetically encoded, photoactivatable, fluorescent pro-

teins allow the lifetime and behavior of any protein to be studied independently
of other newly synthesized proteins.
Another way to exploit GFP fused to a protein of interest is known as fluores-
cence recovery after photobleaching (FRAP). Here, a strong focused beam of
light from a laser is used to extinguish the GFP fluorescence in a specified region
of the cell, after which one can analyze the way in which remaining unbleached
fluorescent protein molecules move into the bleached area as a function of time.
This technique, like photoactivation, can deliver valuable quantitative data about
a protein’s kinetic parameters, such as diffusion coefficients, active transport
rates, or binding and dissociation rates from other proteins (Figure 9–20). 100 µm
Fluorescent Biosensors Can Monitor Cell Signaling Figure 9–21 Visualizing intracellular Ca21
concentrations by using a fluorescent
Extracellular signals cause rapid and transient changes in the concentration indicator. The branching tree of dendrites
of intracellular signaling molecules that play an important role in how cells of a Purkinje cell in the cerebellum receives
more than 100,000 synapses from other
respond. But how to see and measure such dynamic and rapid changes remains a neurons. The output from the cell is
challenge. Changes in the concentration of some of these molecules, for exam- conveyed along the single axon seen
ple Ca2+ ions, can be analyzed using simple ion-sensitive indicators, whose light leaving the cell body at the bottom of the
emission reflects the local Ca2+ ion concentration (Figure 9–21, and see also picture. This image of the intracellular
Figure 15–31). However, the most sensitive indicators available are a range of Ca2+ concentration in a single Purkinje
cell (from the brain of a guinea pig) was
genetically encoded biosensors, all based on the growing family of fluorescent taken with a low-light camera and the
proteins described earlier. These sensors can be synthesized by the specific cells Ca2+-sensitive fluorescent indicator
of interest and easily fused with protein tags that target them to specific desti- fura-2. The concentration of free Ca2+
nations within the cell. Here they can act as molecular informants, reporting is represented by different colors, red
back like spies on transient signaling events to the careful observer. To convert being the highest and blue the lowest.
The highest Ca2+ levels are present in the
information about changes in the level of a signaling molecule into changes in thousands of dendritic branches. (Courtesy
observable fluorescence intensity requires two key components: a sensing com- of D.W. Tank, J.A. Connor, M. Sugimori,
ponent that responds to the target signaling event, and a reporting component and R.R. Llinas.)
blue fluorescent Figure 9–22 Genetically encoded

protein fluorescent biosensors. (A) Here we show
FRET one strategy for constructing a fluorescent
yellow fluorescent
protein
biosensor for calcium ions. A sensor, in
this case calmodulin (see Figure 15–34),
undergoes a large conformational change
on binding Ca2+. This change brings
together the blue and yellow fluorescent
proteins to which each end of the sensor is
attached, close enough to undergo Förster
Ca2+ cAMP resonance energy transfer (FRET) and to
change the wavelength of the fluorescence
emission to yellow in response to a violet
FRET excitation light. By measuring the ratio of
fluorescence intensity at two wavelengths,
blue and yellow, we can determine the
concentration ratio of the Ca2+-bound
indicator to the Ca2+-free indicator, thereby
providing an accurate measurement of
the free Ca2+ concentration. (B) This
(A) sensor (B) sensor panel illustrates a similar strategy used
to construct a biosensor for cAMP. In
this case the sensor is a cAMP-regulated
time 0 1 sec 4 sec 18 sec
guanine nucleotide exchange factor, which
again undergoes a large conformational
change, enough to move the two attached
fluorescent proteins farther apart, thus
abolishing their FRET. Hence the emitted
light is switched from yellow to blue. (C) A
calcium biosensor, similar to that shown in
A, is genetically encoded and expressed
meristem poked in an Arabidopsis seedling. When a cell
with a glass needle 20 µm
in the epidermis, on the side of the shoot
(C) apical meristem, is pricked with a small
glass needle, calcium enters the cell
from the extracellular environment, and
this response is rapidly propagated as a
that translates that response into a visible and quantifiable output. Many biosen- wave of calcium entering cells across the
sors use two connected fluorescent proteins that can be brought close enough entire surface of the meristem. Mechanical
together to undergo Förster resonance energy transfer (see Figure 9–19). Bring- signals help pattern plant morphogenesis,
ing them together, or indeed moving them apart, is a connecting sensor module. and transient calcium responses affect cell
The sensor is usually a protein or protein domain that undergoes a large confor- polarity. (C, from T. Li et al., Nat. Commun.
10:726–735, 2019. Reproduced with
mational change on binding to the target molecule. The general principle used permission of SNCSC.)
to construct a genetically encoded biosensor is shown in Figure 9–22. Measur-
ing the ratio of the intensities of light emitted by the two fluorescent proteins in
the biosensor provides a quantitative measure of the concentration of the target
molecule of interest. Many hundreds of such biosensors have been created. Some
can monitor and measure small molecules in living cells, such as Ca2+, cAMP, IP3,
NADPH (and hence redox state), H+ ions (and hence pH), and neurotransmitters
such as acetylcholine and glutamate. Others can measure the activity of kinases,
phosphatases, active caspases, and even temperature.
Imaging of Complex Three-dimensional Objects Is Possible with

the Optical Microscope
For ordinary light microscopy, as we have seen, a tissue has to be sliced into thin
sections to be examined; the thinner the section, the crisper the image. Because
information about the third dimension is lost upon sectioning, how, then, can we
get a picture of the three-dimensional architecture of a cell or tissue, and how can
we view the microscopic structure of a specimen that, for one reason or another,
cannot first be sliced into sections? Although an optical microscope is focused on
a particular focal plane within a three-dimensional specimen, all the other parts
of the specimen, above and below the plane of focus, are also illuminated, and
the light originating from these regions contributes to the image as out-of-focus
blur. This can make it very hard to interpret the image in detail and can lead to fine
image structure being obscured by the out-of-focus light.
(A) (B)
10 µm
Figure 9–23 Image deconvolution. (A) A light micrograph of a Caenorhabditis elegans embryo,
fluorescently labeled for microtubules (green), mitochondria (red), and DNA (blue). Detail at any one
level of focus is blurred by light from out-of-focus levels of the specimen. (B) After deconvolution
of the three-dimensional stack of images, an optical section at the same level of focus shows a
much crisper image with more contrast and much reduced blurring. (A and B, from D. Sage et al.,
Methods 115:28–41, 2017, doi 10.1016/j.ymeth.2016.12.015. With permission from Elsevier.)
Two distinct but complementary approaches help to solve this problem: one
is computational, the other optical. These three-dimensional microscopic imag-
ing methods make it possible to focus on a chosen plane in a thick specimen
while rejecting the light that comes from out-of-focus regions above and below
that plane. Thus, one sees a crisp, thin optical section. From a series of such optical
sections taken at different depths and stored in a computer, a three-dimensional
image can be reconstructed (Movie 9.1). The methods do for the microscopist
what the computed tomography (CT) scanner does (by different means) for the
radiologist investigating a human body: both machines give detailed sectional
views of the interior of an intact structure.
The computational approach is often called image deconvolution. To under-
stand how it works, remember that the wave-like nature of light means that the
microscope lens system produces a small blurred disc as the image of a point
source of light (see Figure 9–4), with increased blurring if the point source lies
above or below the focal plane. This blurred image of a point source is called the
point spread function (see Figure 9–29). An image of a complex object can then
be thought of as being built up by replacing each point of the three-dimensional
specimen by a corresponding blurred disc, resulting in an image that is blurred
overall. For deconvolution, a computer program uses the measured point spread
function of a point source of light from that particular microscope to determine
what the effect of the blurring would have been on the image, and then applies an
equivalent “deblurring” (deconvolution), turning the blurred three-dimensional
image into a series of clean optical sections, albeit still constrained by the diffrac-
tion limit (Figure 9–23).
The Confocal Microscope Produces Optical Sections

by Excluding Out-of-Focus Light
The confocal microscope achieves a result similar to that of deconvolution, but
does so by manipulating the light before it is measured; it is an analog technique
rather than a digital one. The optical details of the confocal microscope are com-
plex, but the basic idea is simple, as illustrated in Figure 9–24, and the results are
far superior to those obtained by conventional light microscopy.
The confocal microscope is generally used with fluorescence optics (see
Figure 9–10C), but instead of illuminating the whole specimen at once, in the usual
way, the optical system at any instant focuses a spot of light onto a single point at
a specific depth in the specimen. This requires a source of pinpoint illumination
(A) (B) (C) (D)
detector
detector
detector
confocal
B
pinholes
dichroic
mirror
laser
objective
3D specimen
point of
focus
fluorescent specimen is illuminated emitted fluorescent emitted light from out- Figure 9–24 The confocal fluorescence
with a focused point of light from light from in-focus of-focus point is out of
a pinhole point is focused at focus at pinhole and microscope. (A) This simplified diagram
pinhole and reaches is largely excluded shows that the basic arrangement of
detector from detector optical components is similar to that of the
standard fluorescence microscope shown
in Figure 9–10C, except that a laser is used
that is usually supplied by a laser whose light has been passed through a pinhole. to illuminate a small pinhole whose image
The fluorescence emitted from the illuminated material is collected at a suitable is focused at a single point in the three-
light detector and used to generate an image. A pinhole aperture is placed in front dimensional (3D) specimen. (B) Emitted
fluorescence from this focal point in the
of the detector, at a position that is confocal with the illuminating pinhole; that specimen is focused at a second (confocal)
is, precisely where the rays emitted from the illuminated point in the specimen pinhole. (C) Emitted light from elsewhere
come to a focus. Thus, the light from this point in the specimen converges on this in the specimen is not focused at the
aperture and enters the detector. pinhole and therefore does not contribute
By contrast, the light emitted from regions of the specimen that are out of to the final image. By scanning the beam
of light across the specimen, a very sharp
focus is also out of focus at the pinhole aperture and is therefore largely excluded two-dimensional image of the exact plane
from the detector. To build up a two-dimensional image, data from each point of focus is built up that is not significantly
in the plane of focus are collected sequentially by scanning across the field from degraded by light from other regions of
one side to the other in a regular pattern of pixels and are displayed on a com- the specimen. (D) Commercial versions of
laser scanning confocal microscopes can
puter screen. Although not shown in Figure 9–24, the scanning is usually done
be configured for both upright and inverted
by deflecting the beam with an oscillating mirror placed between the dichroic microscopes. Shown here is a standard
(beam-splitting) mirror and the objective lens in such a way that the illuminating upright confocal microscope. (D, courtesy
spot of light and the confocal pinhole at the detector remain strictly in register. of Andrew Davis.)
Variations in design now allow the rapid collection of data at video rates.
The confocal microscope has been used to resolve the structures of numer-
ous complex three-dimensional objects (Figure 9–25), from large multicellular
Figure 9–25 Confocal fluorescence

microscopy produces clear optical
sections and three-dimensional data
sets. (A) The elaborate cup-shaped trap
of the carnivorous water plant, Utricularia
gibba. A stack of 452 separate confocal
images using a fluorescent label for the
cell walls was assembled to produce the
image. (B) A reconstruction of an object
can be assembled from a stack of optical
sections. In this case, and at a vastly
different scale, the complex branching
structure of the mitochondrial compartment
in a single live yeast cell is shown. (A,
(A) (B) courtesy of Karen Lee, Claire Bushell, and
50 µm 2 µm Enrico Coen; B, courtesy of Stefan Hell.)
Figure 9–26 Multiphoton imaging.

