Final Exam, 150 points PMB 185: Techniques in Light Microscopy

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1 Final Exam, 150 points Name PMB 185: Techniques in Light Microscopy Point value is in parentheses at the end of each question. Note: GFP = green fluorescent protein ; CFP = cyan fluorescent protein ; YFP = yellow fluorescent protein 1) In order to best visualize yeast plasma membrane proteins, which of the following optical technique would you use: (2) A) Fluorescence Recovery After Photobleaching microscopy B) Total Internal Reflection Fluorescence microscopy C) Differential Interference Contrast microscopy D) Two-Photon microscopy 2) A Spinning disk confocal microscope is the best technique for imaging: (2) A) Imaging of GFP expression in a chemically fixed fibroblast nucleus B) Imaging of a bacterium plasma membrane probed with a fluorescent dye C) Imaging of a GFP-expressing living fish embryo heartbeat D) Fluorescence lifetime imaging of a neuronal synapse 3) Your research project involves protein-protein interactions. Which microscope technique should you use, and with which fluorescent probes? (2) A) FRAP using YFP and PI B) TIRF using DAPI and Calcofluor M2R C) FRET using GFP and Fluorescein D) FLIM using CFP and YFP 4) Visualizing DiI- (green)and DiO-labeled (red) axons in the mouse brain may be best accomplished using: (2) A) 1-Photon confocal imaging B) 2-Photon fluorescence imaging C) 3-Photon Structured Illumination imaging D) 4-Photon transmitted light imaging 5) You wish to identify starch contamination in various Parmesan Cheese samples. What type of microscope would be best for evaluating this sample? (2) A) Brightfield B) Hoffman Modulation Contrast C) Phase Contrast D) Polarized Light Microscopy Page 1

2 6) Which of the following is NOT a fundamental property of a Charge Coupled Device (CCD) detector? (2) A) Fixed spatial sampling B) Variable scan frequency C) Variable gain settings D) Often Cooled 7) You are interested in comparing the fluorescence intensity between CFP-labeled organelles in living mammalian cells. What microscope detector would be best to use? (2) A) 8-bit Bayer filter color camera B) 12-bit grayscale EMCCD camera C) 16-bit PMT detector D) 24-bit color CCD camera 8) A white powder suspected of containing highly refractive Anthrax spores may be best imaged microscopically using what microscope technique? (2) A) Phase Contrast B) Polarized Light Microscopy C) Differential Interference Contrast D) Darkfield 9) In order for two fluorescent molecules to interact via Förster resonance energy transfer they must (2) A) be less than 10nm apart B) have a Stokes shift less than 10nm C) within 10Å of the coverslip D) be excited by photons arriving at less than 10ps apart 10) Regarding TIRF microscopy, which of the following is FALSE: (2) A) The Evanescent wave decays exponentially B) The Evanescent wave is a standing wave 100nm into the sample C) The Evanescent wave forms at the angle of total reflection D) The Evanescent wave energy is a function of the emission wavelength 11) When visualizing a particle using Negative Phase Contrast microscopy, it appears bright white on a gray background. What is the approximate phase displacement of that particle: (2) A) + λ B) - λ C) +560nm D) 1040Å Page 2

3 12) Which of the following parameters determine the optical section thickness of a laser scanning confocal microscope image? (2) A) X/Y sample rate and refractive index of the immersion medium B) Z-sampling rate and pinhole diameter C) Objective NA and pinhole diameter D) Refractive index of the mounting medium and pinhole diameter 13) Which of the following is NOT important for accurate Restoration/deconvolution microscopy? (2) A) An empirically derived PSF B) A 16-bit PMT detector C) A maximum number of iterations required for optimum deconvolution D) A coverslip that is precisely 170µm thick 14) FRAP is a useful fluorescence microscope technique because it (2) A) determines the transport rate of fluorescently labeled cell components B)...measures the total time a population of fluorophores stay in the excited state C) reassigns out-of-focus light to its correct geometric position D) indicates whether a fluorophore is very near a coverglass surface 15) Which of the following pairs of microscope components are in conjugate planes? (2) A) Condenser diaphragm & intermediate image B) Objective back focal plane & eyepiece front focal plane C) Field diaphragm & intermediate image D) Sample plane & condenser diaphragm 16) The Nyquist sampling theorem states that the optimal spatial sampling rate is at least: (2) A) ½ smallest object you want to visualize B) Just a little bit less than what you re trying to visualize C) 2 8 lowest frequency you want to visualize D) 2 smallest object you want to visualize 17) When imaging a GFP-labeled sample, you used a poorly constructed GFP fluorescence filter cube. What would happen if (6) A) filter A was omitted B) filter B was omitted Color beneath the objective Color seen through the eyepieces A detector C B C) filters A and C were switched sample Page 3

