Techniques in Light Microscopy
PMB 185, F 2014
Midterm Exam
28 questions/ 85 points


Name: ____________________________

The point value of the question is in parentheses.

1) In which conjugate plane groups are the following microscope optical devices placed? (4)

A: Imaging Planes; B: Illumination Planes

Wollaston Prism I       B

Condenser Iris             B

Reticle                         A

Exit Pupil                    B

Field Iris                      A

DF Stop                      B

Phase Plate                  B

PlasDIC prism                        B


2) Define and describe Chromatic Aberration. (2)

When white light is deconstructed into its component wavelengths by dispersion, each wavelength realizes a different refractive index (n). Therefore each wavelength focuses at a different position along the optical axis. This produces rainbow fringes around objects and decreases resolution.

3) Define Infinity Correct Optics and note any special optical components that may be present? (2):

Modern objectives project sample light to infinity, and do not form a real intermediate image. A third lens the “tube lens” must be present within the body. This is a positive, converging lens that creates the Real image at the IIP. This image is, of course, used as the object by the eyepiece.

4) State why is it important to first adjust for Köhler Illumination before successfully using the following microscope imaging techniques? (5)


A few words explaining why Köhler is required


Even sample Illumination, Condenser iris modulates contrast


Insures that light from the DF Stop “misses” the AA


Insures that the cond. Ph. Annulus is conjugate to the obj. Ph Plate


Insures that W/Nom1 in Cond is conjugate to W/Nom2 in/above the obj


Insures that scattered sample light passes correctly through Amp Modulator


5) Define Numerical Aperture and its relationship to light collection and resolution. (3)

NA=nsin(aa/2); where n=the ref index of the immersion medium, AA=obj angular aperture.

As NA increases 1) light collection increases, 2) resolution increases (d gets smaller).


6) How does the Rayleigh Criterion of Resolution differ from the Abbé definition? (2)


Rayleigh defined resolution as a function of the closeness of two objects (first Airy superimposed over 1st minimum).

Abbe defined resolution as a function of the diffraction of one object. Included NA and wavelength

7) How does the use of Immersion Oil effect microscope resolution? (2)

Look at (5): NA=nsin(aa/2). Immersion oil has a higher n (refractive index) than water (1.515 vs 1.33). Therefore NA will increase when oil is used. As stated in #5, as NA increases both light collection and resolution also increase.

8) What is the name and function of the “Last” (top) polarizing filter when used in Quantitative Polarized Light Microscope? (2)

“Analyzer”: 1) It is set 90° to the orientation of the Polarizer. 2) For QUANTITATIVE PLM a Retardation Plate is also required. It is set at 45° to Pol and Ana. The lambda-plate creates two orthogonal wavefronts that BOTH interact with the Analyzer. The function of which is to “fold together” (vector addition) the E- and O-rays from the lambda plate. Colors generated at ANA relate to the birefringence number of the sample.

9) Recently, when trying to image bacteria at 100x you were unsuccessful in finding the focal plane (because they are so small). Remembering your basic microscopy you switched to a 10x objective and DF imaging, successfully found the bacteria, then switched back to 100x and proceeded to complete your work. Why was this possible? (2)

1) 10x obj has a greater Depth of Field than the 100x. Makes it easier to find the sample.

2) DF is a good technique to DETECT sub-resolution objects (bacts at 10x).

3) Once the sample plane is found using 1 & 2, parfocality predicts that the 100x objective will also be at the correct focal plane.


10) When viewing cells grown in plastic petri dishes why do you see rainbow colors when trying to use DIC? (2):


DIC is sensitive to birefringence. Additional e- and o-rays created by the plate, as well as the plate’s variable OPL contribute to the production of rainbow colors due to inappropriate wave interference.



11) How would you know whether a thumbprint seen through the eyepiece is on the Reticle or the Wollaston Prism 2? (2)


If you can see it this means that the print is in an Imaging Optical Plane. Only the Reticle is in the Imaging planes.

12) What is the fundamental difference between the generation of 3D between PlasDIC versus HMC? (2)

PlasDIC: Interference of Difference Images

HMC: Sample light refracted to different %T sectors, only light intensity (amplitude) is changed.

13) What is the function of WP2 in Differential Interference Contrast Microscopy? (2)

Modulate BIAS, or phase differential between sample difference images. Subsequent interference (at ANA) produces contrast.


14) Compare the following compound microscope types. (6)






Example Specimen


Typical Microscope Slide

Live cells in a petri plate

Whole plate or organism

Appropriate Illumination Technique


All except DIC

Usually Oblique


15) In the following Microscope Techniques in what optical plane does the image contrast get generated (or created)? (6)


IIP/Imaging Plane




IIP/Imaging Plane





Phase Contrast



16) Match the typical sample type to the Preferred Microscope Optical Technique. (3)

a) In Vitro experiment in a plastic culture dish ___F, C_                                  A) BF

b) Unknown powder found under a park bench ___D_                                   B) DF

c) Sub-resolution nano-particles adhered to a coverslip  __B__                         C) PC

d) Lactobacillus (a bacterium) in yogurt  __E, C__                                          D) PLM

e) Safranin & Fast Green-stained plant tissue sections __A__                         E) DIC

f) Mammalian cells grown on glass coverslips. __E, C_                                    F) HMC



17) Compare and Contrast Critical Illumination and Köhler Illumination? (4)


Illumination source conjugate with Sample plane. Image of filament visible.