Infrared laser light causes less damage
to living cells than does visible light and
can also penetrate farther, allowing
microscopists to peer deeper into living
tissues. The two-photon effect, in which
a fluorochrome can be excited by two
coincident infrared photons instead of a
single high-energy photon, allows us to see
nearly 0.5 mm inside the cortex of a live
structures to subcellular structures; for example, the networks of cytoskeletal mouse brain. A dye, whose fluorescence
fibers, the dynamics of organelles, and the arrangements of chromosomes and changes with the calcium concentration,
genes in the nucleus. reveals active synapses (yellow) on the
dendritic spines (red) that change as a
The relative merits of deconvolution methods and confocal microscopy for function of time; in this case, there is a day
three-dimensional optical microscopy depend on the specimen being imaged. between each image. (Courtesy of Thomas
Confocal microscopes tend to be better for thicker specimens with high levels Oertner and Karel Svoboda.)
of out-of-focus light. They are also generally easier to use than deconvolution
systems, and the final optical sections can be seen quickly. In contrast, the
complementary metal-oxide semiconductor (CMOS) cameras that are used for
deconvolution systems are extremely efficient at collecting almost every photon
emitted, and they can be used to make detailed three-dimensional images from
specimens that are too weakly stained or too easily damaged by the bright light
used for confocal microscopy.
Both methods, however, have another drawback; neither is good at coping
with very thick specimens. Deconvolution methods quickly become ineffective
any deeper than about 40 μm into a specimen, while confocal microscopes can
only obtain images up to a depth of about 150 μm. Special microscopes can now
take advantage of the way in which fluorescent molecules are excited, to probe
even deeper into a specimen. Fluorescent molecules are usually excited by a
single high-energy photon, of shorter wavelength than the emitted light, but they
can in addition be excited by the absorption of two (or more) photons of lower
energy, as long as they both arrive within a femtosecond or so of each other. The
use of this longer-wavelength excitation has some important advantages. In addi-
tion to reducing background noise, red or near-infrared light can penetrate deeper
into a specimen. Multiphoton microscopes, constructed to take advantage of this
two-photon effect, can obtain sharp images, sometimes even at a depth of half
a millimeter within a specimen. This is particularly valuable for studies of living
tissues, notably in imaging the dynamic activity of synapses and neurons just
below the surface of living brains (Figure 9–26).
Superresolution Fluorescence Techniques Can Overcome

Diffraction-limited Resolution
The variations on light microscopy we have described so far are all constrained
by the classic diffraction limit to resolution described earlier; that is, to about
0.2 μm, or 200 nm (see Figure 9–5). Yet many cellular structures—from nuclear
pores and ribosomes to nucleosomes and clathrin-coated pits—are much
smaller than this and so are unresolvable by conventional light microscopy. How-
ever, several approaches are now available that bypass the limit imposed by the
diffraction of light, and some can now successfully resolve objects as small as
10 nm, a remarkable, twentyfold improvement.
The first of these so-called superresolution approaches, structured illumi-
nation microscopy (SIM), is a fluorescence imaging method with a resolution of
about 100 nm, or twice the resolution of conventional bright-field microscopy.
SIM overcomes the diffraction limit by using a grated or structured pattern of
light to illuminate the sample. The microscope’s physical setup and operation are
quite complex, but the general principle can be thought of as similar to creating
a moiré pattern, an interference pattern created by overlaying two grids with dif-
ferent angles or mesh sizes (Figure 9–27). The illuminating grid and the sample
features combine into an interference pattern in which features smaller than the
grid spacing are transformed into larger patterns. This results in original features
Figure 9–27 Structured illumination

microscopy. The principle, illustrated here,
is to illuminate a sample with patterned light
and measure the moiré pattern. Shown are
(A) the pattern from an unknown structure
and (B) a defined grid pattern. (C) When
these are combined, the resulting moiré
pattern contains more information than
is easily seen in A, the original pattern. If
(A) (B) (C) the known pattern (B) has higher spatial
frequencies, then better resolution will
result. However, because the spatial
patterns that can be created optically
beyond the classical limit being transformed so that they can now be imaged by are also diffraction-limited, SIM can only
the optical system. Computer image processing can then be used to restore them improve the resolution by about a factor
into an image that has a resolution up to twice the classical limit. Illumination of 2. (From B.O. Leung and K.C. Chou,
by a grid means that the parts of the sample in the dark stripes of the grid are not Appl. Spectrosc. 65:967–980, 2011. With
permission from SAGE.)
illuminated and therefore not imaged, so the imaging is repeated several times
(usually three) after translating the grid through a fraction of the grid spacing
between each image. As the interference effect is strongest for image components
close to the direction of the grid bars, the whole process is repeated with the grid
pattern rotated through a series of angles to obtain an equivalent enhancement
in all directions. Finally, mathematically combining all these separate images by
computer creates an enhanced superresolution image. SIM is versatile because
it can be used with any fluorescent dye or protein, and combining SIM images
captured at consecutive focal planes can create three-dimensional data sets
(Figure 9–28).
To get around the diffraction limit, two other superresolution techniques
exploit aspects of the point spread function, a property of the optical system
mentioned earlier. The point spread function is the distribution of light inten-
sity within the three-dimensional, blurred image that is formed when a single
point source of light is brought to a focus with a lens. Instead of being identical
to the point source, the image has an intensity distribution that is approximately
described by a Gaussian distribution, which in turn determines the resolution
of the lens system. Two points that are closer than the width at half-maximum
(A) (B) (C)

2 µm
Figure 9–28 Structured illumination microscopy can be used to create three-dimensional data. These three-
dimensional projections of the meiotic chromosomes at pachytene in a maize cell show the paired lateral elements of the
synaptonemal complexes. (A) The chromosome set has been stained with a fluorescent antibody to cohesin and is viewed
here by conventional fluorescence microscopy. Because the distance between the two lateral elements is about 200 nm,
the diffraction limit, the two lateral elements that make up each complex are not resolved. (B) In the three-dimensional SIM
image, the improved resolution enables each lateral element, about 100 nm across, to be clearly resolved, and the two
chromosomes of each separate pair can be seen to coil around each other. (C) Because the complete three-dimensional data
set for the whole nucleus is available, the path of each separate pair of chromosomes can be traced and artificially assigned a
different color. (Courtesy of C.J. Rachel Wang, Peter Carlton, and Zacheus Cande.)
point source of light

Y
X
200 nm
lens Z
intensity
intensity
Y
X
diffraction-limited
image of the point X X
source 200 nm
(A) 200 nm (B) (C)
Figure 9–29 The point spread function of a lens determines resolution. (A) When a point
source of light is brought to a focus by a lens system, diffraction effects mean that, instead of being
imaged as a point, it is blurred in all dimensions. As shown, the point spread function is elongated,
meaning that the resolution is better in the XY axes than along the Z axis. (B) In the plane of the
image, the distribution of light approximates a Gaussian distribution, whose width at half-maximum
height under ideal conditions is about 200 nm. (C) Two separate point sources that are about
200 nm apart can still just be distinguished as separate objects in the image, but if they are any
nearer than that, their images will overlap and not be resolvable.
height of this distribution will become hard to resolve because their images
overlap too much (Figure 9–29).
In fluorescence microscopy, the excitation light is focused to a spot on the
specimen by the objective lens, which then captures the photons emitted by any
fluorescent molecule that the beam has raised from a ground state to an excited
state. Because the excitation spot is blurred according to the point spread func-
tion, fluorescent molecules that are closer than about 200 nm will be imaged as
a single blurred spot. One approach to increasing the resolution is to switch all
Figure 9–30 Superresolution microscopy
the fluorescent molecules at the periphery of the blurry excitation spot back to can be achieved by reducing the size of
their ground state or to a state where they no longer fluoresce in the normal way, the point spread function. (A) The size of
leaving only those at the very center to be recorded. This can be done in practice a normal focused beam of excitatory light.
by adding a second, very bright laser beam that wraps around the excitation beam (B) An extremely strong superimposed
like a torus. The wavelength and intensity of this second beam are adjusted so as laser beam, at a different wavelength
and in the shape of a torus, or doughnut,
to switch the fluorescent molecules off everywhere except at the very center of the depletes emitted fluorescence everywhere
point spread function, a region that can be as small as 20 nm across (Figure 9–30). in the specimen except right in the center
of the beam, reducing the effective width
effective fluorescence of the point spread function (C). As the
excitation spot depletion beam spot specimen is scanned, this small point
spread function can then build up a crisp
image in a process called STED (stimulated
emission depletion) microscopy. (D) Here,
STED microscopy is used to examine
the structure of the nuclear pore. Fixed
samples of the nuclear envelope have been
stained by indirect immunofluorescence,
using antibodies to different nuclear pore
(A) (B) (C) components. Membrane ring proteins (see
200 nm Figure 12–55) have been stained red while
the FC repeat proteins that form fibrils in
the center of the pore are stained green.
(E) An enlargement of the boxed region
shows the clear eightfold symmetry of the
membrane ring proteins and the central
fibrillar region with a resolution of about
20 nm. [A, B, and C, from G. Donnert
et al., Proc. Natl. Acad. Sci. USA 103:
11440–11445, 2006. Copyright 2006
National Academy of Sciences. With
permission from National Academy of
Sciences; D and E, from F. Gottfert et al.,
(D) Biophysical Journal 105(1):PL01–L03,
(E)
500 nm 150 nm 2013. With permission from Elsevier.]
The fluorescent probes used must be in a special class that is photoswitchable:

their emission can be reversibly switched on and off with lights of different wave-
lengths. As the specimen is scanned with this arrangement of lasers, in much
the same way as in a confocal microscope, fluorescent molecules are switched
on and off, and the small point spread function at each location is recorded. The
diffraction limit is breached because the technique ensures that similar but very
closely spaced molecules are in one of two different states, either fluorescing or
dark. This approach is called STED (stimulated emission depletion) microscopy,
and various microscopes using versions of the general method are now in wide
use. Resolutions of 20 nm have been achieved in biological specimens (see
Figure 9–30).
Single-Molecule Localization Microscopy Also Delivers

