Discovery Talk Questions and Answers: Developing PALM microscopy

Eric Betzig & Harald Hess
Assessments created by Dr. Kassandra Ori-McKenney

Questions

1. In their talk, Drs. Betzig and Hess talk about a new class of optical highlighters (07:59). What does PA-FP stand for?
   1. Photoactivatable fluorescent probe
   2. Photoavoidance fluorescent protein
   3. Photoactivatable fluorescent protein
   4. Photoavoidance fluorescent precursor
2. Why is it difficult to resolve individual fluorescent molecules within a cell?
   1. The cameras cannot detect single photons.
   2. The molecules are very densely packed together.
   3. The molecules are spaced too far apart.
   4.  The background noise is too high.
3. In their talk, Drs. Betzig and Hess explain that PA-FPs were the ‘missing link’ for super resolution microscopy (08:26).
   Explain why the PA-FPs were so fundamental to their idea of imaging single molecules.
4. In their paper (p. 1642), they reveal that acquiring one single superresolution image could take between 2 and 12 hours.
   Why would it take so long to generate one image?
5. Drs. Betzig and Hess originally used the quantum well experiment where they separated the exciton recombination sites in terms of X, Y and spectral (or wavelength) dimensions in order to see individual luminescent centers.  How did their new photoactivatable localization microscopy differ in terms of the dimensions they were observing?
6. What are the photophysical characteristics for a PA-FP that one must consider when using it for PALM? (select all that apply)
   1. The photobleaching half-life of the PA-FP.
   2. The contrast between its activated and inactivated state.
   3. Its propensity to blink.
   4. None of the above.
7. Explain why it would be useful to use PALM and transmission electron microscopy (TEM) in tandem to study a particular protein.
8. Why is PALM well suited for studies of proteins bound to a membrane? (Select all that apply)
   1. Thin sections permit the study of a membrane region without using harsh, invasive conditions.
   2. PALM can resolve smaller associated membranes and their corresponding proteins.
   3. Membrane proteins are easier to image than non-membrane bound proteins.
   4. none of the above
9. In Figure 4 E-F, examine the differences between the TIRF image (E) and the PALM image (F).  Explain what “V”, “R”, and “P” are and how PALM helps to distinguish between “R” and “P”.
10. Explain one limitation of the PALM system and whether the authors address how this limitation could be overcome.

