Final Exam, 176 points PMB 185: Techniques in Light Microscopy

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1 Final Exam, 176 points Name PMB 185: Techniques in Light Microscopy Point value is in parentheses at the end of each question. 1) Order the steps in setting up Köhler illumination. It is not necessary to use all of the steps. (6): A: Focus condenser to see sharp field iris F: Focus on the sample edges B: Adjust contrast with the condenser iris G: Close the Field iris C: Center Phase Annulus H: Center the image of the field iris D: Focus on the Exit Pupil I: Open the Field Iris to just outside the FOV E: Focus at Infinity J: Adjust contrast with the field iris 2) How is a sample image formed when the detector is either a PMT or scmos? Describe one microscope/optical technique that uses each type of detector (4). PMT scmos Image Formation Describe Technique 3) What is Nyquist Sampling in microscopy? Show the mathematical relationship, describe each term, and give an example. (2). Page 1

2 4) Under what optical conditions will an Evanescent Wave be created? What microscope technique depends on its formation, and what is it used for? (3) Creation Dependent Microscope Technique Use 5) Compare and contrast 2P and Spinning Disk Confocal microscopy. (4) 2P SD confocal Light Source Parameters that determine X/Y resolution Detector Type and placement Highest axial resolution Page 2

3 6) Match these important numbers to their meaning in microscopy (4). # Answer Definition A: Wavelength of 2P Ti:Sapphire pump laser 50 B: IR wavelength for 2-Photon imaging of GFP 364 C: Numerical Aperture of a SIM objective 2.4 D: Working distance (in µm) of a 20x objective 1.46 E: Average maximum phase retardation of living cells 170 F: Mechanical tube length of pre1975 microscopes 960 G: Thickness (µm) of a #1 coverslip (you hope) -¼ H: DAPI Ex Wavelength peak I: Refractive index of coverslip glass J: STED lateral resolution K: Wavelength of X-Rays used in XRT at the ALS L: Refractive index of water 7) A scientist wants to capture a movie of living, stem cell-based cardiac cells that have formed beating cell clusters (50µm in diameter). What microscope technique and environmental conditions would you use to capture this movie? Explain your answer. (4) 8) Compare and contrast SIM and PALM microscopy (3). SIM PALM Typical Sample Type Highest achievable resolution Allowable fluorophore types 9) In the following FRET fluorescence filter cube label components A C (3): Page 3 Possible Answers: 1) Donor Em 2) Donor Ex

4 3) Donor Dichroic 4) Acceptor Ex 5) Acceptor Em 6) Acceptor Dichroic 10) Explain why the lateral (X/Y) resolution attained by laser scanning confocal microscopy is better than that which can be attained using Widefield fluorescence microscopy. (2) 11) In a laser scanning confocal microscope what imaging parameters can increase Signal-to-Noise? (3) 12) The following diagram describes several characteristics of filter sets for WF fluorescence microscopy (3). A) Plot A shows what kind of Emission Filter? B) Which filter set would you use to image GFP-bacteria? C) Which filter set would be used for DAPI-probed bacteria? Page 4

5 13) In this diagram of a compound microscope label the optical planes A D (4): A: B: C: D: 14) Show the calculation for determining the refraction angle of a beam of light coming from air and entering water at a 23 angle. (You don't need to calculate the answer.) (2) 15) Match the following microscope techniques to the appropriate optical basis (5). Technique Restoration microscopy Confocal microscopy 2P Microscopy TIRF Microscopy STED Microscopy Optical Basis A) Airy disc centroid B) Refractive index boundary C) Non-Linear fluorescence D) Fourier mathematical computation E) Optical rejection of blur F) Moiré pattern deconvolution G) PSF shaping 16) Match these scientists with the statement regarding microscopy (6). Ernst Orville Scientist Answer Statement Abbe Golub A) Invented the compound microscope B) Nobel laureate for describing the non-linear photon absorption effect C) Invented the gimbal mirror Page 5

6 Maria Mats Stefan Zacharias August A.v. Marvin Paul Georges Willebrord Goeppert-Mayer Gustafsson Hell Janssen Köhler Leeuwenhoek Minsky Nipkow Nomarski Snell D) Invented STED microscopy E) Invented the Field Lens F) Law of Refraction G) Invented the confocal microscope H) First description of bacteria I) Donated 160 antique microscopes to UCB J) Defined Numerical Aperture K) Differential Interference Contrast Microscopy L) Invented the Structured Illumination microscope M) Transmitted light sample illumination N) Invented a spinning disk imaging system O) Saw the Homunculus in human sperm cells 17) For the following descriptions of microscope samples, state which microscope imaging technique should be used (5)? A) Fluorescent aerogel from Deep Space B) H&E-stained liver biopsy C) GFP-expressing transparent mouse brain D) Fluorescent cell division protein in bacteria E) Explosion residue 18) Match the following Transmitted microscope techniques to the appropriate optical basis. (5) BF DIC HMC PLM DF A) Light scattering B) Wave Interference C) Light Absorption D) Birefringence E) Refractive index gradients 19) Compare STED and STORM. (4) Light Source Sample types STED STORM Page 6

Image Processing Attainable resolution 20) Regarding the following figure: (4) A: What phenomenon does A demonstrate? B: What does the curve B represent?

