Imaging Bio Molecules

Imaging of
Biomolecules
Bright/Dark Field
Phase Contrast Transmitted Light
Polarization
Differential Interference Contrast (DIC)
Fluorescence (epi, confocal, TIRF, super-resolution etc)
Friday 27 October 17
Microscopes are designed with 3 essential functions
Magnification (to make structural detail visible to the detector)
Resolution (limited by wavelength, not lens aberrations)
Contrast (most biological specimens are transparent at vis wavelengths)
Absorption based contrast:

- color dyes: histology
- epi-fluorescence (modern day fluo. probes)
Refractive Index Differences based contrast:
- in specimen structural details from background (media,
cytosol)
Specimens with a higher Refractive Index than the
background produce retardation of light
Retardations in Transparent Specimens Scatter
(Diffract) Illuminatng Light
Bright Field Microscopy
Dark Field Microscopy:
Objective Collects Only Scattered Light; Rejects
Illumination Light
Angle of the illuminating cone
of light (high numerical
aperture) is bigger than the
angle of the scattered cone of
light collected by the
objective (low numerical
aperture).
- Cardioid condenser
Dark Field Microscopy:
Illumination Light
Advantages:
- High sensitivity - black background provides high contrast to see scattered
light from small objects.
- excellent for low magnification outlines of individual cells (such as flagella).
- It can be effectively used at high magnifications to image living bacteria, or at
low magnifications to view and image cells, tissues, and whole mounts.
Disadvantages:
- Resolution limited by need for NAcondenser > NAobjective
- Scattered light in thick specimens lowers contrast of fine structural detail
- Poor depth of field
- Images of internal cellular structure often inaccurate coz of the missing
interference with the undiffracted light (loss of signal)
Phase Contrast Microscopy:
Illumination Light
Phase Contrast Microscopy:
Lateral shifts in phase of wavefronts passing through
sample and passing undiffracted
- Positioned within the objective rear focal
plane is a phase ring or plate that not only
attenuates the bright, direct light originating
from the phase stop in the condenser, but also
adds a constant phase shift to this light.
- If the specimen contains sub-structures that

have mixed refractive indices, these entities
guide the light from the direct waves into new
paths (red dotted line).
- Wavefronts diffracted by the specimen (in

effect, those containing structural
information) will not pass through the phase
ring in the objective, meaning that they will
not be attenuated or retarded.
- All of the wavefronts are ultimately

recombined to form the intermediate image by
the tube lens.
Polarization Contrast Microscopy:
Optical anisotropy based capture of scattered
wavefronts from the specimen
The polarizer beneath the condenser (near the
aperture diaphragm) ensures that the specimen is
illuminated with linearly polarized wavefronts
that pass through the condenser.
The analyzer (a second polarizer), which is

oriented at an angle of 90 degrees to that of the
polarizer, is located behind the objective.
If no specimen is placed on the microscope

stage, the scene observed through the eyepieces
will remain completely dark.
When illuminated, many specimens turn the

vibration direction of the polarized light out of
the plane produced by the polarizer and
eventually the diverted planes are allowed to pass
through the crossed polarizer (analyzer), showing
up as images.
Phase Polarized
- difference of n of
- difference of n
specimen in different
between specimen
directions
and background
- optical anisotropy
Differential Interference Contrast Microscopy:
A polarizing microscope with condensor and two
DIC prisms
DIC prisms
DIC prisms
DIC
Ph Con
Pros Cons
Fluorescence
Framework of Fluorescence
Microscopy
Excitation Filter
- The excitation filter is the

optical element that passes only
the wavelength of light necessary
for excitation from the excitation
light source (usually a mercury
lamp) to the fluorophore.
- As shown in Figure 2, only the
excitation wavelength passes
through the filter, usually a "band
pass filter".
Dichroic Mirror
- The dichroic mirror is the optical

element that separates the excitation
light from the fluorescence. Dichroic
mirrors are special mirrors that reflect
only a specific wavelength of light,
allowing all other wavelengths to pass
through.
- Dichroic mirrors used in epi-

fluorescence microscope filter blocks
are placed in a 45° incidence angle to
light, creating a "stop band" of
reflected light and a "pass band" of
transmitted light. Light passing
through the excitation filter is reflected
90° toward the objective and the
specimen
Emission Filter
Barrier filters are optical elements that

separate fluorescence emanating from
the fluorophore from other background
light. As shown in Figure 4, the barrier
filter transmits light of the fluorescence
wavelength which passes through the
dichroic mirror while blocking all other
light leaking from the excitation lamp
(reflected from the specimen or optical
elements)
This is necessary because the strength

of the fluorescent light is weaker than
the excitation light by a factor of
more than 100,000:1.
Organic dyes
Classics
Coumarin Fluorescein Rhodamine
332/456 490/520 550/575
Systems of conjugated bonds that share electrons