Superresolution
If a single fluorescent molecule is imaged, it appears as a circular blurry disc
about 200 nm across, but if sufficient photons have contributed to this image,
then the precise mathematical center of the disc-like image, and therefore the
position of that fluorescent molecule, can be determined very accurately, often
to within a few nanometers (Figure 9–31). But the problem with a specimen
that contains a large number of adjacent fluorescent molecules, as we saw ear-
lier, is that they each contribute blurry, overlapping point spread functions to
the image, making the exact position of any one molecule impossible to resolve.
Another way around this limitation is to arrange for only a very few, clearly sep-
arated molecules to actively fluoresce at any one moment. The exact position
of each of these can then be computed, before subsequent sets of molecules
are examined.
In practice, this can be achieved by using lasers to sequentially switch on
a sparse subset of fluorescent molecules in a specimen containing switch-
able fluorescent labels. There are now hundreds of such labels, and they fall
into three classes: photoactivated labels, which switch for example from dark
to green; photoconvertible labels, which switch for example from green to red;
and photoswitchable labels, which switch back and forth. Labels are activated,
for example, by illumination with near-ultraviolet light, which modifies a small
subset of molecules so that they fluoresce when exposed to an excitation beam
at another wavelength. These are then imaged before bleaching quenches their
fluorescence, and a new subset is activated. Each molecule emits a few thousand
photons in response to the excitation before switching off, and the switching pro-
cess can be repeated tens or even hundreds of thousands of times, allowing the
exact coordinates of a very large set of single molecules to be determined. The full
set can be combined and digitally displayed as an image in which the computed
exact position of the

100 photons 1000 photons 10,000 photons fluorescent molecule
200 nm
Figure 9–31 Single fluorescent molecules can be located with great accuracy. Determination
of the exact mathematical center of the blurred image of a single fluorescent molecule becomes
more accurate as more photons contribute to the final image. The point spread function described
in the text dictates that the size of the molecular image is about 200 nm across, but in very bright
specimens, the position of its center can be pinpointed to within a nanometer or so. (From A.L.
McEvoy et al., BMC Biol. 8:106, 2010. With permission of the authors.)
successive cycles of activation and bleaching allow well-separated single fluorescent molecules Figure 9–32 Single-molecule localization
to be detected microscopy (SMLM). (A) In this imaginary
specimen, sparse subsets of fluorescent
molecules are individually switched on
briefly and then bleached. The exact
positions of all these well-spaced molecules
can be gradually added together and built
up into an image at superresolution. (B) In
this portion of a cell, the microtubules have
the exact center of each fluorescent molecule is determined and its position added to the map been fluorescently labeled and imaged (left)
in a TIRF microscope (see Figure 9–38)
and (right) at superresolution in a PALM
microscope. The diameter of each
microtubule on the right now resembles
its true size, about 25 nm, rather than the
250 nm for each microtubule in the blurred
diffraction-limited image on the left.
(B, courtesy of Shinsuke Niwa.)
a superresolution image of the fluorescent structure is built up as the positions of tens of thousands of
successive small groups of molecules are added to the map
(A)
(B)
1 µm
location of each individual molecule is exactly marked (Figure 9–32). The two
main methods of single-molecule localization microscopy (SMLM) have been
variously termed photoactivated localization microscopy (PALM) or stochastic
optical reconstruction microscopy (STORM).
By switching the fluorophores off and on sequentially in different regions
of the specimen as a function of time, all the superresolution imaging methods
described above allow the resolution of molecules that are much closer together
than the 200-nm diffraction limit. In STED, the locations of the molecules are
determined by using optical methods to define exactly where their fluorescence
will be on or off. In PALM and STORM, individual fluorescent molecules are
switched on and off at random over a period of time, allowing their positions
to be accurately determined. PALM and STORM techniques have depended on
the development of novel fluorescent probes that exhibit the appropriate switch-
ing behavior. STORM originally relied on photoswitchable dyes, while PALM
used photoswitchable fluorescent proteins, but the general principle is the same
for both. All these methods can incorporate multicolor imaging (Figure 9–33),
Figure 9–33 Multiple structures that are below the diffraction-limited resolution can be
imaged by single-molecule localization microscopy. (A) Two recently divided Escherichia coli
cells imaged in a STORM microscope with a resolution of about 20 nm. The cells are stained (A)
1 µm
with three separate switchable fluorescent labels: the membrane is labeled green, the recently
segregated DNA molecules are blue, and the ends of the two replicated chromosomes are seen
as two bright white dots. (B) In this nerve cell, evenly spaced ring-like structures of actin (red) are
wrapped around the circumference of the axon with a periodicity of about 190 nm, just smaller than
the diffraction limit to resolution. In between are similarly spaced structures of spectrin (blue). This
periodic actin–spectrin cytoskeletal framework helps support the long thin axons of nerve cells.
Such images depend heavily on the development of new, very fast-switching, and extremely bright
fluorescent probes. (A, from C.K. Spahn et al., Sci. Rep. 8:14768, 2018, doi.org/10.1038/s41598- (B)
018-33052-3; B, from K. Xu et al., Science 339:452–456, 2013, doi 10.1126/science.1232251.) 1 µm
fluorescent labels
gel monomers
gel
fix cell gel proteinase gel

formation digestion expansion
in water
(A) (×4)
Figure 9–34 Expansion microscopy. (A) Although the technique has numerous variations, the
essential features are that the fluorescently labeled sample is embedded in a polymer gel to
which the fluorescent labels are covalently attached. After a proteinase digestion step, the gel is
immersed in water and everything in the sample expands equally in every direction, usually by
between 4 and 10 times, thus allowing details to be seen far more easily. (B) A peroxisome, whose
membrane has been labeled with a fluorescent probe, appears in a confocal microscope as a
blurred, diffraction-limited disc. (C) After expansion by a factor of 10, the image is captured with a
standard epifluorescence microscope and, after deconvolution, shows the peroxisomal membrane
well resolved and with a resolution of 25 nm. (From S. Truckenbrodt et al., EMBO Rep. 19:e45836, (B) (C)
2018, doi 10.15252/embr.201845836.) 200 nm
and to some extent live-cell imaging in real time. Ending the long reign of the
diffraction limit has reinvigorated light microscopy and its place in cell biology
research.
Expanding the Specimen Can Offer Higher Resolution,

but with a Conventional Microscope
All the approaches to improvement of resolution that we have discussed so far
have centered on increasingly sophisticated and expensive developments of the
microscope itself. Looking at the problem from the other end, the specimen end,
swelling the sample to physically make it larger would in theory allow higher-
resolution imaging, while still using a conventional fluorescence microscope.
A new specimen preparation technique, called expansion microscopy (ExM),
does exactly that. The process starts by staining the fixed sample with fluorescent
labels such as antibodies that target the molecules of interest. The labeled speci-
men is then treated with a chemical cross-linker and incubated with acrylate and
acrylamide monomers. These monomers then polymerize to form a polyelectro-
lyte gel that simultaneously incorporates the cross-linked labels. With the labels
covalently cross-linked to the polymer gel, and locked in their original relative
positions, cellular material in the sample, predominantly proteins that might hin-
der subsequent expansion, is then carefully digested away. The gel containing the
labeled specimen is now gently swollen by removing the buffer salts with water, so
that it expands equally in all directions by between 4 and 10 times (Figure 9–34A).
Two fluorochromes that were initially 100 nm apart, and consequently below the
diffraction-limited resolution of a standard microscope, will now be 0.4–1.0 μm
apart and are therefore easily resolved (Figure 9–34B and C). “Blowing-up” the
sample allows effective superresolution to be enjoyed at up to 25 nm and without
costly hardware (Figure 9–35A and B).
Expansion microscopy is proving valuable for detecting and quantitating which
RNA transcripts are expressed in which individual cells in the brain. If all RNA
molecules present are anchored firmly to the polymer gel before the expansion
step, then the sample can be washed and re-probed sequentially with multiple
fluorescent RNA probes using in situ hybridization (Figure 9–35C and D). Expan-
sion takes place in all directions, and so depth information is also retrievable at
higher resolution. Expansion microscopy samples can still be imaged by either
confocal or light-sheet microscopy (discussed next), and deconvolution methods
can still be used on the images—both help to improve three-dimensional imaging
of large specimens.
Figure 9–35 Expansion microscopy.

(A and B) Two orthogonal views of the
same cultured human nasal epithelial
cells that have been stained with a
fluorescent dye, expanded by ten times,
and then imaged by conventional confocal
microscopy. The hollow centers of ciliary
basal bodies, which are not resolvable
by conventional microscopy, are clearly
visible in both top view (A) and side view (B)
(red arrows) (see Movie 9.1). (C and D)
A segment of mouse brain lateral
hypothalamus, 800 × 800 × 300 μm,
that has been expanded by a factor of
(C) 2, probed by sequential rounds of in situ
200 µm RNA hybridization, and imaged by light-
(A) sheet microscopy. The cellular expression
patterns of six different genes are shown:
Gad1 (red), Slc17a6 (green), Hcrt (blue),
Trh (yellow), Calb2 (magenta), and Meis2
(cyan). (A and B, courtesy of Hugo
Damstra, Lukas Kapitein, and Paul Tillberg;
C and D, courtesy of Yuhan Wang, Mark
Eddison, Scott Sternson, and Paul Tillberg.)
(B) (D)
2 µm 30 µm
Large Multicellular Structures Can Be Imaged Over Time