Answers

1. c. photoactivatable fluorescent protein
   p. 1642: “Here, we developed a method for isolation of single molecules at high densities based on the serial photoactivation and subsequent bleaching of numerous sparse subsets of photoactivatable fluorescent protein (PA-FP) molecules.”
2. b. The molecules are very densely packed together.
   (09:22) “Right there you see a lot of molecules and they are normally very densely packed, impossible to resolve them clearly, but if you put in a little bit of blue light, you can turn on very small subset and they are far enough apart, that you can see each one glow independently localize it’s center and you repeat this until you exhaust all of the molecules and you can then resolve the complete super-resolution image whereas if they all glowed at once you would just see a massive blur that looks like this.”
   p. 1642: “In brief, individual molecules densely packed within the resolution limit of a given instrument (as defined by its point-spread function (PSF)) are first isolated from one another on the basis of one or more distinguishing optical characteristics.”
3. Short Answer: Because one can turn on these single PA-FP molecules one at a time, then resolve an entire image that is comprised of these individual fluorescently activated molecules.
   Long Answer: PA-FPs are normally dark, but we can turn them on by shining a light on them, and control the number of FPs turned on by modifying the intensity (or brightness) of the light. Therefore, we can turn on a subset of individual FP molecules at one time, then wait for them to bleach, then turn on another subset of FPs until we’ve generated many subsets that can then be combined.  This way, we can generate a final image in which we can distinguish each individual fluorescent molecule.
   (08:26) “So the thought is that, rather than bathing the entire specimen with blue light until it all glows, just turn on the blue or purple light for a very brief period of time, so only a few molecules turn on at once. Then, since they are isolated from one another, we can find their centers, and plot those. Then, they burn out and bleach and we turn on a new set of molecules by pulsing light on again, and repeat this process for many frames until we determine the coordinates of every molecule inside of the sample.”
   (09:15) “Just in case you didn’t quite get Eric’s explanation, let me just restate it in plain English. Basically (…), right there you see a lot of molecules and they are normally very densely packed, impossible to resolve them clearly, but if you put in a little bit of blue light, you can turn on very small subset and they are far enough apart, that you can see each one glow independently localize it’s center and you repeat this until you exhaust all of the molecules and you can then resolve the complete super-resolution image. Whereas, if they all glowed at once you would just see a massive blur that looks like this.”
4. Because they can only photo-activate a subset of individual molecules at a time, therefore, in order to generate a single image, they needed to perform multiple rounds of photoactivation and bleaching until every PA-FP has been activated.
   p. 1642: Initial image frames typically consisted of sparse fields of individually resolvable single molecules on a weaker background presumably dominated by the much larger population of PA-FP molecules still in the inactivated state. When necessary, excitation and thus bleaching was maintained until such sparse fields were obtained. Additional image frames were then captured until single-molecule bleaching resulted in a mean molecular separation considerably larger than that required for isolation (Fig. 1, A and C). At that point, we applied a light pulse from a second laser at a wavelength capable of activating the remaining inactive PA-FPs, at a duration and intensity chosen so that the overall density of activated PA-FPs was increased back to a higher, but still resolvable, level (Fig. 1, B and D). This process of photoactivation, measurement, and bleaching was then repeated for many cycles over 10^4 to 10^5 image frames (depending on the expression level and spatial distribution of the PA-FPs) until the population of inactivated, unbleached molecules was depleted.
5. Using PALM, they were observing data in the dimensions of X, Y and Time (t). This is because using PA-FPs, they could more easily identify the luminescent centers of the molecules. However, because they are spaced so sparsely, they had to take many images over time and combine them.
   p. 1642: “When the xy frames from any such image stack are summed across time t, the molecular signals overlap to produce a diffraction-limited image (Fig. 1, E and F).”
   (09:02) “So instead of that original quantum well experiment, where we separated the exciton recombination sites in terms of X, Y, and wavelength now it’s in terms of X, Y, and time.”
6. a, b and c
   p. 1643: “Longer photobleaching half-life leads to more photons per molecule, but for a given excitation intensity, it also requires longer data acquisition times between activation pulses to maintain an appropriate density of individually resolvable molecules . . . PA-FPs that remain activated until bleached ensure that all possible photons are extracted. Finally, PA-FPs less prone to blinking are desirable, given that it can be difficult to distinguish a single blinking molecule from multiple molecules that are serially activated and bleached in the same DLR.”
7. By using a combination of these microscopy methods, one could determine the exact localization of a particular protein in relation of the rest of the cellular environment.
   PALM would allow to precisely localize a particular protein to nanometer resolution using PA-FPs, while TEM would reveal all of the cellular structures. Therefore, by combining these methods, we would be able to localize the protein within the broader cellular context.
   p. 1644: “Such comparative PALM/TEM imaging permits the nanometer-scale distribution of a specified protein to be determined in relation to the rest of the cellular ultrastructure at much higher molecular density than in immunolabeled TEM.”
8. a. and b.
   p. 1644: “demonstration of PALM on fixed cultured cells in phosphate-buffered saline (Fig. 4) is also notable both as a means to study proteins at or near the plasma membrane under minimally invasive conditions and as a precursor to eventual three-dimensional (3D) PALM imaging.”
   p. 1644: “A TIRF image shows the outlines of the limiting membrane (Fig. 2A) but only hints at the intricate structure that is resolved by PALM, such as smaller associated membranes that may represent interacting lysosomes or late endosomes.”
   There is no evidence that membrane bound proteins are easier to image than non-membrane bound proteins.
9. V is a void of protein, R is a region where there is a higher density of protein, and P is an area of clustered proteins that are probably budding from the membrane. TIRF reveals the R and P regions as being equal in brightness, but different in size, while PALM reveals a difference in both brightness and size between R and P, because P is a much brighter smaller region, while R is a region that is not necessarily brighter, but there are more molecules in that region.
   p. 1645: “In whole cells, PALM with TIRF excitation is well suited to studies of proteins bound to the plasma membrane, such as the dEosFP-fused Gag protein of human immunodeficiency virus 1 imaged by TIRF and PALM in Fig. 4, E and F, respectively. Gag, a retroviral protein that mediates the assembly of virus-like particles (VLPs), is revealed by PALM in various stages of organization: voids (arrows marked V), one high-density region (arrow R), and several tight clusters probably indicative of budding VLPs  (arrows P).”
10. There are two limitations to this system, one that the authors mention in the discussion and one that they do not.  Either is acceptable as an answer, but the point of the question is to make the students think about how this microscope could be used.
    Answer #1: One limitation of the PALM system is using this microscope to compose a 3D image.  All of the resolved images in the paper are from extremely thin cryostat sections of cells, where very few molecules are stacked in the z dimension. With a thicker section, it may be more difficult to resolve individual molecules in the z dimension and there may be cellular autofluorescence. The authors address this by proposing to use cryogenic PALM of vitrified cells.
    p. 1645: In the last paragraph: “Bulk cellular autofluorescence complicates the extension of PALM to 3D, but the improved single-molecule sensitivity predicted for a proposed optical lattice microscope may help. However, the most promising path to 3D may involve cryogenic PALM of vitrified cells.”
    Answer #2: One limitation of the PALM system is using this microscope for live cell imaging. Due to the long period of time it takes to collect data for a single, fixed image, there is no conceivable way to image dynamic molecules within the cell even on the order of minutes. The authors do not address this.
    p. 1645: In the last paragraph: “As such, it could be widely adopted in short order for the near-molecular resolution imaging of specified proteins for in vitro preparations and fixed cells.”