9) Cell Visualization

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.

Answer: 1 The force required to unfold the domains is expected to be higher in the presence of the chaperone protein

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.

Answer: 4 Each peak normally represents one unfolding event. The last peak corresponds to the rupture of the polypeptide and loss of tension at high extensions.

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: B 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.

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

Answer: B The photon emitted by a fluorescent molecule is necessarily of lower energy and longer wavelength than the absorbed photon. Therefore the excitation light has greater energy and a shorter wavelength than the emitted light.

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: C 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.

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.

Answer: C On excitation with violet light, such a protein would shift its emission peak toward longer wavelengths, from blue to green, upon a calcium concentration increase.

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.

Answer: CCC Confocal microscopy provides a rapid way of eliminating blurring, but requires brighter illumination.

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

Answer: D Contrast in the electron microscope depends on the electron-scattering power of the atoms in the specimen, which in turn depends on their atomic number. Thus all the atoms here have high atomic numbers, and high electron contrast, except carbon, which has an atomic number of 6 and is a major element in biological specimens to start with.

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.

Answer: D Cryoelectron microscopy reveals the electron density of entire particles, not just their surface.

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.

Answer: D In a total internal reflection fluorescence (TIRF) microscope, an evanescent wave excites only those fluorescent molecules that lie within an extremely thin plane adjacent to the cover slip.

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

Answer: D In the two-photon effect, which is employed by multiphoton microscopes, fluorescent molecules are excited almost simultaneously by two (or more) photons of longer wavelength compared to one-photon excitation. The longer wavelength provides advantages including the ability to image deep within live tissues.

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.

Answer: D Quantum dots have several advantages over organic fluorescent dyes, including higher brightness, stability, and emission tunability.

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.

Answer: D The diameter of a typical globular protein is on the order of nanometers, about ten million times smaller than a tennis ball, which has a diameter on the order of centimeters. Ten million multiplied by the diameter of a typical animal cell (10 to 20 micrometers) yields 100 to 200 meters, comparable to the dimensions of a stadium. It should be noted that a plant cell can be 200 µm across, corresponding to the size of a town in our analogy; on the other hand, a bacterium at 2 µm would be only as large as a tennis court.

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

Answer: E All of these methods can be used for flash-freezing of the samples.

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: E The pulse of laser light photoactivates the incorporated tubulins in the region of the spindle irradiated. After about 5 minutes, most of the activated tubulin molecules near the pole will have quickly moved toward the poles and left the spindle, which no longer fluoresces, while those activated near the equator will have moved along the spindle microtubules toward the poles and they still fluoresce.

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.

Answer: FTFT The highest resolution achieved by superresolution techniques has been about 20 nm, an order of magnitude past the limit set by light diffraction. Different methods overcome the limit in various ways. In structured illumination microscopy (SIM), this is done by using a patterned light source that is translated and rotated with respect to the sample to collect a series of interference images that are then processed. Stimulated emission depletion microscopy (STED) overcomes the limit by changing the shape of the point spread function, whereas localization methods approximate the center of the point spread function by stochastic sampling.

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.

Answer: FTFT Total destructive interference (complete canceling) only occurs when the two waves are out of phase by exactly 180 degrees. The formal resolution limit of light microscopy is about 0.2 µm.

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.

Answer: HOHOP Sequence-specific staining of nucleic acids can be achieved by in situ hybridization of probes that are attached to fluorescent dyes. If general DNA staining is desired, a fluorescent organic molecule such as DAPI can be used. Fluorescent or luminescent ion-sensitive indicators (many of which are small molecules) can be used to measure cellular ionic concentrations in real time. A protein can be genetically engineered as a fluorescent fusion protein, and then imaged in living cells by fluorescence microscopy.

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.

Answer: N If the gene encoding the fusion cannot rescue a loss-of-function mutation of the original gene, the presence of the fused protein might be interfering with gene function.

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.

Answer: PSST Different superresolution fluorescence techniques overcome diffraction-limited resolution in different ways. For example, through illumination by a grid, structured illumination microscopy (SIM) can improve the resolution by about a factor of two. Stimulated emission depletion microscopy (STED) uses a bright torus-shaped lased beam to switch the fluorescent molecules off at the periphery of the point spread function. Single-molecule localization methods such as photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) use lasers to sequentially switch on a sparse subset of fluorescent molecules in a specimen.

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.

Answer: SEM Often an entire plant part or small animal can be put into the microscope with very little preparation.

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.

Answer: SSST Scanning electron microscopy is generally simpler and has a greater depth of field. It is used to see the surface of specimens, which can be entire plant parts or small animals.

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.

Answer: TTTT The practical resolving power of an electron microscope can be about 0.05 nm. However, problems in specimen preparation, contrast, and radiation damage can limit the effective resolution to 1 nm for biological samples. Emission gun in electron microscopy is analogous in function to a light source in light microscopy; the magnetic coils focus the electron beams, just as glass lenses focus the light; and the use of electron-dense material to achieve contrast in electron microscopy is similar to the use of light-absorbing dyes in light microscopy.