Deck 9: Visualizing Cells

Question 1

Tubulin labeled with caged fluorescein can be introduced into dividing cells by microinjection. Various small regions in the mitotic spindle (made up of tubulin subunits) are briefly irradiated with laser light that uncages the fluorescent tubulin. Five minutes after irradiation, the highest spindle fluorescence is observed when the irradiated region is close to the chromosomes near the cell equator, and the lowest fluorescence is observed when regions near the spindle poles are irradiated. Based on this observation, do you think tubulin subunits are incorporated into the spindle mostly near the poles (P) or near the equator (E)? Write down P or E as your answer.

Answer 1

E

Question 2

In which of the following microscopy techniques are oblique rays of light focused on the specimen?

A) Bright-field microscopy
B) Dark-field microscopy
C) Phase-contrast microscopy
D) Differential-interference-contrast microscopy

Answer 2

B
Explanation: In dark-field microscopy, oblique rays of light do not enter the objective directly. Instead, some of the scattered rays from objects (such as cells and their components) enter the objective to create a bright image against a dark background.

Question 3

Which of the following is correct regarding aequorin?

A) It is a small molecule used to detect calcium ions in vivo.
B) It is a fluorescent dye.
C) It emits blue light in the presence of calcium ions.
D) It can be used in animal cells but not plant cells.
E) All of the above.

Answer 3

C
Explanation: Aequorin is a calcium-sensitive luminescent protein that emits blue light in the presence of the ion. It can be expressed in various cells to monitor changes in calcium concentration.

Question 4

When the gene encoding a certain cytoskeleton protein is deleted, the resulting mutant cells round up and do not form their normal appendages. These mutants can be rescued when a gene encoding an N-terminal green fluorescent protein (GFP) fusion of the protein is expressed, but not when a gene encoding a C-terminal GFP fusion is expressed. Which fusion protein (N or C) is appropriate to use in studying cellular localization and activity? Write down N or C as your answer.

Question 5

The following schematic diagram shows the path of light rays passing through in a light microscope. If the angular width of the cone of rays collected by the objective lens (2?) is increased, would the resolution improve (I; i.e. the resolution limit decreases) or deteriorate (D)? Write down I or D as your answer.

Question 6

Single-molecule detection by fluorescence microscopy is limited by the presence of an excess of out-of-focus fluorescent molecules. How does a TIRF microscope uniquely overcome this limitation?

A) By removal of the out-of-focus molecules by selective destruction.
B) By using a deconvolution algorithm that reverses the convolution of signals due to the out-of-focus molecules.
C) By a confocal set-up that eliminates out-of-focus signals.
D) By exciting only in-focus molecules via an evanescent field.
E) By using an objective lens with extremely high numerical aperture.
20-21 Atomic Force Microscopy
Atomic force microscopy (AFM) is used in an experiment to unfold a multidomain protein by applying mechanical force. The protein contains several copies of an immunoglobulin domain that are unfolded one by one as the two ends of the molecule (one attached to a cover slip, and the other to the AFM tip) are being pulled apart, resulting in the "sawtooth" force-extension curves shown below. The same experiment is done twice, once in the presence and once in the absence of a chaperone protein that stabilizes the immunoglobulin domains. Answer the following questions based on this graph.

Question 7

If an average globular protein was of the size of a tennis ball, a typical animal cell would be as large as …

A) a cubicle.
B) a room.
C) a tennis court.
D) a stadium.
E) a city.

Question 8

Consider an engineered chimeric protein made from fusion of three proteins: a blue fluorescent protein (BFP), a calmodulin-binding peptide, and a green fluorescent protein (GFP). Calmodulin is an abundant calcium-binding protein in eukaryotes. Once bound to calcium ions, it can recognize the calmodulin-binding peptide in the fusion protein, change conformation, wrap around the peptide, and bring the BFP and GFP components in close proximity. This results in fluorescence resonance energy transfer (FRET) between BFP and GFP. Accordingly, the fusion protein …

A) is a luminescent ion-sensitive indicator that red-shifts its emission wavelength in the presence of calcium.
B) is a luminescent ion-sensitive indicator that increases its emission in the presence of calcium.
C) is a genetically encoded calcium indicator that red-shifts its emission wavelength in the presence of calcium.
D) is a genetically encoded calcium indicator that increases its emission in the presence of calcium.

Question 9

Two approaches have been devised to deal with the problem of blurring in light microscopy with thicker samples. Indicate whether each of the following descriptions better applies to confocal design (C) or image deconvolution (D). Your answer would be a three-letter string composed of letters C and D only, e.g. CCD.
( ) It is normally faster.
( ) It requires a higher degree of sample illumination.
( ) It can be used to obtain images from relatively deeper parts of the specimen.

