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Imaging Unit 1

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7 views

Imaging Unit 1

Uploaded by

swathi manian
Copyright
© © All Rights Reserved
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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LIGHT AS A PROBE OF MATTER

Dr. Mandhakini
Asst Professor
CNST, Anna University
LIGHT AS A PROBE OF MATTER
• Visible light, the agent used as the
analytic probe in light microscopy, is
a form of energy called
electromagnetic radiation.
• This energy is contained in discrete
units or quanta called photons that
have the properties of both particles
and waves.

Photons as electromagnetic waves exhibit oscillating electric and


magnetic fields, designated E and B, respectively, whose amplitudes
and directions are represented by vectors that oscillate in phase as
sinusoidal waves in two mutually perpendicular planes.
Dual particle and wave-like properties
• The properties of energy, frequency, and wavelength are related through the
following equations, which can be used to determine the amount of energy
associated with a photon of a specific wavelength:

The first equation defines the velocity of light as the product of its frequency and
wavelength.
We will encounter conditions where velocity and wavelength vary, such as when
photons enter a glass lens.
The second equation relates frequency and energy, which becomes important when
we must choose a wavelength for examining live cells.
The third equation relates the energy of a photon to its wavelength.
Since E ∼ 1/λ, 400-nm violet wavelengths are twice as energetic as 800-nm infrared
wavelengths.
Wave Phenomena

• Refraction • Reflection • Dispersion

• Interference • Diffraction
Corpuscular Phenomena
LENSES AND
GEOMETRICAL OPTICS
Geometrical optics of simple lenses.
Negative lenses diverge and positive lenses converge incident beams of
light.
REFLECTION OF LIGHT
The object’s surface is planar, the law of
reflection may be observed, whereby the
angle of incidence equals the angle of
reflection (Fig. ).
Both angles are described by the angles
subtended between the incident and
reflected rays and the normal (an imaginary
line perpendicular to the surface).

Reflection of light, showing ϴi = ϴr


Refraction
In refraction, as in the case of light entering a lens, light rays become bent
(refracted) at the air-glass interface and follow a different path in the second
medium.

The ratio of the velocities (or of the wavelengths) of light in a


vacuum (c, λ) and in the medium (νm, λm) define what is known as
the refractive index n:

For example, the refractive index of water is 1.333, meaning that light travels 1.333
times slower in water than in a vacuum.
Increasing the refractive index corresponds to decreasing the speed of light in the
material.
Snell’s Law
Refraction and Snell’s law: n1sinϴ1 = n2sin ϴ2.
(a) When n2 > n1, light rays become bent in the direction of the normal.
(b) When n2 < n1, light rays bend away from the normal.
If ϴ1 is adjusted so that ϴ2 > 90° (greater than the critical angle), rays are totally reflected
back into the first medium (total internal reflection).
Principles governing ray tracing for a thin lens
lens equation

The well-known lens equation describes the relationship between focal length f and
object and image distances, a and b:

Further, the magnification factor M of an image is described as:


Real Vs virtual image
• Depending on the location of the object relative to the focal point
of the lens, the image may be real or virtual, magnified or
demagnified
Human eye optical system
Optical microscope
Optical microscope
EFFECTS OF LIGHT ON LIVING CELLS

UV wavelength (200nm- 400nm) Near IR radiation (700nm -1400nm)


Damage the cells, because photons of Photons of near-IR radiation are less
UV light are powerful enough to energetic than those of visible
break covalent bonds, thereby light, but are absorbed by carbon
creating reactive free radicals that bonds in macromolecules, such as
chemically alter and denature DNA, and by water (moderately in
macromolecules, such as proteins, the range from 750 to 1000 nm,
nucleic acids, lipids, and small and strongly at wavelengths >1000
metabolites. nm), leading to accumulation of
Damage to DNA and membrane kinetic energy (heat) and
proteins, such as ion channels and denaturation of molecules.
gates, is a particular concern.
EFFECTS OF LIGHT ON LIVING CELLS
• Visible light itself is unique because it is absorbed relatively poorly by
living cells, particularly at green and yellow wavelengths.
• For the most part, cellular absorption of visible light is considerably less
than for the flanking UV and IR wavelengths.
• Since green light is relatively nontoxic and marks the peak sensitivity for
human color vision, the 546-nm green line of the mercury arc lamp is
commonly used for monochromatic illumination of living cells
(Khodjakov and Rieder, 2006)

IR- and UV-blocking filters, such as Schott filters BG38 (for IR) and GG420 (for
UV), are especially useful, since the spectra generated by mercury, metal
halide, and xenon arc lamps used in microscopy are rich in UV and IR
radiation (for mercury, 30% UV, 40% IR, 30% visible; for metal halide, 15%
UV, 45% IR, 40% visible; for xenon, 5% UV, 70% IR, and 25% visible).
THE PRINCIPAL ABERRATIONS OF LENSES

The solution is to make compound lenses made of glasses having different color-
dispersing properties.
For example, glass types known as crown and flint are paired together to make an
achromatic doublet lens that focuses blue and red wavelengths in the same image
plane.
Spherical abberation-

One common solution is to use a combination of


positive and negative lenses of different thicknesses in a
compound lens design.
Example of two asymmetric lenses (left
and right) and one symmetric lens (in
the middle)
Resolution of an optical microscope
DIFFRACTION AND INTERFERENCE IN
IMAGE FORMATION
In the microscope, light from the illuminator is diffracted (literally broken up in the sense of
being scattered or spread) by the specimen,collected by the objective (a second site for
diffraction), and focused in the image plane, where waves constructively and destructively
interfere to form a contrast image.

DIFFRACTION AND INTERFERENCE

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