Forensic Microscopy Test 4

Polarized Light Microscopy and Fluorescence

What does the objective lens say in PLM?

Pol, P, PO, SF

What is different about the PLM?

• Analyzer below the head with vibrational direction up/down

• Polarizer bottom of condenser with vibrational direction east/west

• Rotating stage

What is polarized light?

Light with the same vibrational direction

What happens to light when it hits the filter?

The light splits into a fat ray and the slow ray because 2 refractive indices. One ray across the width and one across the length.

Retardation Value

Distance between the slow ray and fast ray.

What are the colors caused by in PLM?

They are called Interference colors and caused by interference between fast and slow ray, different color because of thickness.

Anisotropic

More than one refractive index

Isotropic

One refractive index, can’t be seen with PLM

Extinction

Fiber turns black when aligned with polarized light, cotton doesn’t undergo extinction

Delusterant

Dark fine flecks in fiber, decrease shine

Sign of Elongation

Tells you whether the fast or slow ray is aligned with the slow ray, Negative or Positive

Positive Sign of Elongation

More intense, fast ray of fiber aligned with slow ray of retardation plate, decrease in retardation

Negative Sign of Elongation

Less intense, slow ray of fiber is aligned with slow ray of retardation plate, greater retardation

Retardation Plate must Include:

Retardation value and direction of slow ray

Refractive Index

Refractive Index affects speed of light, Higher RI=slower speed of light

Polarized Filters

Reduce glare, only lets polarized light through, most objectives made this way to avoid birefringence

Birefringence

B=R/1000T

Michel-Levy Chart

Color chart which relates sample thickness, birefringence, and retardation

What does the fiber going black when putting the quartz wedge condenser mean?

It means the retardation value of the quartz wedge condenser = retardation value of the fiber

How does the quartz wedge condenser work?

Thicker for greater retardation value and thinner for lower retardation value.

What is different about the objective lens in Fluorescence?

• says Fl

• made of fluorite that doesn’t fluoresce

3 filters located under head of microscope in Fluorescence?

• in one cube, not separate

• Excitation Filter

• Dichroic Filter

• Emission Filter

Excitation filter

excites electrons, bandpass filter

Dichroic Filter

Long pass edge filter

Emission Filter

blocks excitation light, long pass edge filter

Types of Filters in Fluorescence?

• Edge filters: Long Pass and Short Pass

• Bandpass Filter

Long Pass Filter

Lets wavelengths longer than x nm pass through

Short Pass Filter

Lets wavelengths shorter than x nm pass through

Quenching(Fluorescence)

Absorption of emitted fluorescence by neighboring bonds/molecules

Photobleaching(Fluorescence)

Photon induced chemical damage

Primary/Auto-Flourescence

Fluoresces in its natural form

Secondary Fluorescence

Fluoresces after treatment with chemicals

Differences in Fluorescent Microscope v. Others

• Light source is very intense

• gives option of light sources in both visible and UV region

Fluorochromes

things that fluoresce, tend to have electrons in double/conjugated bonds