Larger systems >> longer wavelength
Mitotracker DAPI
Oxidized in Stains nucleic acid
mitochondria to a
fluorescent compound
Fluorescent Proteins
GFP
O Shimomura
DC Prasher
M Chalfie
R Tsien
Fluorescence
Epi Confocal
Fundamental problem with conventional epifluorescence
is that fluorescence is emitted along the entire
illumination cone in the sample and not just at focus
Fluorescence Illumination of a single point
Camera
Tube lens
Excitation light
Emission light
Objective lens
Sample
Widefield Illumination Point Illumination
The confocal microscope
Scan excitation spot point-
Detector
by-point to build up image
Pinhole
Tube lens
Excitation light
Emission light
Objective lens
Sample
The pinhole is conjugate to the focal point of the lens

Light sources
Excitation light must be focused to a

diffraction limited spot
Excitation light
Could be done with an arc lamp
and pinhole – but very inefficient
Enter the laser:

Perfectly collimated and Objective lens
high power
Sample
Scanning
Changing entrance
angle of illumination
moves illumination spot
on sample
Objective lens
The emission spot

Sample
moves, so we have to
make sure pinhole is
coincident with it
Confocal optical path
Dichroic Pinhole
Scanning mirrors
x
Detector
y
Laser
Relay lens
Objective
there never is a complete image of the sample -- at any given

instant, only one point of the sample is observed. The
detector is attached to a computer which builds up the image,
one pixel at a time. In practice, this can be done perhaps 3
times a second, for a 512x512 pixel image. The limitation is
in the scanning mirrors.
Downside - Slow
The confocal microscope

Scan excitation spot point-
Detector
by-point to build up image
Pinhole Problems:
Slow (~1 sec to acquire an
image)
Tube lens Low light efficiency (due to
Excitation light use of PMT as detector)
Solution:
Emission light
Use multiple pinholes and
a camera
Objective lens
Sample
Optical Sectioning and 3D reconstruction
x y
x y
x y
Some state-of-the-art variations of
fluorescence microscopy
Total Internal Reflection Fluorescence (TIRF microscopy) -
for problems at the membrane interfaces
Breaking the diffraction barrier
Breaking the diffraction barrier - Super-resolution
Stimulated Emission Depletion Microscopy
Which imaging technique should I use?
1-5 m TIRF (for samples at the coverslip)
Wide-field (+deconvolution)
Fast
1-20 m
Sample Thickness
Spinning Disk Confocal
Sensitivity
10-100 m Line-scanning confocal
>20 m Point scanning Confocal
Slow
>50-100 m 2-photon confocal

Imaging Bio Molecules

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Imaging of

Fluorescence (epi, confocal, TIRF, super-resolution etc)

Magnification (to make structural detail visible to the detector)

Resolution (limited by wavelength, not lens aberrations)

Contrast (most biological specimens are transparent at vis wavelengths)

Absorption based contrast:

Bright Field Microscopy

- If the specimen contains sub-structures that

- Wavefronts diffracted by the specimen (in

- All of the wavefronts are ultimately

The analyzer (a second polarizer), which is

If no specimen is placed on the microscope

When illuminated, many specimens turn the

- The excitation filter is the

- The dichroic mirror is the optical

- Dichroic mirrors used in epi-

Barrier filters are optical elements that

This is necessary because the strength

Coumarin Fluorescein Rhodamine

332/456 490/520 550/575

Systems of conjugated bonds that share electrons

Widefield Illumination Point Illumination

The pinhole is conjugate to the focal point of the lens

Excitation light must be focused to a

Enter the laser:

The emission spot

there never is a complete image of the sample -- at any given

The confocal microscope

1-5 m TIRF (for samples at the coverslip)

Spinning Disk Confocal

>20 m Point scanning Confocal

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