Many problems in cell biology involve being able to follow the movement and
behavior of cells in multicellular living organisms, in early embryo development
for example. Other problems require the ability to disentangle the complexity of
cellular interactions in large and dense tissues, for example the millions of con-
nections between the neurons of the brain. The side effects of prolonged exposure
to high levels of light in the first case, and depth and out-of-focus fluorescence in
the second, mean that most of the techniques we have discussed so far cannot
help. One way of eliminating a lot of the out-of-focus fluorescence is to arrange for
the beam of light from the excitation laser to illuminate the specimen from a direc-
tion perpendicular to the axis from which the emitted fluorescence is viewed. In
this arrangement, called light-sheet microscopy, a thin sheet of laser light, less than
a micrometer thick, is scanned through the specimen, exciting only the labeled
molecules at that depth in the sample to emit their fluorescence (Figure 9–36).
There are many advantages to this method: it results in high-contrast images
with very low photobleaching or photodamage, and three-dimensional informa-
tion is readily obtained. It is also quick. Variants of the method allow ultrathin
light sheets to scan through successive planes of a sample at a rate of hundreds
of planes a second. The long-term, three-dimensional observation of living cells
is a major application, for example in following early embryonic development
in flies or zebrafish over a period of days. In fixed brain samples, the complex
architecture of all the cells and their interconnections can be disentangled
Figure 9–36 Light-sheet microscopy. A
simple diagram showing how a very thin
fluorescence observed by
detection objective sheet of light that is projected (usually from
a special cylindrical microscope objective
specimen lens) through a large specimen excites
only those fluorescent labels in the thin
plane that is illuminated. The resulting
only fluorescent fluorescence is observed by an objective
thin sheet of labels in this lens that is placed perpendicular to the light
laser light from plane are excited sheet. This means that, by progressively
cylindrical lens
moving the specimen stage, multiple,
sequential, and very sharp optical sections
can be obtained rapidly and then digitally
combined into a three-dimensional image.
Figure 9–37 Light-sheet microscopy

in the brain. A 1-mm-thick portion
of a mouse brain has been prepared
axon for expansion microscopy and then
imaged with a light-sheet microscope.
Reconstructions of thousands of optical
sections allow the tracing of individual
neurons and all their connections, such as
cell body this pyramidal neuron (left) from the visual
cortex. On the right is shown the complex
cellular context (green) for a short region of
one of the neuron’s basal dendrites (orange
basal dendrite with its spines shown in yellow).
dendrite (From R. Gao et al., Science 363:245–261,
2019, doi 10.1126/science.aau8302. With
permission from AAAS.)
100 µm 5 µm
(Figure 9–37 and Movie 9.1). Light-sheet microscopy can also be combined with
other techniques. Coupled with STED imaging, for example, superresolution is
attainable, and higher-resolution images can also be obtained by preparing the
sample for expansion microscopy.
Single Molecules Can Be Visualized by Total Internal Reflection Figure 9–38 TIRF microscopy allows
Fluorescence Microscopy the detection of single fluorescent
molecules near the cell surface.
As we have seen, the strong background fluorescence due to light emitted or scat- (A) TIRF microscopy uses excitatory laser
tered by out-of-focus molecules tends to blot out the fluorescence from any one light to illuminate the cover-slip surface
at the critical angle at which all the light
particular molecule of interest. This problem can be solved by the use of a special
is reflected by the glass–water interface.
optical technique called total internal reflection fluorescence (TIRF) microscopy. Some electromagnetic energy extends a
In a TIRF microscope, laser light shines onto the cover-slip surface at the precise short distance across the interface as an
critical angle at which total internal reflection occurs (Figure 9–38A). Because of evanescent wave that excites just those
total internal reflection, the light does not enter the sample, and the majority of molecules that are attached to the cover
slip or are very close to its surface.
fluorescent molecules are not, therefore, illuminated. However, electromagnetic (B) TIRF microscopy is used to follow the
energy does extend, as an evanescent field, for a very short distance beyond the formation of an individual clathrin-coated
surface of the cover slip and into the specimen, allowing just those molecules in pit and its subsequent endocytosis. In
the layer closest to the surface to become excited. When these molecules fluo- this image of the surface of the plasma
resce, their emitted light is no longer competing with out-of-focus light from the membrane of an Arabidopsis root cell,
a clathrin adaptor protein is tagged with
overlying molecules and can now be detected. TIRF has allowed several dramatic GFP. Individual pits can be followed over
experiments, for instance imaging of single motor proteins moving along micro- time. (C) The pit ringed in B is shown at
tubules or actin filaments. At present, the technique is restricted to a thin layer 1-second intervals, demonstrating that
about 200 nm below the cell surface. Although not strictly TIRF, decreasing the its appearance and its removal at the
angle of the incident light so that it is almost parallel to the cover slip can increase plasma membrane by endocytosis takes
place in about 10 seconds. (B and C,
the depth into the cell that can be examined, albeit not so uniformly, a feature from A. Johnson and G. Vert, Front.
useful in cells with an outer wall, such as those of plants and fungi (Figure 9–38B Plant Sci. 8:612, 2017, doi 10.3389
and C). /fpls.2017.00612.)
cell containing only those molecules in the

fluorescent molecules evanescent field fluoresce
~200 nm
cover slip
critical angle α
for total internal immersion oil
reflection
(A) laser light objective lens of (B) (C)

inverted microscope 5 μm 1 μm
Summary
Many light-microscope techniques are available for observing cells. Cells that
have been fixed and stained can be studied in a conventional light microscope,
whereas antibodies coupled to fluorescent dyes can be used to locate specific
molecules in cells in a fluorescence microscope. Living cells can be seen with phase-
contrast, differential-interference-contrast, dark-field, or bright-field microscopes.
All forms of light microscopy are facilitated by digital image-processing techniques,
which enhance sensitivity and refine the image. Confocal microscopy and image
deconvolution both provide thin optical sections and can be used to reconstruct
three-dimensional images.
Techniques are now available for detecting, measuring, and following almost
any desired molecule in a living cell. Fluorescent labels can be introduced to mea-
sure the concentrations of specific ions or signaling molecules in individual cells
or in different parts of a cell. Virtually any protein of interest can be genetically
engineered as a fluorescent fusion protein and then imaged in living cells by fluo-
rescence microscopy. The dynamic behavior and interactions of many molecules
can be followed in living cells by variations on the use of fluorescent protein tags, in
some cases at the level of single molecules. Various superresolution techniques can
circumvent the diffraction limit in different ways and resolve molecules separated
by distances as small as 20 nm.
LOOKING AT CELLS AND MOLECULES IN THE

ELECTRON MICROSCOPE
Light microscopy is limited in the fineness of detail that it can reveal. Microscopes
using other types of radiation—in particular, electron microscopes—can resolve
much smaller structures than is possible with visible light. This higher resolu-
tion comes at a cost: specimen preparation for electron microscopy is complex 0.14 nm
and it is harder to be sure that what we see in the image corresponds precisely
to the original living structure. It is possible, however, to use very rapid freezing
to preserve structures faithfully for electron microscopy. Digital image analysis
can be used to reconstruct three-dimensional objects by combining information
either from many individual particles or from multiple tilted views of a single
object. Together, these approaches extend the resolution and scope of electron
microscopy to the point at which we can faithfully image the detailed structures of
individual macromolecules and the complexes they form, even inside cells.
The Electron Microscope Resolves the Fine Structure of the Cell

The formal relationship between the diffraction limit to resolution and the wave-
length of the illuminating radiation (see Figure 9–5) holds true for any form of
radiation, whether it is a beam of light or a beam of electrons. With electrons,
however, the limit of resolution is very small. The wavelength of an electron Figure 9–39 The resolution of the
decreases as its velocity increases. In an electron microscope with an acceler- electron microscope. This transmission
ating voltage of 100,000 V, the wavelength of an electron is 0.004 nm. In theory, electron micrograph of a monolayer of
graphene resolves the individual carbon
the resolution of such a microscope should be about 0.002 nm, which is 100,000 atoms as bright spots in a hexagonal
times that of the conventional light microscope. Because the aberrations of an lattice. Graphene is a single isolated
electron lens are considerably harder to correct than those of a glass lens, how- atomic plane of graphite and forms the
ever, the practical resolving power of modern electron microscopes is, even basis of carbon nanotubes. The distance
with careful image processing to correct for lens aberrations, about 0.05 nm between adjacent bonded carbon atoms
is 0.14 nm (1.4 Å). Such resolution can
(0.5 Å) (Figure 9–39). This is because only the very center of the electron lenses only be obtained in a specially built
can be used, and the effective numerical aperture is tiny. Furthermore, problems transmission electron microscope in which
of specimen preparation, contrast, and radiation damage have generally lim- all lens aberrations are carefully corrected,
ited the normal effective resolution for biological objects to 1 nm (10 Å). This is and with optimal specimens; it is rarely
nonetheless about 200 times better than the resolution of the light microscope. achieved with most conventional biological
specimens. (From A. Dato et al., Chem.
Moreover, the performance of electron microscopes is improved by electron Commun. 40:6095–6097, 2009. With
illumination sources called field-emission guns. These very bright and coherent permission from the Royal Society
sources substantially improve the resolution achieved. of Chemistry.)
LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE 589
Figure 9–40 The principal features of

light source electron an inverted light microscope and a
gun transmission electron microscope.
These drawings emphasize the similarities
of overall design. Whereas the lenses in the
condenser lens light microscope are made of glass, those
in the electron microscope are magnetic
specimen coils. The electron microscope requires that
the specimen be placed in a vacuum. The
objective lens inset shows a routine transmission electron
microscope in use. (Photograph courtesy
of Andrew Davis.)
projector
eyepiece lens
lens
direct
viewing or
digital digital
camera camera
inverted transmission
light electron
microscope microscope
In overall design, the transmission electron microscope (TEM) is similar to an

inverted light microscope, albeit much larger (Figure 9–40). The source of illu-
mination is a filament or cathode that emits electrons at the top of a cylindrical
column about 2 m high. Because electrons are scattered by collisions with air
molecules, air must first be pumped out of the column to create a vacuum. The
electrons are then accelerated from the filament by a nearby anode and allowed
to pass through a tiny hole to form an electron beam that travels down the col-
umn. Magnetic coils placed at intervals along the column focus the electron
beam, just as glass lenses focus the light in a light microscope. The specimen is
put into the vacuum, through an airlock, into the path of the electron beam. As in
light microscopy, the specimen can be stained—in this case, with electron-dense
material. Some of the electrons passing through the specimen are scattered by
structures stained with the electron-dense material; the remainder are focused to
form an image. The image can be observed on a monitor or is typically recorded
with a sensitive CMOS electron detector. Because the scattered electrons are lost
from the beam, the dense regions of the specimen show up in the image as areas
of reduced electron flux, which look dark.
Biological Specimens Require Special Preparation

for Electron Microscopy
In the early days of its application to biological materials, the electron microscope copper or gold grid covered
with carbon film
revealed many previously unimagined structures in cells. But before these discov-
eries could be made, electron microscopists had to develop new procedures for specimen in ribbon
embedding, cutting, and staining tissues. of thin sections
Because the specimen is exposed to a very high vacuum in the electron

