Polarized Light Microscopy

Visible light has a spectrum of what?

400nm to 700nm

Propagation direction (ray direction) is what?

The way the wave is moving

Vibration direction is what?

The direction of displacement (oscillation direction)

Wavelength is what?

Distance from wavetop to wavetop

Height of wave is what?

amplitude

Unpolarized light is what?

light with rays vibrating in multiple directions

Polarized light is what?

rays vibration in only one direction (one plane)

Incident light is what?

Non-reflected light

Reflection is what?

reflected light

What does the law of reflection state?

The angle the light is reflected at is equal to the angle it struck the surface at.

Ray angle (the normal) is measured from what?

Measured from a line perpendicular to the surface it reflected off of

Reflected light will be what?

Partially polarized

Reflected light preferentially vibrates in what plane?

The plane parallel to the surface it reflected off of

Smooth surfaces give what type of reflection?

Regular (specular) reflection

Rough surfaces give what type of reflection?

Diffuse reflection

What is refraction?

The change in direction of incident light as it passes from one substance into another, and is related to the light’s change in speed.

Only occurs if the incident angle of the light is >0 degrees

Wave front is what?

a line connecting equivalent parts of two waves that are in sync and travelling parallel

Wave normal is what?

perpendicular to the wave front

Refractive index (RI) is what?

how much light bends when passing between two substances

What must happen for light to refract (bend)?

Both substances must have different RIs and have an incidence angle greater than 0 degrees

Difference in refractive indexes affects what?

The amount and direction of the bend

Low Refraction to High Refraction means what?

The light bends normal

What is dispersion?

variation in the RI

Shorter wavelength equals what?

more interaction with the substance, higher RI

Does temperature affect the density and RI of solids?

Yes.

Minor change in density, minor change in RI

Does temperature affect the density and RI of liquids?

Yes.

Major change in density, major change in RI

What composes a compound lens?

Two sets of lenses —> Objective and Ocular

Polarized Light Microscope (PLM) is what?

a compound microscope with a polarizer and an analyzer

What is a lens?

a transparent material with at least one curved surface

What is a lens line?

a line connecting the physical center of the lens and the center of the curvature of the lens’ surface

What is a biconvex lens?

a lens with two focal points that are equidistant from the center of the lens

What type of image is formed by the ocular lens?

a virtual image

What is resolving power?

the ability to virtually distinguish between two objects

Higher resolving power equals what?

The closer two details can be examined while still being distinct

Empty magnification equals what?

An increase in size of an image with no additional resolving power

What is resolving power dependent on?

Numerical Aperture (NA) of objective and condenser lens

NA is equal to what?

Angular aperture as well as refractive index.

Angular aperture is equal to what?

angle at which light can be collected by the lens

Higher NA equals what?

the wider the angle of light the lens can collect

Wider angle of light equals what?

more resolving power of the lens

What happens when resolution goes up?

Contrast goes down

What can adjusting the aperture’s size do?

Can change both resolution and contrast

Aperture Plane

• Lamp filament

• Condenser Aperture

• Objective rear focal plane

  • Iris diaphragm of eye

Image Forming Plane (Field Plane)

• Field Diaphragm

• Specimen

• Fixed eyepiece diaphragm

  • Retina

Kohler Illumination Steps

• Focus on sample

• Shut down field diaphragm so you can see edges

• Adjust height of the condenser so that the edges of the field diaphragm are in sharp focus

• Use the condenser centering screws to center the field diaphragm

• Open the field diaphragm so the aperture is just out of view

• Set condenser to the same NA as the objective lens

What does Kohler Illumination produce?

Unidirectional oblique illumination which will provide max resolution at the cost of contrast

Isotropic Substance RI

one (1) RI- Like glass

What happens if the sample RI is different than the mounting media RI?

Lots of contrast

What happens if the sample RI is the same as the mounting media RI?

No contrast- invisible

What is the Becke line?

A halo of light that moves around or into the substance being viewed

What is the Becke Line method?

Raising the focus by lowering the stage so that the becke line can move into the substance with the HIGHER RI

Uniaxial Substances

• Light with two components and separate RIs

• Anisotropic

Like fibers

Anisotropic

Having different physical properties in different directions.

When does retardation of a substance occur?

When light enters an anisotropic substance, one ray will be fast (lower RI), and one ray will be slow (higher RI). The difference between the two is the substance’s retardation.

Thicker samples will have increased differences, which affects the retardation

What is birefringence?

The difference between the two RI’s of the material being tested.

The larger the birefringence, the bigger the difference between the speed of the rays

How do you calculate birefringence?

Retardation divided by sample thickness times 1000

Extinction Characteristics

• Some fibers will go completely dark under crossed poles when rotated either N-S or E-W

• Cotton will NOT do this due to internal structure

Objective Lens Markings: FL

Lens = fluorite, or a semi-apochromat lens

Objective Lens Markings: Achro/Blank

An achromat lens

Objective Lens Markings: Apo

An apochromat lens

Objective Lens Markings: Plan

Lens is corrected for field curvature

Objective Lens Markings: 4x/10x/20x/40x

Lateral magnification

Objective Lens Markings: P

Optimized/polarized light

Objective Lens Markings: ∞

the objective lens is designed for an infinite tube length

Objective Lens Markings: FN(NUMBER)

Field Number.

EX: FN26.5. Dividing this by the lens’ magnification give you the lens’ field of view in mm:

• 26.5/20=1.325mm

Objective Lens Markings: 0.17 (or between ∞ and FN)

Cover slip thickness

Positive Sign of Elongation

Fiber that has a parallel RI that is greater than it’s perpendicular RI

Negative Sign of Elongation

Fiber that has a parallel RI that is less than it’s perpendicular RI

How to know if a fiber has positive or negative elongation?

• Insert analyzer and position the fiber parallel to the direction of the compensator’s slow wave length

• Insert full wave compensator

• Positive = blue

• Negative = orange