Question 10

Two segments (S1 and S2) in a viral protein are suspected to be responsible for the nuclear localization of the protein in infected human cells. You have engineered a plasmid to encode the green fluorescent protein (GFP) fused to either or both of these peptide segments, and have introduced the plasmid into the cells. After the expression of the fusion protein is induced, you visualize the cells using a fluorescence microscope equipped with filters appropriate for detection of GFP. Your results are presented in the following schematic drawings in which the GFP signal is represented in green. Which of the following is more consistent with these observations?

A) Both S1 and S2 are required for nuclear localization.
B) S1 is required and sufficient for nuclear localization.
C) S2 is required and sufficient for nuclear localization.
D) S1 is required but not sufficient for nuclear localization.
E) S2 is required but not sufficient for nuclear localization.

Question 11

What is the advantage of using quantum dots as an alternative to organic fluorochromes such as Cy3 and Alexa dyes?

A) They are brighter.
B) Their emission spectra can be precisely fine-tuned.
C) They have a longer lifetime and bleach more slowly.
D) All of the above.

Question 12

Given the absorption and emission spectra of three fluorescent dyes in the simplified diagrams below, which pair of dyes is better suited for a fluorescence resonance energy transfer (FRET) study? Write down AB, BC, or AC as your answer.

Question 13

Which microscopy set-up uses a longer wavelength of light than usually excites a particular fluorophore? Which one allows researchers to peek deeper into biological samples?

A) Single-photon; single-photon
B) Single-photon; two-photon
C) Two photon; single photon
D) Two-photon; two-photon

Question 14

Atomic force microscopy (AFM) is used in an experiment to unfold a multidomain protein by applying mechanical force. The protein contains several copies of an immunoglobulin domain that are unfolded one by one as the two ends of the molecule (one attached to a cover slip, and the other to the AFM tip) are being pulled apart, resulting in the "sawtooth" force-extension curves shown below. The same experiment is done twice, once in the presence and once in the absence of a chaperone protein that stabilizes the immunoglobulin domains. Answer the following questions based on this graph.


-According to the force-extension graph, how many immunoglobulin domains are unfolded in each of these experiments? Write down the number as your answer, e.g. 9.

Question 15

A certain GTP-binding protein can exist in two main states. When bound to GDP, it is mostly cytosolic. In its GTP-bound form, however, it associates with the cytosolic face of the endoplasmic reticulum (ER) membrane, where it hydrolyzes the bound GTP after a short delay and is released again into the cytosol. You have created and expressed green fluorescent protein (GFP) fusions of the wild-type protein, as well as that of a mutant protein that does not bind GTP as readily as the wild type. You then perform a fluorescence recovery after photobleaching (FRAP) experiment by photobleaching a small area of the ER membrane and measuring GFP fluorescence recovery over time. According to the results below, which curve (1 or 2) do you think corresponds to the wild-type fusion protein? Write down 1 or 2 as your answer.

Question 16

Indicate true (T) and false (F) statements below regarding light and light microscopy. Your answer would be a four-letter string composed of letters T and F only, e.g. FFFF.
( ) Two light waves of the same amplitude and frequency will completely cancel each other out if not perfectly in phase.
( ) If the refractive index of a medium is 1.1, light travels in a vacuum 1.1 times faster than it does in the medium.
( ) The limit of resolution for conventional light microscopy is approximately 0.4 µm, corresponding to the wavelength of violet light.
( ) A light-emitting particle can be detected with a light microscope even if it is several times smaller than the resolution limit of the microscope.

Question 17

The light used to excite a fluorescent molecule carries … energy and has a … wavelength compared to the light that is then emitted from the molecule.

A) greater; longer
B) greater; shorter
C) the same amount of; shorter
D) less; longer
E) less; shorter

Question 18

Indicate whether you would use a fluorescent organic molecule (O), in situ hybridization (H), or a coupled fluorescent protein (P) to visualize the cells and their molecules in each of the following cases. Your answer would be a five-letter string composed of letters O, H, and P only, e.g. OHOOO.
( ) You would like to see where in the early Drosophila embryo the mRNA encoding a certain transcription regulator is located.
( ) You would like to see the nuclei and count them in an early mouse embryo.
( ) You would like to visualize chromosome 3 in a human cell culture derived from a patient's tissue, based on specific sequences present on this chromosome.
( ) You would like to observe the oscillations in Ca²? ions inside a fertilized frog egg.
( ) You would like to compare the localization of two transcription regulatory proteins in cultured human T cells.

Question 19

Imagine a transcription regulatory protein (X) that is known to shuttle back and forth between nucleus and cytosol in an oscillatory pattern. Protein Y is a nuclear protein that can bind to X to create a dimer that binds to DNA. You have fused protein X to green fluorescent protein (GFP) and protein Y to blue fluorescent protein (BFP), and have measured fluorescence resonance energy transfer (FRET) and non-FRET signals in the nucleus at different time points, as indicated in the following simplified plot. At which time period (1 or 2) do you think protein X is in the nucleus? BFP can be excited at 440 nm, and emits maximally at 470 nm. GFP is excited at 470 nm and emits maximally at 500 nm.