7 Image Processing Attainable resolution 20) Regarding the following figure: (4) A: What phenomenon does A demonstrate? B: What does the curve B represent? C: The cell images in C are not fluorescent images in the traditional sense. What are they? 21) How is the action of an Antifade molecule similar to Förster Resonance Energy Transfer? (2) 22) What do the following molecules probe for? (6) SYTO BC MitoTracker FM4-64 Propidium Iodide Fluorescein Diacetate IKI Page 7

8 23) What is the theoretical basis for the following fluorescence microscope concepts: (12) Concept Basis SYTO/PI viability detection Confocal microscope correct Z-sectioning distance Restoration microscopy computation PALM Imaging of cell membrane proteins SPIM Imaging of Arabidopsis root tips SIM imaging of single-cell algae 24) Come up with a Biological Question that requires fluorescence microscopy, then design an experiment that uses that microscope method. Show your Question, Technique required, experimental protocol, and expected results. Include resolution in those results. Finally explain why your particular microscope method was required. (10) Biological Question Microscopy required Page 8

9 Experimental protocol Expected results, including resolution required Why was the microscopy method required? 25) What is the best microscope technique that one should use to visualize polymerizing actin (20nm) on coverslips (2)? A) Laser Scanning Confocal microscopy B) STORM C) WF Fluorescence Microscopy D) Restoration microscopy 26) The resolution of Spinning Disk confocal microscopy is limited because (2): A) the fixed pinhole diameter decreases resolution at high magnification B) the fast scan rate greatly increases S/N C) the excitation wavelength is necessarily twice as long D) Well-size of the EMCCD detector is too small 27) You are interested in determining whether fluorescently-labeled spindle microtubule subunits are newly synthesized or are recycled within the cell. Which microscope technique should you use (2)? A) FRET using confocal microscopy Page 9

10 Page 10 B) TIRF using Structured Illumination microscopy C) PALM using an inverted microscope D) FRAP using a laser scanning confocal microscope 28) You want to identify whether a fluorescent tag is in the Plasma Membrane or adjacent ER. What microscope technique should you use? (2): A) Structured Illumination microscopy B) STED microscopy C) TIRF microscopy D) Spinning Disk confocal microscopy 29) You have Ziehl-Nielsen-stained (red) bacteria on a microscope slide. What type of microscope would be best for imaging this sample (2)? A) Brightfield microscopy B) Phase Contrast microscopy C) HMC microscopy D) Nomarski Interference Contrast microscopy 30) Detecting contaminating Insect Parts in breakfast cereal may be best detected using (2): A) Widefield fluorescence microscopy B) Phase Contrast Microscopy C) Brightfield microscopy D) Polarized Light microscopy 31) You wish to compare the amount of DNA of the interphase nuclei of two SYTO-probed protozoans using Spinning Disk confocal microscope. What microscope detector would be best to use (2)? A) 8-bit scmos B) 12-bit CCD C) 16-bit EMCCD D) 16-bit PMT 32) Which of the following optical techniques can indicate whether as unprobed white powder may have Bacillus anthracis endospores? (2) A) Brightfield microscopy B) Phase Contrast microscopy C) Darkfield microscopy D) Polarized Light microscopy 33) The following criterion is required for achieving Förster resonance energy transfer: (2) A) two fluorophores have overlapping Ex spectra B) two fluorophores alternate between S 3 and S 0 states C) two fluorophores are close enough to experience nonradiative energy transfer D) two fluorophores can be excited non-linearly 34) In Structured Illumination Microscopy computationally processing the moiré image pattern can reveal (2): A) stochastic emission of the sparse population of PA-fluorophores B) the diameter of the laser-depleted Ex Airy disc

