Techniques in Light Microscopy
PMB 185, F 2017
Midterm Exam
28 questions/ 100 points

 

Name: ____________________________

The point value of the question is in parentheses.

1) Match the device to its Conjugate Plane in this microscope diagram. Note whether the plane is Imaging (A) or Illuminating (B). (6)

A: Imaging Planes; B: Illumination Planes

 

Match

Plane

PlasDIC prism

C       

B

Reticle            

B

A

DF Stop         

D

B

Field Iris                     

E

A

Nomarski Prism I

D

B

Eye Relief

A

B

 

 

 

2) Name and describe the physical property of light and lenses that is responsible for Chromatic and Spherical Aberration of lenses. (4)

CA

Dispersion, spreading if wavelengths due to wavelength differences in refractive index

 

SA

Outer radii of lens focus light to a different point from inner radii

 

 

 

 

 

3) Compare Nomarski DIC and PlasDIC. (6)

 

Components

Contrast generated here

Good for this sample type

NDIC

ANA
WII
Sample
WI
POL

ANA

Cells
Single layers
No birefringence

PlasDIC

ANA
W
POL

Sample

IIP

Ditto, but tolerates birefringence

 

 

 

4) Explain why Köhler Illumination is required for these microscope imaging techniques to work? (I am interested in conjugate planes.) (4)

 

DIC

Places WI and WII in conjugate planes

 

 

PC

Places Phase Annulus and Phase Plate in CPs

 

 

BF

Allows Köhler to work

 

 

DF

Allows light from source to miss AA, but scattered light from sample to be collected.

 

 

 

5) What is the difference between Negative and Positive Phase Contrast Microscopy? Discuss components and why the samples look the way they do. (4)

 

 

Components

Sample Contrast (compared to the background)

 

Negative PC

 

S-Wave RETARDED ¼ wavelength

Bright

 

Positive PC

 

S-Wave ADVANCED ¼ wavelength

Dark

 

 

6) In one of your lab exercises you calculated the Principal Focus of a Negative (biconcave) lens using another lens as the imaging system. Under what conditions could you have measured the focal length using only the Concave lens and no other optic? (2)

 

Place the Negative (diverging) lens in a medium of HIGHER n

 

 

 

 

 

 

 

7) Describe the fundamental difference between Critical and Köhler Illumination? (2)

 

CI: Places the light source in the imaging plane. You see the source as well as the sample

KI: Places the light source in an illuminating plane. You do not see the source.

 

 

 

 

8) In DIC why are WI and WII oriented 45° to the privileged axes of the Polarizer and Analyzer? (2)

 

So BOTH polarized difference images interact at ANA.

 

 

 

 

9) A beam of light travels through transparent medium X with a velocity of 2.3×108 m/sec. (2) (Cvac = 3.0 ×108 m/sec)

 

·      Calculate the index of refraction of medium X.

n=Cvac/Cmed = 3/2.3 = 1.3


·      What could medium X be?

 

Water

 

 

 

 

10) If a transparent sample is placed in a medium with n=1.54 and it becomes invisible in BF, what does that tell you about the refractive index of the sample? Explain you answer. (2)

 

 

Invisible means there is NO refraction, so the light goes straight through and the sample is invisible. Thus nobj = nmed

 

 

 

 

 

 

11) Regarding the Full Wave Retardation Plate: (4)

a.     When in place, why is the background Magenta?

550nm (green) light remains plane polarized and is absorbed by ANA. White light – green = magenta



b.      What does the resulting color of a sample tell you about that sample?

 

Sample color shows the BIREFRINGENCE NUMBER of the sample. BN = Sample (crystal) ID

 

 

 

 

12) Why can’t PCM be used to accurately measure the size of small samples? (2)

 

Phase Halo obscures actual sample boundry

 

 

 

 

 

 

 

 

13). Distinguish Brewster’s Angle from the Critical Angle. (2)

BA: Angle of reflection where reflected light is polarized (parallel to the reflecting surface)

 

CA: Angle of total internal reflection. At this angle, all incident light is reflected from the surface.

