Exam 9: Visualizing Cells

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.

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

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

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.

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.

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?

Which of the following is NOT correct regarding cryoelectron microscopy?

Which of the following is correct regarding aequorin?

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.