microscope, living tissue is usually killed and preserved by chemical fixation. As
electrons have very limited penetrating power, the fixed tissues normally have
to be cut into extremely thin sections (25–100 nm thick, about 1/200 the thick-
ness of a single cell) before they are viewed. This is achieved by dehydrating the 3 mm
specimen, permeating it with a monomeric resin that polymerizes to form a
solid block of plastic, then cutting the block with a fine glass or diamond knife
Figure 9–41 Specimen support. The
on a special microtome. The resulting ultrathin sections, free of water and other metal grid that supports the thin sections
volatile solvents, are supported on a small metal grid for viewing in the micro- of a specimen in a transmission electron
scope (Figure 9–41). microscope.
Figure 9–42 Thin section of a cell. This

thin section is of a yeast cell that has been
very rapidly frozen and the vitreous ice
cell wall
replaced by organic solvents and then
by plastic resin (freeze substitution). The
nucleus, mitochondria, cell wall, Golgi
Golgi stack stacks, and ribosomes can all be readily
seen in a state that is presumed to be as
lifelike as possible. (Courtesy of Andrew
Staehelin.)
nucleus
mitochondrion
ribosomes
100 nm
The steps required to prepare biological material for electron microscopy are
challenging. How can we be sure that the image of the fixed, dehydrated, resin-
embedded specimen bears any relation to the delicate, aqueous biological system
present in the living cell? The best current approaches to this problem depend on
rapid freezing. If an aqueous system is cooled fast enough and to a low enough
temperature, the water and other components in it do not have time to rearrange
themselves or crystallize into ice. Instead, the water is supercooled into a rigid but
noncrystalline state—a “glass”—called vitreous ice. This rapid freezing is usually
performed by plunging the sample into a coolant such as liquid ethane or by cool-
ing it at very high pressure.
Some rapidly frozen specimens can be examined directly in the electron
microscope using a special cooled specimen holder. In other cases, the frozen
block can be fractured to reveal interior cell surfaces or the surrounding ice can
be sublimed away to expose external surfaces. However, we often want to examine
thin sections, and the frozen tissue can be sectioned directly in a cooled micro-
tome. A compromise is to rapidly freeze the tissue, replace the water with organic
solvents, embed the tissue in plastic resin, and finally cut sections. This approach,
called freeze substitution, stabilizes and preserves the tissue in a condition very
close to its original living state (Figure 9–42).
Molecules in all kinds of thin sections can be labeled to identify and local-
ize them. We have seen earlier how antibodies can be used in conjunction with 200 nm
fluorescence microscopy to localize specific macromolecules. An analogous Figure 9–43 Localizing proteins in
method—immunogold electron microscopy—can be used in the electron micro- electron microscopy. Immunogold
scope. The usual procedure is to incubate a thin section first with a specific electron microscopy is used here to find the
primary antibody, and then with a secondary antibody to which a colloidal gold specific location of a protein that is targeted
particle has been attached. The gold particle is electron-dense and can be seen as to the Golgi apparatus. The protein has
been tagged with a genetically encoded
a black dot in the electron microscope (Figure 9–43). Different antibodies can be fluorescent protein and is localized to the
conjugated to different-sized gold particles so multiple proteins can be localized trans-Golgi network. The protein is seen
in a single sample. in this thin section using an antibody to
the fluorescent protein coupled to 10-nm
colloidal gold particles, seen in the electron
Heavy Metals Can Provide Additional Contrast microscope as black dots. The cell has
been frozen under high pressure and
Although phase contrast can make unstained specimens more visible, image freeze substituted before embedding and
clarity in an electron micrograph usually depends on having a range of electron sectioning. (Courtesy of Charlotta Funaya
densities to provide amplitude contrast within the specimen. Electron density in and M. Teresa Alonso.)
(A) (B)
100 nm 100 nm
Figure 9–44 Heavy metals provide contrast in the electron microscope. (A) This transmission
electron micrograph shows RecA protein together with E. coli DNA adsorbed to flakes of mica,
frozen, carefully dried, and then shadowed with evaporated platinum atoms. The RecA protein
clearly forms tight, right-handed helices around the bacterial DNA molecules, some of which can be
seen free at the top of the image (see also Figure 5–48). (B) In this transmission electron micrograph
of actin filaments, negatively stained with uranyl acetate, each filament is about 8 nm in diameter
and is seen, on close inspection, to be composed of a helical chain of globular actin molecules (see
also Figure 16–8). (A, from J. Heuser, J. Electron Microsc. Tech. 13:244–263, 1989; B, courtesy of
Roger Craig.)
turn depends on the atomic number of the atoms that are present: the higher
the atomic number, the more electrons are scattered and the darker that part of
the image. Biological tissues are composed mainly of atoms of very low atomic
number (primarily carbon, oxygen, nitrogen, and hydrogen). To make them more
readily visible, tissues are often impregnated (before or after sectioning) with the
salts of heavy metals such as uranium, lead, and osmium. The degree of impregna-
tion, or “staining,” with these salts will vary for different cell constituents. Lipids,
for example, tend to stain darkly after osmium fixation, revealing the location of
cell membranes (see, for example, Figure 12–2 or Figure 12–15).
Alternatively, if isolated molecules are “shadowed” by platinum or other
heavy metals evaporated from a heated filament, macromolecules such as DNA
or large proteins can be visualized with high contrast in the electron micro-
scope (Figure 9–44A). Negative staining is a similar approach that also allows
fine detail to be seen in isolated molecules or macromolecular machines. In this
technique, the molecules are supported on the thin film of carbon on a grid and
mixed with a solution of a heavy-metal salt such as uranyl formate or acetate.
After the sample has dried, a very thin film of metal salt covers the carbon film
everywhere except where it has been excluded by the presence of an adsorbed
macromolecule. Because the macromolecule allows electrons to pass through
it much more readily than does the surrounding heavy-metal stain, a reverse or
negative image of the molecule is created. Negative staining is especially use-
ful for quickly and cheaply viewing large macromolecular aggregates such as
viruses or ribosomes and for seeing the subunit structure of protein filaments
(Figure 9–44B). Shadowing and negative staining can provide high-contrast
surface views of small macromolecular assemblies, but the size of the smallest
metal particles in the shadow or stain limits the resolution of both techniques to
about 2 nm.
Images of Surfaces Can Be Obtained by Scanning Electron

Microscopy
A scanning electron microscope (SEM) directly produces an image of the
three-dimensional structure of the surface of a specimen. The SEM is usually
smaller, simpler, and cheaper than a transmission electron microscope. Whereas
electron
gun
condenser
lens
beam deflector
scan
generator
objective
lens
video electrons from

screen specimen
detector
specimen
Figure 9–45 The scanning electron microscope. In an SEM, the specimen is scanned by a beam of electrons brought to
a focus on the specimen by the electromagnetic coils that act as lenses. The detector measures the quantity of electrons
scattered or emitted as the beam bombards each successive point on the surface of the specimen and records the intensity
of successive points in an image built up on a screen. The SEM creates striking images of three-dimensional objects with
great depth of focus and a resolution between 0.5 nm and 10 nm depending on the kind of instrument. (Photograph courtesy
of Andy Davis.)
the TEM uses the electrons that have passed through the specimen to form an
image, the SEM uses electrons that are scattered or emitted from the specimen’s
surface. The specimen to be examined is usually either fixed, dried, and coated
with a thin layer of heavy metal or alternatively rapidly frozen and then transferred
to a cooled specimen stage for coating and direct examination in the microscope
(Figure 9–45). The specimen is scanned with a very narrow beam of electrons.
The quantity of electrons scattered or emitted as this primary beam bombards
each successive point of the metallic surface is measured and builds up an image
on a computer screen. Often an entire plant part or small animal can be put into
the microscope with very little preparation (Figure 9–46).
The SEM technique provides great depth of field, thus objects both near and
far in the field of view are imaged sharply. Moreover, because the amount of
electron scattering depends on the angle of the surface relative to the beam, the
image has highlights and shadows that give it a three-dimensional appearance
Figure 9–46 The scanning electron

microscope produces surface
images with great depth of field. SEM
micrographs taken at a wide range of
magnifications. (A) A developing wheat
flower, or spike. This delicate flower spike
was rapidly frozen, coated with a thin metal
film, and examined in the frozen state with
an SEM. This low-magnification micrograph
demonstrates the large depth of focus
of an SEM, even with a large specimen
like this. (B) These pollen grains from a
hellebore flower reveal their sculpted cell
walls in the SEM. The shapes and patterns
are specific for each species of pollen
grain. (C) Chains of bacteria living in the
(A) (B) (C) blue veins of a Stilton cheese. (A, B, and C,
1 mm 50 µm 1 µm courtesy of Kim Findlay.)
(A)
50 nm
(B)
100 nm
Figure 9–47 Higher-resolution SEM. Macromolecular assemblies, shadowed with a very thin
coating of tungsten and imaged in an SEM equipped with a field-emission electron gun. (A) An actin
filament showing the helical arrangement of actin monomers. (B) Clathrin-coated vesicles. [A and
B, from R. Wepf et al., in Biological Field Emission Scanning Electron Microscopy (R. Fleck and B.
Humbel, eds.), pp. 269–298. Hoboken, NJ: Wiley, 2019.]
(see Figure 9–46). Only surface features can be examined, however, and in most
forms of SEM, the resolution attainable is not very high (about 10 nm). As a result,
the technique is usually used to study whole cells and tissues rather than subcel-
lular organelles (see Movie 21.3). However, very-high-resolution SEMs have been
developed with a bright, coherent, field-emission gun as the electron source. As
resolution in the SEM depends not on the wavelength of the electron beam but on
the size of the electron spot that is scanned across the specimen, this type of SEM
can produce images that rival the resolution possible with a negatively stained
specimen in a TEM (Figure 9–47).
Electron Microscope Tomography Allows the Molecular