11 C) fluorescence of sub-resolution structures within the sample D) the average lifetime of fluorescence of the population of fluorophores 35) For a STED microscope to image at super-resolution the following must be true (2): A) The depletion laser wavelength must be 2 the probe Ex wavelength B) The depletion laser must surround the probe excitation laser C) The depletion laser pulse must alternate with the excitation laser pulse D) The depletion laser must be a femtosecond pulse laser 36) Which of the following determines the optical section thickness of a laser scanning confocal microscope (2)? A) Objective NA and lateral scan rate B) Immersion medium refractive index and excitation wavelength C) Emission wavelength and pinhole diameter D) Excitation wavelength and pinhole diameter 37) The Point Spread Function of a fluorescence microscope (2): A) is used for deconvolving TIRF images B) can be used to calculate the lateral scan rate C) determines the noise (ξ) for reiterative deconvolution algorithms D) is the visualization of the blur function of microscope optics 38) FRET is a useful fluorescence microscope technique because it (2)? A) can visualize particles as small as 10nm B) can show whether two fluorophores are <10nm apart C) detects changes in the fluorophore environment in 0.10 ph units D) indicates whether a fluorophore is within 10nm of the coverslip surface 39) Which of the following pairs of microscope components are in conjugate planes (2)? A) Condenser diaphragm and Intermediate Image Plane B) Wollaston I and objective back focal plane C) Field iris and Phase plate D) Sample and exit pupil 40) The Michel-Lévy Color Chart is used to (2): A) determine the birefringence number of an unknown crystal B) determine the optical path length of an isotropic sample C) determine the of shear distance generated by a Nomarski Prism D) identify whether phase-bright objects are Bacillus endospores or 1µm glass spheres. 41) Label the following diagram of a laser scanning confocal microscope (5): A) Page 11 B) C)

12 D) E) 42) Give three (3) reasons why NA is King. Be specific. (6) i) ii) iii) 43) Label the components in this diagram (5): A) B) C) D) What type of Microscope Imaging is this? E) 44) FLIM measures which of the following sample properties (2)? A) Average length of time a population of molecules fluoresce B) Proximity of two complementary fluorophores C) Quantum efficiency of a fluorophore D) Photobleachability of a fluorophore 45) STED microscopy is best used for what sample type (2): A) Neurons deep inside a cleared mouse brain B) GFP-expressing whole Zebra Fish embryos Page 12

13 C) Fluorescent nuclear pores of mammalian cells D) Highly light-scattering gold particles 46) Live-cell imaging of fluorescently-labeled cells can best be performed using (2): A) a system that illuminates the sample using an IR pulse laser B) a microscope enclosed in a temperature-controlled environmental chamber C) a high-power red depletion laser to decrease GFP photobleaching D) a microscope with a slow Z-drive so as not to vibrate the living cells 47) FRAP is a technique that can determine if (2) A) sample fluorophores exist in different environments B) a sample has dynamic movement of fluorescent proteins C) a sample has few imaging modalities D) a sample fluorophores have reached the S 3 excited state 48) What is one function of the Full Wave Red Plate in Quantitative PLM (2). A) Fold together the difference image vibrational planes allowing them to interfere B) Produce the Magenta background to distinguish the Fast and Slow sample waves C) Pair with W1 to negate non-axial path length difference D) Absorb circular polarized light from the sample to determine birefringence number 49) Restoration microscopy can (2). A) restore fluorescence of samples 100nm from the coverglass B) restore photobleached fluorophores using 2 the standard Ex wavelength C) restore actual fluorescent sample movement at 100 frames/sec D) reassign voxel position and intensity to restore 200nm resolution 50) Which of the following statement is true for 2-photon imaging (2): A) The Ti:Sapphire laser is usually set to 2 the probe Ex wavelength B) Imaging requires both Green and Red lasers for sample excitation C) The pinhole is often set to a diameter of 2 the Airy disc diameter D) Sample excitation uses two different lasers 51) Which of the following statements is true for Photoactivated Localization Microscopy (2): A) 3D imaging is not possible B) A lateral resolution of 20nm can be achieved Page 13

14 C) It is a good optical technique for imaging living cells D) Can be an alternative approach to image FRET 52) The Numerical Aperture of an objective determines: (2) A) The type of microscope that it can be used on B) The refractive index of the sample mounting medium C) The amount of light the lens can collect D) The chromatic characteristics of the lens 53) In 2-Photon microscopy lateral resolution is determined by (2) A) the diameter of the probe Em Airy Disc B) the proximity of the NDD C) the wavelength of the Ti:Sapphire excitation laser D) the diameter of the pinhole 54) Which of the following is a property of Restoration microscopy? (2) A) The Nipkow disc has diffraction-limited pinhole sizes B) It is used for imaging diffraction-limited fluorescent samples C) It uses cylindrical lens to create a diffraction-limited sheet of Ex light D) The excitation grid has diffraction-limited spacing Page 14

A Brief History of Light Microscopy And How It Transformed Biomedical Research

A Brief History of Light Microscopy And How It Transformed Biomedical Research Suewei Lin Office: Interdisciplinary Research Building 8A08 Email: sueweilin@gate.sinica.edu.tw TEL: 2789-9315 Microscope