Basic Light Microscopy Flashcards
Quiz 1 Start from the basics of light and microscopes
What techniques are standard for analysis of Trace Evidence?
- polarized light microscopy (fibers, minerals)
- comparison transmitted light microscopy (hairs, fibers)
- index of refraction measurements (glass, fibers)
What techniques are standard for analysis of Drug Analysis?
- crystal tests
What techniques are standard for analysis of Forensic Biology?
- crystal tests for hemoglobin
- identification of sperm
What techniques are standard for analysis of Firearms examinations?
- comparison reflected light microscopy (bullets, cartridges, took marks)
What techniques are standard for analysis of Questioned Document Examination?
- reflected light microscopy of handwriting and printing
What are some examples of hyphenated microscopic methods?
- microspectrophotometry
- micro FTIR
- micro Raman
Who invented the 1st microscope? i.e. Father of Microscopes
Zacharias Janssen 1590
What was Robert Hook’s contribution to science?
- developed and used a compound microscope to observes cells in cork in 1665
What was Van Leeuwenhoek’s contribution to science?
- developed a simple microscope with very good lens to examine small organisms
List the type of Lens Aberrations
- chromatic aberration
- spherical aberration
- coma
- astigmatism
- curvature of field
- lateral color
Define Chromatic Aberration
- 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)
What can counteract Chromatic Aberration?
- 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
Define Spherical Aberration
- 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)
Define Coma
- 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
Define Astigmatism
- 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
Define Curvature of Field
- causes a flat object to appear sharp only at certain parts instead of being uniformly sharp
Define Lateral Color
- 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
Define Plan
- 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)
What are Achroplans?
- a type of objective lens
- corrects curvature of field
- best for transmitted light
What are Epiplans?
- a type of objective lens
- corrects for curvature of field
- designed for reflected light use
What are Achromat lenses?
- lenses that have good color correction for 2 wavelength of light
(they are easy on the wallet)
What are Planachromats?
- achromats with correction for flatness of field as well as color correction at 2 wavelengths
What are Plan-Neofluar lens?
- 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
What are Apochromatic (planapochromat) lenses?
- 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
What does Oel mean on an objective lenses?
- type of immersion medium
- for oil
What does W mean on an objective lenses?
- type of immersion
- water
What does Imm mean on an objective lenses?
- type of immersion medium
- multi-immersion for oil, water and glycerin
What does Ph or Phaco mean on an objective lenses?
- 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
Define Numerical Aperture (N.A.)
- 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
Equation for N.A.
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
Define Resolution
- defined as the ability of a lens to distinguish between small objects
- different from magnification
Abbe’s Law of Limiting Resolution
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
Rayleigh Resolution
- 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
Where on the objective lenses marks the Tube Length?
- 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
Where on the objective lenses marks the coverslip thickness?
- 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)
What # coverslips are not recommended?
0, 1, & 2
Coverslip Thickness
Some lenses will have a - sign instead of a number. What does this mean?
- means that the objective is meant to be used with no coverslip
What does LD mean on an objective lenses?
hint: in the place of the coverslip thickness #
- long working distance
- up to 1.5mm
What does Korr mean on an objective lenses?
hint: in the place of the coverslip thickness #
- some lenses will have a rotating ring that allows one to correct for a coverslip thickness