Light very bright but uneven

Poor contrast control




Illumination source conjugate with Condenser plane. Light source image not visible.

Light very bright and even.

Contrast control using the Condenser Iris



18) Why are Bacillus subtilis endospores bright white when viewed using Phase Contrast Microscopy? (2)

Endos retard light nearly 1/4lambda. When using Negative PC, the background is also retarded 1/4lambda. Constructive interference caused by in-phase sample and bkgnd light causes the Amplitude to greatly increase. Thus the endospores are Phase Bright.

19) What is the major discovery or invention that is associated with the following Optical Pioneers? (4)


Discovery or Invention


DIC; modified DIC prism


Law of Refraction


Phase Contrast microscopy


Condenser producing even sample illumination


Achromatic Doublet


Tripod microscope; gimbal mirror


NA; immersion oil; resolution based on diffraction


First compound (“Double”) microscope



20) Would DIC be a useful technique to visualize broken glass coverslip bits mounted in water? If so explain why, if not explain why not and give an alternate optical technique that would be better. (2)


No: DIC images continuous OPL gradients well, and “flat”, no gradient, poorly.

DF would be best as it will show clearly the edges of the glass. PC will also work.


21) In Phase Contrast Microscopy a) what device do you use to align the Phase Plate and Phase Annulus, b) why does it work, and c) why is it necessary to use it? (3)

a) The device is: __Phase Telescope____________________________

b) It works because: _____It focuses on the BFP, where the phase Plate resides. ____________________________

c) It is necessary because: ____you need to align both rings, and to do so you have to see them. _____________


22) What is the meaning of the following designations associated with microscope components: (6)


63x/1,35          Oil objective Mag/NA

160: Mechanical Tube Length

Plan-Achromat: Objective flat field, corrected for 2 colors

į: ICS, Infinity corrected objective

10x/19: Eyepiece mag/FN

45mm: Parfocal distance (for 3 mfgrs)



23)  Define the following (4):

1) Parfocal Distance: Distance between Obj mount and sample plane

2) Working Distance: Distance between front obj element and sample plane

3) Angular Aperture: Collection/cone angle of an objective.

4) Mechanical Tube Length: Distance between Eyepiece and objective mounts.

24) How does one use a Full-Wave Retardation Plate to identify a particular birefringent material? (3)


1) Determine sample thickness
2) Insert lambda plate 45° between Pol and Ana in a scope

3) Rotate sample to get most/darkest saturated color. This aligns the Fast axes of the sample and Lambda plate
4) Note sample color
5) Find color on Michel-Levy chart
6) Find intersection of Sample Thickness and Color

7) Follow closest M-L Ray to determine the Birefringence Number of the sample


25) This aberration of lenses, which describes how light passing through the outer radii of a lens differs from light that passes the inner radii, has been overcome by the addition of that. (2)

This aberration: ___Spherical__________

That addition: __Stop/Aperture/Doublet______








26) Define Optical Path Length and its effect on light as it pertains to microscopy imaging. (2)


OPL=nl, where n=the refractive index of the object; l=geometric path length in Ķm.

OPL affects phasing of light (either polarized or non polarized). Subsequent wave interference between phased light (via OPL) and background light generates contrast. Techniques that exploit OPL phasing are PC, DIC, PlasDIC, PLM


27) In a compound microscope, why must the objective lens (or objective + Tube lens) be a converging lens (or lens system)? (2)


A converging lens creates a Real Image (as opposed to a virtual image). The eyepiece must “see” the Real Intermediate Image in order to complete the magnification of the image. This is a compound microscope. Modern Infinity corrected objectives use a body optic, Tube Lens, to create the Real intermediate image at the IIP.



28) How does Nomarski DIC differ from PlasDIC? Discuss hardware, illumination, and generation of contrast. (4)


N-DIC: 1) uses two prisms: a modified Wollaston prism allowing the Nomarski prism to be placed distant from the BFP, and another in a conjugate plane in the condenser.
2) Arrangement of optics (from light source): Pol, N1, sample, N2, Ana
3) N2 modulates Bias and modifies phase offset.
4) Interference at the ANA between difference images produces contrast.


PlasDIC: 1) Uses only one prism.
2) Pol is directly attached to the prism, and downstream from the sample.
3) The single prism creates the difference images (shear) as well as modulate bias.
4) The Analyzer folds vibrational planes of the difference images together, however
5) Interference occurs at the IIP where the two image wavefronts create a real image