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)

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)?

 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