4 18) Which of the following microscopes would be the most useful for screening (counting) populations of GFP-expressing fruit flies? (2) A) Widefield fluorescence on a stereo microscope B) Widefield fluorescence on an upright microscope C) Laser scanning confocal on an inverted microscope D) Brightfield imaging on a dissecting microscope 19) In this diagram of Hoffman Modulation Contrast (right) describe the function of: (4) A A) B) B 20) In Structured Illumination Microscopy, which of the following is true: (2) A) A Moiré pattern can reveal sample intensity B) A resolution of 2.4nm can be achieved C) A Moiré image pattern can reveal 50nm objects D) Individual fluorescent molecules can be statistically localized! 21) Which of the following is TRUE for Restoration/deconvolution microscopy: (2) A) It photobleaches samples severely B) Spatial resolution is poor C) It reassigns the intensity of out-of-focus voxels to different Z-positions D) It cannot be used for thin samples 22) A major advantage of a Nipkow disk for fluorescence imaging is (2) A) High imaging bit-rate B) Fast sample scan rate C) High light throughput D) Fixed pinhole size Page 4

5 23) Arrange in the appropriate experimental order from first to last the following steps in a FRAP experiment (5) A) Collect a time series of images using non-photobleaching conditions B) Image sample under non-photobleaching conditions C) Purposely photobleach the sample ROI D) Select an appropriate ROI E) Compare ROI time series data to control intensity values 24) Order the steps in setting up Köhler illumination: (6) A: Center the condenser F: Focus on the sample B: Close the Field iris to the optical G: White-balance the CCD detector diameter of the FOV C: Adjust Shear of Wol II H: Adjust the eyepiece to the Red Dot D: Focus on the Field Iris I: Adjust contrast with the condenser iris E: Adjust contrast with the Field iris J: Focus at Infinity Hint: It is not necessary to use all of the above steps. 25) Compare spatial sampling when using a CCD versus a PMT detector? (4) CCD PMT X/Y-sampling Z-sampling 26) What parameters define a VOXEL of a 3D data set of a sample probed with two different fluorescent probes? (2) Page 5

6 27) Calculate the theoretical X/Y spatial resolution attainable when imaging GFP using a 2P microscope (1.4NA objective). (2) 28) Compare and contrast Deconvolution and Confocal. Discuss light source, sampling rate, detectors, out-of-focus light (OOF): (5) Deconvolution Confocal Light Source X/Y Sampling Detector OOF light 3D Image reconstruction 29) Match these important numbers to their microscope meaning. (8) # Ans Definition 170 A: Wavelength of X-Rays used in X-Ray microscopy 100 B: Critical angle of a glass/water interface C: Main Argon ion laser line 488 D: IR wavelength for 2-Photon imaging 1.47 E: Maximum extent (nm) of the Evanescent wave in TIRF 2.4 F: Thickness (µm) of a #1 coverslip 960 G: Refractive index of immersion oil 60.1 H: Numerical Aperture of a TIRF objective Page 6

7 30) What is the function of the Tube Lens in an epifluorescence microscope? (2) 31) Compare and contrast 2P fluorescence and STED microscopy: Discuss photobleaching, resolution, excitation light source. (5) 2P STED Photobleaching X/Y Resolution Z-Resolution Light source λ Light source power 32) The Raleigh criterion defines: (2) A) The distance between an Airy disc and its first order maximum B) The minimum distance allowing resolution of two adjacent Airy discs C) The wavelength of highest resolution when imaging two adjacent Airy discs D) The magnification required to visualize two adjacent Airy discs 33) One of the deconvolution algorithms, Maximum Likelihood Estimation, uses probability to compute: (2) A) theoretical instrument noise values B) theoretical sample intensity values C) theoretical point spread function D) theoretical coverslip thickness 34) Name one condition under which the Effective NA will be different from the stated Objective NA. (2) Page 7