Question 20

In the diagram below, a logarithmic scale of sizes is shown. Indicate which of the sizes indicated (A to H) better corresponds to the dimensions of each of the following. Your answer would be a three-letter string composed of letters A to H only, e.g. HCG.

( ) A bacterium
( ) An animal cell
( ) A globular protein

Question 21

The presence of which of the following provides the sample with the lowest electron density?

A) Osmium
B) Lead
C) Uranium
D) Carbon
E) Gold

Question 22

Indicate true (T) and false (F) statements below regarding superresolution fluorescence microscopy. Your answer would be a four-letter string composed of letters T and F only, e.g. FFFF.
( ) Resolutions of 5 nm or better can be readily achieved by superresolution fluorescence techniques.
( ) SIM overcomes the limit imposed by the diffraction of light by a computational analysis on images obtained from interference patterns.
( ) PALM and STORM techniques reduce the width (or "spread") of the point spread function.
( ) Success of STED depends on fluorescence probes that are reversibly switched off and on.

Question 23

Which of the following is NOT correct regarding cryoelectron microscopy?

A) It does not require shadowing or negative staining.
B) It involves obtaining images from many-sometimes tens of thousands of-individual molecules that may or may not all be in the same orientation.
C) Subnanometer resolutions (e.g. 0.5 nm or better) can be achieved by this technique.
D) It can only be used to see the exterior surface of molecules and complexes.
E) Atomic models obtained from x-ray crystallography can be fitted into the density envelope obtained from this technique.
Answers

Question 24

Indicate true (T) and false (F) statements below regarding electron microscopy. Your answer would be a four-letter string composed of letters T and F only, e.g. FFFF.
( ) Depending on acceleration voltage, the resolution limit of an electron microscope can be as small as 0.05 nm.
( ) The emission gun and the magnetic coils in an electron microscope are analogous to the light source and the glass lenses in a light microscope, respectively.
( ) Contrast in specimens for electron microscopy can be achieved using electron-dense material.
( ) For biological samples, the effective resolution of electron microscopy is about 1 nm.

Question 25

You have generated strains of Drosophila melanogaster that are expected to show interesting developmental phenotypes such as misplaced organs in the adult fly. However, some of these phenotypes are not readily seen with light microscopy. You therefore fix each mutant fly, dry it, coat it with a thin layer of gold, and place the entire fly into an electron microscope for viewing. What type of microscope are you using? Write down SEM or TEM as your answer.

Question 26

Electron microscopy samples are often chemically fixed before dehydration, resin-embedding, and sectioning. But they can also be "fixed" by rapid freezing, in a way that precludes ice-crystal formation, to ensure minimal damage to the original cell structures. How can this be done?

A) High-pressure cooling
B) Plunging into liquid nitrogen
C) Spraying with a jet of liquid propane
D) Contact with a copper block cooled by liquid helium
E) All of the above

Question 27

Indicate whether each of the following descriptions better applies to SIM (S), STED (T), or STORM/PALM (P) superresolution techniques. Your answer would be a four-letter string composed of letters S, T, and P only, e.g. PPTS.
( ) It switches on and off individual fluorophores at random over time to accurately determine their position.
( ) It creates a moiré pattern from the interference of the illuminating pattern and the sample features.
( ) It doubles the resolution of conventional fluorescence microscopy.
( ) It limits excitation to the fluorophores that are located at the center of the focal point by using a doughnut-shaped beam in addition to the excitation beam.

Question 28

Atomic force microscopy (AFM) is used in an experiment to unfold a multidomain protein by applying mechanical force. The protein contains several copies of an immunoglobulin domain that are unfolded one by one as the two ends of the molecule (one attached to a cover slip, and the other to the AFM tip) are being pulled apart, resulting in the "sawtooth" force-extension curves shown below. The same experiment is done twice, once in the presence and once in the absence of a chaperone protein that stabilizes the immunoglobulin domains. Answer the following questions based on this graph.


-According to the force-extension graph, which curve (1 or 2) would you expect to correspond to the reaction in the presence of the chaperone protein? Write down 1 or 2 as your answer.

Question 29

Indicate whether each of the following descriptions better applies to the scanning (S) or transmission (T) electron microscopy techniques. Your answer would be a four-letter string composed of letters S and T only, e.g. TSTS.
( ) It generally has a greater depth of field.
( ) It is usually smaller, cheaper, and simpler.
( ) It detects electrons that are scattered or emitted from the specimen.
( ) It is used to create electron-microscope tomograms.