Architecture of Cells to Be Seen in Three Dimensions
The SEM can only provide a surface view of an object, which tells us little about the
important three-dimensional relationships between macromolecules and organ-
elles within a living cell. Moreover, thin sections viewed in a TEM also often fail
to convey the three-dimensional arrangement of cellular components, and the
images can be misleading. It is possible to reconstruct the third dimension from
serial sections, but this is a lengthy and tedious process. But even thin sections
have a significant depth compared with the resolution of the electron micro-
scope, so the TEM image can also be misleading in an opposite way, through the
superimposition of objects that lie at different depths.
Because of the large depth of field of electron microscopes, all the parts of the
three-dimensional specimen are in focus, and the resulting image is a projection
(a superimposition of layers) of the structure along the viewing direction. The
lost information in the third dimension can be recovered if we have views of the
same specimen but from many different directions. The computational methods
for this technique are widely used in medical CT scans. In a CT scan, the imaging
equipment is moved around the patient to generate the different views. In electron
microscope (EM) tomography, the specimen holder is tilted in the microscope,
which achieves the same result. The specimen is usually tilted to a maximum of
60° in every direction, and in this way we can arrive at a three-dimensional recon-
struction, in a chosen standard orientation, by combining different views of a single
object. Each individual view will be very noisy, but by combining them in three
dimensions and taking an average, the noise can be significantly reduced. Thick
plastic sections of embedded material have been used to create three-dimensional
reconstructions, or tomograms (Movie 9.2), of cells, but increasingly microscopists
are applying EM tomography to unstained, frozen, hydrated sections, and even to
rapidly frozen whole cells or organelles. Individual macromolecular assemblies that
594 PANEL 9–1: Protein Structure Determination Using CryoEM
SINGLE-PARTICLE RECONSTRUCTION BY CRYOEM molecules immobilized in thin film of ice
X-ray crystallography is one way to determine a

protein structure. However, large macromolecular
machines are often hard to crystallize, as are many
carbon film on EM grid
integral membrane proteins, and for dynamic proteins
and assemblies it is hard to access different In this technique, a droplet of the pure protein in water is placed
conformations through crystallography alone. To get on a small EM grid that is plunged into a vat of liquid ethane at
around these problems, investigators are increasingly −180ºC. This freezes the proteins in a thin film of ice and the rapid
turning to cryo-electron microscopy (cryoEM) to solve freezing ensures that the surrounding water molecules have no
macromolecular structures. time to form ice crystals, which would damage the protein’s shape.
beam of electrons
The sample is examined, still frozen, by high-voltage transmission
electron microscopy. To avoid damage, it is important that only a
few electrons pass through each part of the specimen. Sensitive
detectors are therefore deployed to capture every electron that
passes through the specimen. Much EM specimen preparation
and data collection is now fully automated and many thousands
of micrographs are typically captured, each of which will contain
hundreds or thousands of individual molecules all arranged in
random orientations within the ice.
electron detector captures projected image of molecules
Algorithms then sort This crisper two-dimensional

the molecules into image set, which represents
sets where each set different views of the particle,
contains molecules are then combined and
that are all oriented converted via a series of
in the same direction. complex iterative steps into a
The thousands of high-resolution
images in each set are three-dimensional structure.
all then superimposed
and averaged to Model of GroEL
improve the signal-to- (Courtesy of Gabriel Lander.)
noise ratio.
5 nm
CRYOEM STRUCTURE OF 60S large ribosomal subunit at path of an rRNA loop fitted
0.25 nm resolution into the electron-density map
THE RIBOSOME
Courtesy of Joachim Frank.
Mg2+
G C
RNA bases
60S ribosomal subunits randomly
oriented in a thin film of ice 100 nm 5 nm 1 nm
Although by no means routine, big improvements in This resolving power now approaches that of x-ray
image-processing algorithms, modeling tools and sheer crystallography, and the two techniques thrive together, each
computing power all mean that structures of bootstrapping the other to obtain ever more useful and dynamic
macromolecular complexes are now becoming attainable structural information. A good example is the structure of the
with resolutions in the 0.2- to 0.3-nm range. ribosome shown here at a resolution of 0.25 nm.
(A) (B) (C) (D)

500 nm 500 nm 50 nm 10 nm
appear as multiple copies in the tomogram can be identified, and with a compu- Figure 9–48 EM tomography. The COP1
tational process called subtomogram averaging to reduce noise and gain structural coat mediates vesicle traffic within the
Golgi apparatus and retrograde traffic
information, molecular structures inside cells can now be obtained at a resolution
to the endoplasmic reticulum (ER) (see
of better than 2 nm (Figure 9–48). Electron microscopy now provides a robust Figures 13–4 and 13–5). EM tomography
bridge between the scale of the single molecule and that of its cellular environment. has helped visualize the details of COP1
coats in situ on buds and vesicles in rapidly
Cryo-electron Microscopy Can Determine Molecular Structures frozen Chlamydomonas cells. (A) One slice
through a three-dimensional tomogram
at Atomic Resolution of a complete Golgi apparatus. (The
tomogram can be seen in Movie 9.2.)
As we saw earlier (p. 567), noise is important in light microscopy at low light lev- (B) Using the information from several
els, but it is a particularly severe problem for electron microscopy of unstained such tomograms, a portion of the Golgi
macromolecules. A protein molecule can tolerate a dose of only a few hundreds of is shown here, color coded to show ER
electrons per square nanometer without damage, and this dose is orders of mag- dark yellow, the cis vesicles yellow, the
four cis cisternae green, the four medial
nitude below what is needed to define an image at atomic resolution. cisternae red, the trans cisterna blue,
The solution is to obtain images of many identical molecules—perhaps hun- medial vesicles pink, trans vesicles light
dreds of thousands of images of individual particles—and combine them to blue, and the trans Golgi network purple.
produce an averaged image, revealing structural details that are hidden by the Ribosomes can also be seen as small gray
noise in the original images. This procedure is called single-particle reconstruc- blobs. (C) Individual slices through COP1
vesicles in the tomogram; the bottom one
tion (Panel 9–1). Before combining all the individual images, however, they must is partially uncoated. (D) By identifying
be aligned with each other. With the help of a computer, the digital images of ran- and averaging more than 10,000 COP1
domly distributed and unaligned molecules can be processed and combined to subunits on vesicles in the tomograms,
yield high-resolution reconstructions (see Movie 13.1). Although structures that a molecular structure was obtained by
have some intrinsic symmetry, such as dimers or helical repeats, are somewhat subtomogram averaging at a resolution of
2 nm. Structures of the various proteins in
easier to solve (Figure 9–49), this technique has also been used for huge macro- the COP1 coat have been solved, and they
molecular machines, such as ribosomes, that have no symmetry (see Panel 9–1). can be fitted neatly into the electron-density
Cryo-electron microscopy (cryoEM) depends crucially on very rapidly freez- envelope of the EM structure. A surface
ing the aqueous specimen to form vitreous ice, which does not allow ice crystals view of a triad of COP1 subunits on the
surface of a vesicle is shown here together
to form and therefore does not damage the specimen. A very thin (about 100 nm)
with the molecular structures (in color) of
film of an aqueous suspension of purified macromolecular complex is prepared the individual components that have been
on a microscope grid and is then rapidly frozen by being plunged into a coolant. fitted into the EM structure. (Adapted from
A special sample holder keeps this hydrated specimen at –160°C in the vacuum Y.S. Bykov et al., eLife 6:e32493, 2017,
of the microscope, where it can be viewed directly without fixation, staining, or doi 10.7554/eLife.32493.)
drying. Unlike negative staining, in which what we see is the envelope of stain
exclusion around the particle, cryoEM produces an image from the macromolec-
ular structure itself. The specialized transmission electron microscopes required
operate with much higher electron accelerating voltages than that of a routine
TEM and typically run at 300,000 V. However, as very low electron doses are
used to obtain cryoEM images, the intrinsic contrast in the images produced is
very low, and to extract the maximum amount of structural information, special
image-processing techniques must be used. Huge advances in direct electron
detectors and faster, more efficient image-processing techniques that involve
image alignment routines, motion correction, and contrast transfer function cor-
rections mean that the structures of molecules as small as 100 kilodaltons can
now be solved. The smaller the molecule, the noisier the image, and the main
(A) (B) (C)

50 nm 10 nm 2 nm
Figure 9–49 CryoEM structure of microtubules. This cryoEM reconstruction of the structure of
a microtubule was helped by the intrinsic symmetry of the microtubule itself (see Figure 16–37).
This detailed model of the whole microtubule has allowed an examination of the way in which
the protofilaments interact and the way in which the whole lattice and associated proteins
are assembled. (A) CryoEM image of two intact microtubules embedded in vitreous ice. (B) A
reconstruction of the surface lattice of a single microtubule at a resolution of 0.35 nm (3.5 Å).
(C) The detailed electron-density map of the tubulin dimer extracted from the structure of the intact
microtubule. α-Tubulin is darker green, and β-tubulin is lighter green. (From E. Nogales, Mol. Biol.
Cell 27:3202–3204, 2016, doi 10.1091/mbc.E16-06-0372. With permission from Elsevier.)
advantages of the method are best seen with large and sometimes flexible mac-
romolecular complexes such as viruses, ribosomes, and large integral membrane
proteins that are hard to crystallize (Figure 9–50).
A remarkable resolution of 0.12 nm (1.2 Å) has been achieved in a particularly
stable protein by cryoEM, enough to see clearly the detailed atomic structure and
to rival x-ray crystallography in resolution (Figure 9–51). Electron microscopy,
however, also has some very clear additional advantages over x-ray crystallography
(A)
(discussed in Chapter 8) as a method for macromolecular structure determination.
First, it does not require crystalline specimens. Second, it can deal with extremely receptor-binding domains
large complexes—structures that may be too large or too variable to crystallize sat-
isfactorily; for example, membrane proteins. Third, it allows the rapid analysis of
different conformations of protein machines; for example, the different states of the
F1 ATPase proton pump shown in Figure 14–31. Fourth, the glycosylation patterns
and mobile loops on the surface of proteins, which are often impossible to see in
x-ray structures, are more readily resolved in cryoEM structures. And fifth, only a
minute amount of sample is required compared with that needed to make crystals.
The analysis of large and complex macromolecular structures is helped con-
siderably if the atomic structure of one or more of the subunits is known, for
Figure 9–50 The spike protein on the SARS-CoV-2 virus. The SARS-CoV-2 virus was responsible
for the COVID-19 pandemic. Protruding from the viral membrane are many trimeric spike proteins
that mediate binding of the virus to a receptor on cells in our respiratory tract and its subsequent glycan
entry into the cell. The trimeric spike protein is a target both of our immune system and of vaccine side chains
developers. The closed conformation of the trimeric spike protein shown here, both from the top
(A) and from the side (B), was obtained from rapidly frozen intact virus particles. Spike proteins
were identified by computer from multiple tilted images of the viruses and subtomogram averaging
applied to them. The final electron-density map was determined to a resolution of 0.35 nm, good
enough for the molecular model (shown here) to be accurately fitted within its envelope, although
viral
the details of the membrane-spanning portion of the trimeric spike protein are not revealed. The membrane 2 nm
proteins are heavily N-glycosylated, and these surface glycans are shown in green, while the three
spike proteins are shown in dark green, light blue, and light brown. (PDB code: 6ZWV.) (B)
0.2 nm
(C) tyrosine arginine histidine
(A) (B)
50 nm 5 nm
Figure 9–51 Atomic resolution by