8 35) Explain why a DIC image would have no 3D appearance if the Shear angle was set to 0? (3) 36) Give two methods by which one can increase Signal/Noise (S/N) when doing confocal microscopy? (4) 37) The following diagram describes the spectral characteristics of a 2-color filter set for fluorescence microscopy. For the most likely set of spectra (4) A) What are the Excitation ranges for both? B) What are the Emission ranges for both? C) If the sample exhibits autofluorescence what color would you see when exciting for GFP? D) Name two dyes that might work with this filter set. Page 8

9 38) In a Compound Microscope name the Image-forming conjugate planes in order starting from the light source. (2) 39) STED microscopy is ideal for which of the following sample types: (2) A) 100nm GFP viruses in mammalian fibroblasts B) Highly refractive Bacillus anthracis endospores in an unknown white powder C) Imaging YFP-transport proteins the 100nm synapse gap in a living rat brain D) Highly reflective, 5nm colloidal gold particles on a glass surface 40) Refer to the diagrams, then match the following: (4) A) FLIM Fluorescence B) 2P Fluorescence C) FRET Imaging D) WF Fluorescence imaging Page 9

10 41) Matching (4) GFP Chlorophyll Yellow Fluorescent Protein (YFP) MitoTracker Orange DAPI Formaldehyde-fixed fibroblasts Lignin in Xylem Fluorescent PAS A: Intrinsic fluorescence B: Induced fluorescence C: Molecular D: Probed 42) Which of the following statements is true for PALM microscopy: (2) A) Fluorescence Lifetime is calculated B) Photoactivatable fluorophores are required C) The Rayleigh resolution criterion is calculated D) Identifies birefringent samples 43) The Quantum Efficiency of a fluorophore is: (2) A) The number of emitted photons divided by exciting photons B) The number of fluorophore molecules required to see a particular target C) The number of exciting photons divided by emitted photons D) The ratio of Ex/Em wavelength maxima 44) FLIP microscopy is a good technique that could be used to answer which of the following experimental questions? (2) A) Localization of fluorescent proteins to a Leukocyte plasma membrane. B) Localization of bacteria in a thick biofilm C) Quantifying the rate of exchange of organelle membrane proteins D) Evaluating whether two proteins are closely spaced and thus interacting 45) Match the following microscope techniques to the appropriate optical basis. (5) Brightfield DIC HMC Polarized Light Microscopy Confocal Microscopy A) Rejection of out-of-focus light B) Wave Interference C) Light Absorption D) Birefringence E) Refractive index gradients Page 10

11 46) Match these gone-but-not-forgotten scientists with the microscope technique or concept that resulted from their tireless research. (7) August Köhler Ernst Abbe Georges Nomarski Marvin Minsky Paul Nipkow Maria Göppert-Mayer William Nicol A) Numerical Aperture B) Differential Interference Contrast Microscopy C) Transmitted light sample illumination D) Invented an early form of TV, used now for high-speed confocal. E) Invented the first confocal microscope F) Invented the first useful polarizing device for microscopy G) Nobel laureate for describing the Two-Photon effect 47) Describe Dispersion and where in microscopy it becomes important. (3) 48) Put the following optical techniques in their correct chronological order (earliest to most modern): (5) A) Compound Microscope B) Single Lens magnifier C) Phase Contrast microscopy D) Differential Interference Contrast E) Deconvolution microscopy F) Köhler Illumination G) PALM H) Confocal microscopy I) Reflecting mirror for transmitted light J) Epifluorescence microscopy 49) For the following descriptions of microscope images, state which microscope imaging technique was used (3)? A) Gray background, 3D appearance, low magnification B) Black background, sample shows rainbow colors C) Black background, Blue and Red colors only D) White background, multiple colors Page 11

12 50) Match the most appropriate image detector with the microscope technique. (6) Spinning disk confocal microscopy Live-cell WF fluorescence Brightfield Confocal laser scanning microscopy 2-photon fluorescence microscopy Deconvolution microscopy A) EMCCD B) Full-Frame 16-bit CCD C) Bayer filter color CCD D) Photo-multiplier tube E) High-Speed interline CCD F) GaAsP Non-descanned detector (NDD) Page 12