 

 

 

 

 

14) In the following microscope techniques what property of the sample allows Image contrast to be created, and where in the microscope is contrast created? (6)

 

Sample optical property

Where

PLM

Birefringence

ANA

DIC

OPL gradients

ANA

BF

Absorption

Sample

HMC

Refraction Gradients

HAM/BFP

DF

Scattering

Sample

PC

Phase/OPL gradients btwn sample and bkgnd

IIP

 

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

a) Microbial biologists love this technique _____E____                          A) PLM

b) Asbestos (a mineral) determination in your home ____A____            B) DF

c) Bacteria on the surface of a cheek cell___F____                                    C) HMC

d) Cells growing in a plastic dish ___C____                                            D) BF

e) Dye-stained plant leaf sections on a microscope slide ____D___        E) PC

f) Volvox flagella in a living culture  _____B___                                      F) DIC

                                                                                                                    

 

 

 

16) What factors determine microscope resolution (d)? (An equation works here, but please define the terms.) (4)

 

d is related to WAVELENGTH and NA

 

 

 

 

 

 

17) What will be the result of plane polarized light impinging on a birefringent crystal at the following orientations? (2)

i) Parallel to one of the Privileged axes:

No change
           

ii) NOT parallel to one of the Privileged axes (or the OA):

 

Birefringence produces two orthogonally polarized beams vibrating parallel to the Privileged Axes.

 

 

 

18) What is the major discovery or invention that is associated with the following Optical Pioneers? (8)

Pioneer

Discovery or Invention

A. Köhler

Even sample illumination: Köhler Illumination

E. Culpeper

Gimbal Mirror

F. Zernike

Phase Contrast

E. Abbe

NA, Immersion Oil, Dispersion index (Abbe number), resolution based on diffraction

W. Snell

Law of Refraction

H. Janssen

1st Compound Microscope

J. Dollond

Achromatic Doublet

G. Nomarski

Modified Wollaston Prism.

 

 

19) Which would be better, DIC or PC, for visualizing microscopic cubic crystals? Explain your answer. (4)

 

PC because crystals are probably birefringent, and DIC hates that

Cubes have sharp boundaries and DIC hates that too

PC cares only about refractive index differences between the sample and background. It should be OK.







 

 

20) What are the three physical configurations of the compound microscope, and what is each typically used for? (6)

Scope Style

Typical Use

Upright          

Everything, but mostly slides

Inverted

Mainly living cells in some kind of liquid vessel

Stereo/Dissection

dissection

 

 

21)  Define the following: (5)

a) Mechanical Tube Length: 160mm. Distance btwn eyepiece mount and objective mount.

b) Infinity Corrected Optics: Objective focuses to infinity, requires a tube lens to form an II

c) PlanApochromat: Fancy objective chromatic and spherical corrected for 4-5 wavelengths, plus it’s flat field

d) Parfocal Distance: Distance between nosepiece and sample plane for ALL objectives.

e) Field Number: Eyepiece, size of the eyepiece aperture at the IIP. Directly related to the FOV

22) Certain samples like quartz sand and plastic show rainbow colors when viewed through crossed polarizers. Explain this phenomenon.  (2)

 

Continuous, and uneven OPL gradients produces differential phasing of pol light. Subsequent wave interference at ANA accentuates and decreases certain colors producing rainbows.

 

 

 

 

 

 

23) Monochromatic light impinges on a glass block at a 23.4° angle. Describe the two paths the light beam takes. (A diagram or equations work here.) (4)

a) Reflection and the same angle as incidence

b) Refraction through block follows Snell

 

 

 

 

24) What is Optical Path Length (OPL) and why do we, as microscopists, care so much about it? (2)

 

OPL = n•l, where n= refractive index and l=geometric distance in µm. This is the source of sample light phasing, and the basis of so many optical techniques.

 

 

 

 

 

25) What is the difference between Shear and Bias in a DIC microscope? (4)

 

Shear: Distance between DIFFERENCE IMAGES from WI. Always << 1 AU

 

Bias: Difference between Phasing of the DIFFERENCE IMAGES. Can be modulated by WII

 

 

 

 

 

26) What is the orientation of the privileged axis of polarized sunglasses. Explain your answer. (2)

 

 

Reflected light can be polarized (see Brewster’s Angle) parallel to the surface. Polarized sunglasses are polarized perpendicular to the surface to absorb reflected/polarized light. This reduces glare.

 

 

 

 

 

27) What is the difference between a Nicol Prism and a Wollaston Prism? (2)

Nicol Prism

Uses TIR to send one of the two polarized beams (O-ray) out the side. This results in a plane polarized beam (E-Ray) exiting the end of the crystal.

Wollaston Prism

BOTH polarized beams exit the crystal separated by some distance. These are the “difference images” in DIC, or the two images necessary for binocular eyepieces in old-timey scopes.

 

 

28) Which of the following microscopes is Compound (C) and which is Simple (S)? How do you know (4)?  

../Graphics/CmpdvsStereo.pdf

 

Scope

C or S

Explain

Guess the Century

a

C

 

Eyepieces and Objectives, 2 lens systems

20th

 

b

S

Leeuwenhoek’s famous magnifier. Has one tiny lens

17th

 

c

C

(This is a hard one because it’s weird) There are (only) two lenses here

Early 17th

 

d

S

Only one lens above the sample stage

19th