example from x-ray crystallography (Figure 9–52). Molecular models can then be cryoEM. Apoferritin is a cytosolic protein,
mathematically “fitted” or docked into the envelope of the structure determined present in almost all living organisms, that
reversibly stores iron in a nontoxic form. It
at lower resolution using the electron microscope. X-ray and cryoEM approaches is a large (474 kilodaltons) and particularly
often combine profitably together to determine molecular structures. stable molecule. Its hollow globular cage
has 24 symmetrical subunits, which means
that a structure can be determined with
Light Microscopy and Electron Microscopy Are Mutually Beneficial relatively few particles. (A) Cryo-electron
The interior of the cell is a confusing place, with molecules crowded together in micrograph of cage-like apoferritin particles.
(B) By use of every possible new technical
the cytosol and intricate and complex membrane-bounded compartments. To advance in single-particle reconstruction,
discover which molecules are located exactly where and in which tiny vesicles the complete cryoEM structure shown here
or subcompartments of the cell is not straightforward, even with the genetically is at the remarkable resolution of 0.12 nm
encoded labels that can target almost any protein. We have seen that superreso- (1.2 Å). (C) When the known amino acid
lution light microscopy can be used to very accurately locate specific molecules sequence is modeled into the electron-
density map, clear electron densities can be
within a cell. A major disadvantage, however, of all fluorescence imaging tech- seen associated with hydrogen atoms in the
niques is that it is only the tagged molecules that are imaged—their cellular three amino acid side chains. The molecular
context remains invisible. When fluorescence imaging is combined, however, model is fitted into the final electron-density
with looking at the same specimen in the electron microscope, this correlative envelope that is shown as a gray cage.
light microscopy and electron microscopy technique, or CLEM, can allow specific (A, from T. Nakane et al., Nature 587:152–
156, 2020, doi 10.1038/s41586-020-2829-0;
target molecules to be examined in their full cellular context. Although this can B, EMD-11668; C, adapted from K.M. Yip
be achieved using fixed and sectioned material, most such approaches now use et al., Nature 587:157–161, 2020. With
rapidly frozen material to co-localize target molecules both in the light and in the permission from Nature.)
EZH2 nucleosome
Figure 9–52 PRC2, a large
histone core macromolecular machine. Polycomb
repressive complex 2 (PRC2) is a
large protein complex involved in
establishing heterochromatin and the
epigenetic regulation of gene expression
(see Figure 4–40). PRC2 interacts with a
nucleosome through the binding of the
nucleosomal DNA by one of its subunits,
EZH2, which also engages the extended
tail of histone H3 to direct its lysine 27
(K27) to the active site for methylation. The
density map of PRC2 and two essential
cofactors bound to a single nucleosome
DNA
was produced by single-particle cryo-
electron microscopy reconstruction at a
resolution of 0.35 nm. The long arm of
H3 histone tail histone H3 is shown in more detail with
the protein backbone modeled into the
density map. (Courtesy of Vignesh Kasinath
PRC2 and Eva Nogales and based on EMDB-
21707. From V. Kasinath et al., Science
371:eabc3393, 2021. With permission
lysine 27 from AAAS.)
IRE1 assembled in specialized ER normal ER specialized ER
(A) (B) (C)

200 nm
electron microscope, and of these two general approaches are common. The first Figure 9–53 Correlated light and electron
is to freeze the cell or tissue, locate the positions of the target molecule with fluo- microscopy (CLEM). The correct folding
of proteins in the endoplasmic reticulum
rescence light microscopy, and then, after transferring the frozen specimen to an
(ER) is sensed by a major transmembrane
electron microscope, tilting it, using EM tomography to find the exact point in the protein called IRE1 (see Figures 12–36
tomogram that corresponds to the fluorescent signal (Figure 9–53). and 12–37). If IRE1 is activated, it forms
A second approach, and a demanding one too, is again to rapidly freeze the oligomers that are visible in fluorescence
cell and locate fluorescent molecules at high resolution by single-molecule local- microscopy as bright foci. Here, stressed
cells, expressing fluorescent IRE1 and
ization microscopy. The frozen cell is then transferred to a modified SEM that growing on an EM grid, are rapidly
incorporates a separate focused ion beam, usually of gallium ions, that can be frozen and subsequently imaged by EM
scanned across the frozen block face like a miniature milling machine, remov- tomography. The resulting tomograms
ing about 10 nm of the sample at a time. The SEM records a two-dimensional can be directly correlated with the light
image of the scattered electrons from the surface of the block face at each step, micrographs. (A) A fluorescent spot of
labeled IRE1 is shown here precisely
and a three-dimensional image of the cell is gradually built up that can be superimposed on a slice through its
correlated with the original localization data, all with a final resolution of corresponding EM tomogram that contains
about 5 nm (Figure 9–54). The technique is called focused ion beam–scanning a network of ER. (B) Another slice through
electron microscopy, or FIB–SEM for short. The same technique, but without the the tomogram at a different level shows
fluorescent labels, can be used on much larger specimens that have been conven- IRE1 is localized as aggregates in a
complex network of specialized, narrow
tionally fixed, stained with heavy-metal salts, and embedded in plastic. Although ER tubules. (C) The outlines of the ER
the structural preservation may not be so good as with frozen specimens, the membranes in each slice of the tomogram
approach, although very time consuming, is proving useful in analyzing complex are manually defined (in a process called
cellular interactions; for example, in mapping the neural connections in brain segmentation), and the drawing here
shows that the oligomers of IRE1 are
tissue (see Movie 9.1).
concentrated in this convoluted network
of specialized ER tubules. (A, B, and C,
Using Microscopy to Study Cells Always Involves Trade-Offs adapted from S.D. Carter et al., 2021,
doi 10.1101/2021.02.24.432779.)
The history of cell biology has been tightly interlinked with that of microscopy.
What we now know about the structure and function of cells has depended
crucially on being able to image cells, organelles, and the molecules they
contain—seeing is indeed believing. But for the young biologist today, there is,
as we have seen, a bewildering variety of imaging technologies from which to
choose, and knowing which is best suited to solve the problem at hand is not
easy. All imaging approaches have trade-offs to consider. At an obvious level, the
dynamics of cells are only accessible with certain kinds of light microscopies and
with living cells. If higher resolution is required, with either electron microscopes
or light microscopes, then that comes with increasing cost and complexity.
Single-molecule localization microscopy also requires elaborate hardware and
also takes many minutes to acquire each image. The cryoEM-derived structures
of large protein complexes require the use of high-voltage machines that cost
many millions of dollars. Such resources are usually confined to large centralized
microscopy facilities that can be shared by many users. The precise localization of
molecules within the cell requires the use of fluorescent labels, but, because only
the labels themselves can be detected in a fluorescence microscope, the cellular
Figure 9–54 Focused ion beam–scanning

nucleus electron microscopy (FIB–SEM).
Superresolution light microscopy is
combined here with three-dimensional
electron microscopy of rapidly frozen cells
plasma membrane to enable the high-resolution localization
of target molecules throughout the entire
mitochondrion volume of a cell. Sequential slices through
the frozen cell are obtained by steadily
milling the surface of the frozen block face
with a focused ion beam, while images of
the surface are collected at each step in an
SEM. This particular cell has been labeled
with fluorescent markers for the lumen of
the endoplasmic reticulum (green) and
for the outer membrane of mitochondria
(magenta). (A) Three orthogonal slices
through the cell show the combined
electron microscope and fluorescence
light microscope images. (B) A small
endoplasmic region of the same cell imaged with a
(A) reticulum structured illumination microscope (SIM)
is used to define mitochondrion and ER.
(C) The corresponding block face image
in the SEM. (D) The correlated electron
microscope and light microscope images
identify the position of the fluorescent labels
in the electron micrograph. (E, F, and G)
Because the three-dimensional SEM data
set is of the entire cell, different views of the
same area can be readily obtained. Here,
the three corresponding vertical sections
along the yellow dotted lines on the images
above are shown. (From D.P. Hoffman
et al., Science 367:265–277, 2020. With
permission from AAAS.)
(B) (C) (D)
(E) (F) (G)

200 nm
context is sacrificed. Imaging itself involves several trade-offs to be considered. An

improvement in any one parameter—image contrast, resolution, signal-to-noise
ratio, specimen damage by photons or electrons, the depth of specimen that can
be imaged, or the speed of image recording—will inevitably require a sacrifice in
one or more of the others, and understanding these trade-offs will help determine
which approach is best for the cell biology problem being tackled.
Summary
Discovering the detailed structure of cells and their molecules requires the higher res-
olution attainable in a transmission electron microscope. Three-dimensional views
of the surfaces of cells and tissues are obtained by scanning electron microscopy.
Specific macromolecules can be localized by combining electron microscopy with
fluorescence light microscopy. EM tomography enables three-dimensional informa-
tion about cellular architecture to be obtained. The shapes of isolated molecules can
be roughly determined by electron microscopy techniques involving negative staining
or heavy-metal shadowing, but detailed molecular structures require cryoEM and
single-particle reconstruction using computational manipulations of data obtained
from multiple images and multiple viewing angles to produce detailed reconstruc-
tions of macromolecules and molecular complexes. The resolution obtained with
these methods means that atomic structures of individual macromolecules can be
“fitted” to the images derived by electron microscopy. CryoEM can often determine
the structures of molecules that are inaccessible to x-ray crystallography.
PROBLEMS
Which statements are true? Explain why or why not. 9–5 Why do humans see so poorly under water? And
why do goggles help?
9–1 A fluorescent molecule, having absorbed a single
photon of light at one wavelength, always emits it at a lon- 9–6 Explain the difference between resolution and
ger wavelength. magnification.
9–2 Transmission electron microscopy and scanning 9–7 Figure Q9–3 shows a series of modified fluores-
electron microscopy can both be used to examine a struc- cent proteins that emit light in a range of colors. Several
ture in the interior of a thin section: transmission electron of these fluorescent proteins contain the same chromo-
microscopy provides a projection view, while scanning phore, yet they fluoresce at different wavelengths. How do
electron microscopy captures electrons scattered from the you suppose the exact same chromophore can fluoresce at
structure and gives a more three-dimensional view. several different wavelengths?
Discuss the following problems.
9–3 The diagrams in Figure Q9–1 show the paths of

