Basic Light Microscopy

Question 1

What techniques are standard for analysis of Trace Evidence?

Answer 1

• polarized light microscopy (fibers, minerals)
• comparison transmitted light microscopy (hairs, fibers)
• index of refraction measurements (glass, fibers)

Question 2

What techniques are standard for analysis of Drug Analysis?

Answer 2

• crystal tests

Question 3

What techniques are standard for analysis of Forensic Biology?

Answer 3

• crystal tests for hemoglobin

- identification of sperm

Question 4

What techniques are standard for analysis of Firearms examinations?

Answer 4

• comparison reflected light microscopy (bullets, cartridges, took marks)

Question 5

What techniques are standard for analysis of Questioned Document Examination?

Answer 5

• reflected light microscopy of handwriting and printing

Question 6

What are some examples of hyphenated microscopic methods?

Answer 6

• microspectrophotometry
• micro FTIR
• micro Raman

Question 7

Who invented the 1st microscope? i.e. Father of Microscopes

Answer 7

Zacharias Janssen 1590

Question 8

What was Robert Hook’s contribution to science?

Answer 8

• developed and used a compound microscope to observes cells in cork in 1665

Question 9

What was Van Leeuwenhoek’s contribution to science?

Answer 9

• developed a simple microscope with very good lens to examine small organisms

Question 10

List the type of Lens Aberrations

Answer 10

• chromatic aberration
• spherical aberration
• coma
• astigmatism
• curvature of field
• lateral color

Question 11

Define Chromatic Aberration

Answer 11

• caused by the variation of refractive index with wavelength for a lens material
  (this doesn’t happen with curved mirrors)
• the wavelength dependence results in slightly different focal lengths for different wavelengths of light
  (creates a “rainbow” around image)

Question 12

What can counteract Chromatic Aberration?

Answer 12

• achromats (compound lenses) can reduce or eliminate the problem
• components are chosen such that some of the variation in RI as a function of wavelength cancels out

Question 13

Define Spherical Aberration

Answer 13

• a loss of definition in the image arising from the surface geometry of a shperical mirror or lens
• results b/c the actual focal point of a light ray depends on its distance from the optic axis
  (there are multiple focal points causing the imagine to be partially clear)

Question 14

Define Coma

Answer 14

• caused by the distortion of a wavefront as it encounters a lends asymmetrically (distortion of how light hits the lenses)
• light rays farther from the optic axis have severe aberration and the resulting image looks like a comet-shaped series of circles

Question 15

Define Astigmatism

Answer 15

• a defect in lens caused by a deviation from spherical curvature
• results in distorted images, as light rays are prevented from meeting at a common focus
• result is two line images at different points in space

Question 16

Define Curvature of Field

Answer 16

• causes a flat object to appear sharp only at certain parts instead of being uniformly sharp

Question 17

Define Lateral Color

Answer 17

• causes “colors” to focus at different distances from the image center
• most visible near corners of images
• a point source of white light is imaged as a small solar spectrum

Question 18

Define Plan

Answer 18

• stands for flat field
• lenses which are uncorrected for flatness of field will have the center of the field in focus and the outer edges out of focus
  (or vice versa depending on how you focus the lens)

Question 19

What are Achroplans?

Answer 19

• a type of objective lens
• corrects curvature of field
• best for transmitted light

Question 20

What are Epiplans?

Answer 20

• a type of objective lens
• corrects for curvature of field
• designed for reflected light use

Question 21

What are Achromat lenses?

Answer 21

• lenses that have good color correction for 2 wavelength of light
  (they are easy on the wallet)

Question 22

What are Planachromats?

Answer 22

• achromats with correction for flatness of field as well as color correction at 2 wavelengths

Question 23

What are Plan-Neofluar lens?

Answer 23

• semiapochromatic lenses
• have good color correction for at least 3 wavelengths & also have flatness of field
• excellent for polarization microscopic techniques i.e. differential interference
• excellent lenses for all types of fluorescence microscopy (transmit UV very well)
• contains fluorite elements making it good for fluorescence work

Question 24

What are Apochromatic (planapochromat) lenses?

Answer 24

• the most higly color corrected objectives
• corrected for 4 wavelengths and are top of the line in objective lenses
• most often have the highest numerical apertures
• do not transmit UV light
• work very well for visible light excitation in the blue and green ranges

Question 25

What does Oel mean on an objective lenses?

Answer 25

- type of immersion medium | - for oil

Question 26

What does W mean on an objective lenses?

Answer 26

- type of immersion | - water

Question 27

What does Imm mean on an objective lenses?

Answer 27

- type of immersion medium | - multi-immersion for oil, water and glycerin

Question 28

What does Ph or Phaco mean on an objective lenses?

Answer 28

- used for dark phase illumination (usually a number follows it) - generally not as good for fluorescence applications as the light transmission is reduced by the presence phase ring inside the lens

Question 29

Define Numerical Aperture (N.A.)

Answer 29

- appears as a slash followed by a number ranging from 0.035 to 1.4best - # is directly related to the resolution of the lens - related to the amount of light brought into the lens (very important for fluorescence microscopy) - higher N.A. = better resolving power & the brighter the image it can produce

Question 30

Equation for N.A.

Answer 30

N.A. = n(wavelength)*sin(mu) ``` n = index of refraction of the medium in which the lens is working at wavelength mu = half angle subtended by point on specimen at entrance pupil of objective ```

Question 31

Define Resolution

Answer 31

- defined as the ability of a lens to distinguish between small objects - different from magnification

Question 32

Abbe's Law of Limiting Resolution

Answer 32

d = minimum resolved separation v = wavelength of the monochromic light or shortest of mixed wavelengths NA = limiting numerical aperture of objective or condenser d = (0.5v)/NA

Question 33

Rayleigh Resolution

Answer 33

- adopted the criterion that 2 point objects can be discerned as separate if the 1st order diffraction maximum of the Airy Disk of one lies in the 1st minimum of the Airy disk of the 2nd d = (0.61v)/NA

Question 34

Where on the objective lenses marks the Tube Length?

Answer 34

- marks on the line below the magnification and the NA - for older lenses its 160mm to 170mm - for corrected objectives (newer) its usually infinity meaning it can be at any length

Question 35

Where on the objective lenses marks the coverslip thickness?

Answer 35

- the # after the slash after the tube length - it's 0.17 for most objectives for close working distance (use 1 1/2 coverslips which range between 0.16mm & 0.19mm in thickness)

Question 36

What # coverslips are not recommended?

Answer 36

0, 1, & 2

Question 37

Coverslip Thickness Some lenses will have a - sign instead of a number. What does this mean?

Answer 37

- means that the objective is meant to be used with no coverslip

Question 38

What does LD mean on an objective lenses? hint: in the place of the coverslip thickness #

Answer 38

- long working distance | - up to 1.5mm

Question 39

What does Korr mean on an objective lenses? hint: in the place of the coverslip thickness #

Answer 39

- some lenses will have a rotating ring that allows one to correct for a coverslip thickness