Name: ______________________
PIN: __________________________
Biology (188)
E. D. Salmon, K. Bloom
Exam II
HONORS PLEDGE: (sign) _______________________________
1. (5 pts) List 5 major attributes of fluorescence microscopy and give for each a specific example of
the advantages of each:
a. ____________________________________________________________________________
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b. ____________________________________________________________________________
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c. ____________________________________________________________________________
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d. ____________________________________________________________________________
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e. ____________________________________________________________________________
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2. (3 pts) Fluorescence is a form of luminescence. What is the difference between:
Fluorescence:__________________________________________________________________
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Delayed fluorescence:____________________________________________________________
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Phosphorescence:_______________________________________________________________
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3. (5 pts) For 500nm wavelength, what is the number of photons/sec passing through a cm2 area if
the intensity of light is 1 Watt/cm2 (show calculations)
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4. (2pts) What are the advantages of a large stokes shift?
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5. (2 pts) Why is the absorption spectrum of hydrogen a very narrow band of wavelengths
approaching a single wavelength whereas the absorption spectrum of fluorescein is a broad band of
wavelengths?
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6. (5 pts) Fluorescence intensity, I = Io(epsilon)(Phi)cl. For fluorescence microscopy, what is the
significance of each of the terms in this equation?
Io ______________________________________________________________
(epsilon)_________________________________________________________
(Phi)___________________________________________________________
c_______________________________________________________________
l _______________________________________________________________
7. (4 pts) What are the advantages of incident or epi-illumination for fluorescence microscopy over:
Transmitted light illumination: ____________________________________________________
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Darkfield illumination: ___________________________________________________________
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8. (2.5 pts) Why is a 100 W Hg light source typically used for epi fluorescence microscopy over a
100 W Quartz halogen or 100W Xenon light source?
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9. (4 pts) The equation governing image brightness in epi-fluorescence microscopy is objective
NA4/magnification2. Explain the origins of the dependence on:
NA:__________________________________________________________________________
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Magnification:__________________________________________________________________
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10. (9 pts) Common fluorophores used for fluorescence microscopy are Hoechst 33342, fluorescein,
rhodamine, S65T GFP, Cy3, Cy5, X rhodamine, perpidium iodide, Fura 2. What are the peak
absorption and peak emission for each fluorophore:
Hoechst 33342:_____________________________________________
Fluorescein:________________________________________________
Rhodamine:_________________________________________________
S65T GFP:__________________________________________________
Cy3:______________________________________________________
Cy5:_______________________________________________________
X-rhodamine:________________________________________________
perpidium iodide:_____________________________________________
Fura 2:_____________________________________________________
11. (4 pts) How can you avoid the following practical problems in fluorescence microscopy:
Autofluorescence:_______________________________________________________________
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Photobleaching:_________________________________________________________________
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12. (4 pts) Give 4 possible reasons why the intensity of a fluorescent image is unexpectedly dim
when using an objective of 60X/NA=1.4:
1.________________________________________________________
2._______________________________________________________
3._______________________________________________________
4. _______________________________________________________
13. (2 pts) Which is the best type of lens to use for triple labeled fluorescent imaging of DAPI, GFP
and Cy5:
______________________________________________________________________________
14. (2 pts) Explain why a filter wheel is commonly used to remove the DIC analyzer when
obtaining a fluorescent image after obtaining a DIC image, but no filter wheel is needed for
obtaining sequentially a phase contrast image and fluorescent image
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15. (5 pts) How does a laser scanning confocal microscope create an image of a narrow optical
section through a thick specimen? Include role of laser, scanning device, detector, computer,
pinhole and filters
1. __________________________________________________________________________
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2. ____________________________________________________________________________
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3. ____________________________________________________________________________
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4. ____________________________________________________________________________
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5. ____________________________________________________________________________
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16. (4 pts) You need to obtain images of 20 thin optical sections at 0.2 μm intervals at a depth
beginning 50 μm deep within a fluorescent specimen. What key equipment is needed for this
application:
______________________________________________________________________________
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17. (3 pts) List the excitation wavelengths of an Argon -Krypton laser and an example of a
fluorophore excited by each wavelength:
______________________________________________________________________________
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18. (2 pts) Why is photobleaching and photodamage a much bigger problem with laser scanning
confocal microscopy in comparison to multi-photon excitation microscopy or widefield
fluorescence microscopy using a cooled CCD camera
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19.(10 pts) Draw the reaction mechanism for generation of the chromophore in the green
fluorescent protein. What is the structure of the rest of the protein and how does it contribute to the
fluorescence?
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20. (5 pts) GFP has been widely used to study a variety of intracellular events.
Describe a new application that might further extend its utility.
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21. (5pts) You have a CCD camera with 16 um wide pixels. For a wavelength of 510 green
fluorescence from GFP protein, how much magnification to the camera is needed in addition to a
60X objective so the size of the camera pixels does not limit resolution and resolution is only
limited by the wavelength of light? Remember, you need 3 pixel widths per radius of the Airy disk.
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22. (5 pts) You are using a CCD camera with 1000x1000 pixel elements in the detector. If each
pixel well can hold 32,000 photon induced electrons, and the readout noise is 8 electrons, how many
bits will be needed in the analog to digital converter to numerically represent the dynamic range of
the camera? Note that 8bits gives 2^8 gray levels, 10 bits gives 2^10, 12 bits gives 2^12, etc.
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23. (8pts) Some CCD cameras have ability to bin together the amount of photon induced electrons
in their adjacent wells before sending the results to the A/D converter. How much gain in signal and
loss in resolution is obtained by:
Gain
Loss Resolution
2x2 binning _______________________________________________________
4x4 binning _______________________________________________________
8x8 binning _______________________________________________________
16x16 binning _____________________________________________________
24. (5 pts) What are 5 functional features that make cooled CCD cameras an excellent choice of
detector for biological fluorescence microscopy
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25. (2 pts) What happens to the PSF when using a laser scanning confocal for increasing depth in
water beneath the surface of the coverslip when using an oil immersion NA =1.4 objective. How
can this problem be corrected? Suggest two different methods.
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