Biology 177: Principles of Modern Microscopy

Август 20, 2022

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Biology 177: Principles of Modern Microscopy

Содержание

2. The First Contrast Histological stains Still important today
3. The Ultimate Contrast Transparent specimen contrast Bright field 2-5% Phase & DIC 15-20% Stained specimen 25%
4. Polarized light Circular polarization, rarely produced in nature Can see on iridescent scarab beetles and Mantis
5. Polarized light Radial light waves becomes polarized when reflected off surface at Brewster’s angle Brewster’s angle
6. Polarized light We cannot detect the polarization of light very well But some animals can see
7. Polarized light microscopy Highly specific detection of birefringent components Orientation-specific Less radiation than through other techniques
8. Polarized light microscopy With crossed polarizers: Only items that rotate the plane of polarization reach the
9. Polarized light microscopy images Brightfield Background Birefringent Material Polarized Light Pol + Red I Color of
10. Birefringence Material having a refractive index (η) dependent on polarization Responsible for DOUBLE REFRACTION, splitting of
11. Birefringence Augustin-Jean Fresnel first described in terms of polarized light Isotropic solids are not birefringent (glass)
12. Birefringence Light split into extraordinary and ordinary rays Birefringence difference between refractive index of extraordinary ray
13. Birefringence Structural Anisotropic Stress or strain Isotropic
14. Full Wave (First Order) Retardation Plate Also known as: Lambda plate Red plate Red-I plate Gypsum
15. Polarized light microscopy Using full wave retardation plate Phyllite Metamorphic rock aligned under hear and stress
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The First Contrast
Histological stains
Still important today

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The Ultimate Contrast
Transparent specimen contrast
Bright field 2-5%
Phase & DIC 15-20%
Stained specimen

25%
Dark field 60%
Fluorescence 75%

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Polarized light
Circular polarization, rarely produced in nature
Can see on iridescent scarab

beetles and Mantis shrimps
Mantis shrimps can see circularly polarized light

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Polarized light
Radial light waves becomes polarized when reflected off surface at

Brewster’s angle
Brewster’s angle ranges from 50° to 70° depending on surface material.
Used to polarize lasers
Why sunglasses horizontally polarized

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Polarized light
We cannot detect the polarization of light very well
But some

animals can see polarized light
Many insects, octopi and mantis shrimps

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Polarized light microscopy
Highly specific detection of birefringent components
Orientation-specific
Less radiation than through

other techniques such as fluorescence
Linear / circular Polarized Light
Differential Interference Contrast (DIC) uses polarized light

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Polarized light microscopy
With crossed polarizers:
Only items that rotate the plane of

polarization reach the detector
Retardation plate optional
Converts contrast to color

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Polarized light microscopy images
Brightfield
Background
Birefringent Material
Polarized Light
Pol + Red I
Color of

sample and background modified by wave plate

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$Birefringence Material having a refractive index (η) dependent on polarization Responsible$

Birefringence
Material having a refractive index (η) dependent on polarization
Responsible for DOUBLE

REFRACTION, splitting of a ray of light into two with differing polarization

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Birefringence
Augustin-Jean Fresnel first described in terms of polarized light
Isotropic solids are

not birefringent (glass)
Anisotropic solids are birefringent (calcite, plastic dishes)
Splits light into two rays with perpendicular polarization

Augustin-Jean Fresnel
1788-1827

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Birefringence
Light split into extraordinary and ordinary rays
Birefringence difference between refractive index

of extraordinary ray (ηe) and ordinary ray (ηo)

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Birefringence
Structural
Anisotropic
Stress or strain
Isotropic

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Full Wave (First Order) Retardation Plate
Also known as:
Lambda plate
Red plate
Red-I plate
Gypsum

plate
Selenite plate
Retard one wavelength in the green (550 nm) between extraordinary ray and ordinary ray

Cotton

Uric Acid

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Polarized light microscopy Using full wave retardation plate
Phyllite
Metamorphic rock aligned under hear

and stress
Oolite
Sedimentary rock of cemented sand grains

Plane-
Polarized

Cross-
Polarized

Full wave
retardation plate