light rays passing through a specimen into a dry lens or
into an oil-immersion lens. Offer an explanation for why
oil-immersion lenses should give better resolution. Air,
glass, and oil have refractive indices of 1.00, 1.51, and 1.51,
respectively.
Figure Q9–3 A rainbow of colors produced by modified fluorescent
DRY LENS OIL-IMMERSION LENS proteins (Problem 9–7). (Courtesy of Nathan Shaner, Paul Steinbach,
and Roger Tsien.)
9–8 A fluorescent biosensor was designed to report

objective the cellular location of active Abl protein tyrosine kinase.
lens
air oil A blue (cyan) fluorescent protein (CFP) and a yellow
cover slip fluorescent protein (YFP) were fused to either end of
slide
a hybrid protein, which consisted of a substrate pep-
Figure Q9–1 Paths of light rays through dry and oil-immersion lenses tide recognized by the Abl protein tyrosine kinase and a
(Problem 9–3). The red circle at the origin of the light rays is the phosphotyrosine-binding domain (Figure Q9–4A). Stim-
specimen. ulation of the CFP domain does not cause emission by
the YFP domain when the domains are separated. When
9–4 Figure Q9–2 shows a diagram of the human the CFP and YFP domains are brought close together,
eye. The refractive indices of the components in the light however, fluorescence resonance energy transfer (FRET)
path are air, 1.00; cornea, 1.38; aqueous
humor, 1.33; crystalline lens, 1.41; and (A) REPORTER (B) FRET
vitreous humor, 1.38. Where does the 434 nm
main refraction—the main focusing— + phosphatase
476 nm 1.3
occur? What role do you suppose the CF Abl + ATP
P
lens plays? Abl kinase 1.2
YFP/CFP
substrate
peptide
Y
1.1
iris
omit Abl or ATP
YFP
vitreous
phosphotyrosine- 1.0
humor retina
binding domain 0 5 10 15 20 25 30
cornea
time (hours)
lens Figure Q9–4 Fluorescent biosensor designed to detect tyrosine phosphorylation (Problem
9–8). (A) Domain structure of the biosensor. Four domains are indicated: CFP, YFP,
aqueous
humor
tyrosine kinase substrate peptide, and a phosphotyrosine-binding domain. (B) FRET
assay. YFP/CFP is normalized to 1.0 at time zero. The biosensor was incubated in the
presence (or absence) of Abl and ATP for the indicated times. Arrow indicates time of
Figure Q9–2 Diagram of the human eye addition of a tyrosine phosphatase. (From A.Y. Ting et al., Proc. Natl. Acad. Sci. USA
(Problem 9–4). 98:15003–15008, 2001. With permission from National Academy of Sciences.)
REFERENCES 601
allows excitation of CFP to stimulate emission by YFP. to the primary antibodies. You then examine it by elec-
FRET shows up experimentally as an increase in the ratio tron microscopy (Figure Q9–5). Are the gold particles
of emission at 526 nm (from YFP) versus 476 nm (from (black dots) consistently associated with any particular
CFP) when CFP is excited by 434-nm light. structure?
Incubation of the biosensor protein with Abl
Figure Q9–5 An
protein tyrosine kinase in the presence of ATP gave an astrocyte membrane
increase in the ratio of YFP/CFP emission (Figure Q9–4B). labeled with primary
In the absence of ATP or the Abl protein, no FRET occurred. antibodies against
FRET was also eliminated by addition of a tyrosine phos- aquaporin and
phatase (Figure Q9–4B). Describe as best you can how this then with secondary
antibodies to
biosensor detects active Abl protein tyrosine kinase. which colloidal
gold particles have
9–9 Under ideal conditions, with the simplest of spec- been attached
imens (a monolayer of carbon atoms, for example) and (Problem 9-10).
careful image processing, the practical resolving power (From J.E. Rash
of modern electron microscopes is about 0.05 nm, some et al., Proc. Natl.
Acad. Sci. USA
25-fold above the theoretical limit of 0.002 nm. This is 95:11981–11986,
because only the very center of the electron lens can be 1998. With
used, and the effective numerical aperture (n sin θ) is lim- permission from
ited by θ (half the angular width of rays collected at the National Academy
objective lens). Assuming that the wavelength (λ) of the of Sciences.)
electrons is 0.004 nm and that the refractive index (n) is
1.0, calculate the value for θ, where resolution (0.05 nm) =
0.61 λ/n sin θ. How does this value of θ compare with that
for a conventional light microscope (60°)?
9–10 Aquaporin water channels in the plasma 100 nm
membrane play a major role in water metabolism and
osmoregulation in many cells. To determine their struc- 9–11 The technique of negative staining uses heavy
tural organization in the membrane, you use immunogold metals such as uranium to provide contrast. If these heavy
electron microscopy. You prepare a membrane sample, metals do not actually bind to defined biological struc-
incubate it with primary antibodies against aquaporin tures (which they do not), how is it that they can help to
then with gold-tagged secondary antibodies that bind make such structures visible?
REFERENCES
General Chen F, Tillberg PW & Boyden ES (2015) Expansion microscopy.
Science 347, 543–548.
Vale R, Stuurman N & Thorn K (2006–2021) Microscopy Series
(https://www.ibiology.org/online-biology-courses/microscopy- Giepmans BN, Adams SR, Ellisman MH & Tsien RY (2006) The
series/) A major online video series starts with the basics of optical fluorescent toolbox for assessing protein location and function.
microscopy and concludes with some of the latest techniques, Science 312, 217–224.
such as superresolution microscopy. Greenfield EA (ed.) (2014) Antibodies: A Laboratory Manual, 2nd ed.
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Greenwald EC, Mehta S & Zhang J (2018) Genetically encoded
Looking at Cells and Molecules in the Light fluorescent biosensors illuminate the spatiotemporal regulation
Microscope of signaling networks. Chem. Rev. 118, 11707–11794.
Agard DA, Hiraoka Y, Shaw P & Sedat JW (1989) Fluorescence Hell S (2009) Microscopy and its focal switch. Nat. Methods 6, 24–32.
microscopy in three dimensions. In Fluorescence Microscopy Huang B, Babcock H & Zhuang X (2010) Breaking the diffraction
of Living Cells in Culture, part B (Taylor DL & Wang Y-L, eds.). barrier: super-resolution imaging of cells. Cell 143, 1047–1058.
Methods in Cell Biology, Vol. 30. San Diego: Academic Press. Jacquemet G, Carisey AF, Hamidi H . . . Leterrier C (2020) The cell
Boulina M, Samarajeewa H, Baker JD . . . Chiba A (2013) Live imaging biologist’s guide to super-resolution microscopy. J. Cell Sci. 133,
of multicolor-labeled cells in Drosophila. Development 140, jcs240713.
1605–1613. Jonkman J, Brown CM, Wright GD . . . North AJ (2020) Tutorial:
Burnette DT, Sengupta P, Dai Y . . . Kachar B (2011) Bleaching/blinking guidance for quantitative confocal microscopy. Nat. Protoc. 15,
assisted localization microscopy for superresolution imaging using 1585–1611.
standard fluorescent molecules. Proc. Natl. Acad. Sci. USA 108, Klar TA, Jakobs S, Dyba M . . . Hell SW (2000) Fluorescence
21081–21086. microscopy with diffraction resolution barrier broken by stimulated
Chalfie M, Tu Y, Euskirchen G . . . Prasher DC (1994) Green emission. Proc. Natl. Acad. Sci. USA 97, 8206–8210.
fluorescent protein as a marker for gene expression. Science Lippincott-Schwartz J & Patterson GH (2003) Development and use of
263, 802–805. fluorescent protein markers in living cells. Science 300, 87–91.

Alberts Visualizing Cells2

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Molecular Biology of

THE CELL Seventh Edition

THE CELL Seventh Edition

All rights reserved

Editor: Betsy Twitchell

Permission to use copyrighted material is included alongside the appropriate content.

Library of Congress Cataloging-in-Publication Data

Names: Alberts, Bruce, author.

PART I INTRODUCTION TO THE CELL

LOOKING AT CELLS AND MOLECULES

(A) image on retina (B) Figure 9–2 A light microscope.

TWO WAVES IN PHASE TWO WAVES OUT OF PHASE

Figure 9–3 Interference between light

LENSES RESOLUTION: the resolving power of the

the objective lens 0.61 λ

Photon Noise Creates Additional Limits to Resolution When Light

only scattered waves out of phase

Images Can Be Enhanced and Analyzed by Digital Techniques

Figure 9–9 Staining of cell components.

Second, sectioned tissues can be used to visualize specific patterns of differ-

Specific Molecules Can Be Located in Cells by Fluorescence

absorption emission of photon

GROUND STATE 3 3 second barrier filter: cuts out

the cellular distribution and abundance of specific expressed RNA molecules

Figure 9–13 Different fluorescent probes

Antibodies Can Be Used to Detect Specific Proteins

Figure 9–14 Immunofluorescence.

primary antibody: secondary antibodies: Figure 9–15 Indirect immunocytochemistry.

Individual Proteins Can Be Fluorescently Tagged in Living Cells

Protein Dynamics Can Be Followed in Living Cells

these GFP variants. These genetically encoded, photoactivatable, fluorescent pro-

blue fluorescent Figure 9–22 Genetically encoded

Imaging of Complex Three-dimensional Objects Is Possible with

The Confocal Microscope Produces Optical Sections

(A) (B) (C) (D)

Figure 9–25 Confocal fluorescence

Figure 9–26 Multiphoton imaging.

Superresolution Fluorescence Techniques Can Overcome

Figure 9–27 Structured illumination

(A) (B) (C)

point source of light

(A) 200 nm (B) (C)

The fluorescent probes used must be in a special class that is photoswitchable:

Single-Molecule Localization Microscopy Also Delivers

exact position of the

fix cell gel proteinase gel

Expanding the Specimen Can Offer Higher Resolution,

Figure 9–35 Expansion microscopy.

Large Multicellular Structures Can Be Imaged Over Time

Figure 9–37 Light-sheet microscopy

cell containing only those molecules in the

(A) laser light objective lens of (B) (C)

LOOKING AT CELLS AND MOLECULES IN THE

The Electron Microscope Resolves the Fine Structure of the Cell

Figure 9–40 The principal features of

In overall design, the transmission electron microscope (TEM) is similar to an

Biological Specimens Require Special Preparation

Because the specimen is exposed to a very high vacuum in the electron

Figure 9–42 Thin section of a cell. This