STORM/ PALM ANSWER KEY

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1 STORM/ PALM ANSWER KEY Phys598BP Spring 2016 University of Illinois at Urbana-Champaign Questions for Lab Report 1. How do you define a resolution in STORM imaging? If you are given a STORM setup, how would you measure the resolution? Answer this with the concept of FWHM (Full-widthhalf-maximum.) -- (4 points) You may refer to B. Huang, W. Wang, M. Bates, X. Zhuang, "Three-dimensional Superresolution Imaging by Stochastic Optical Reconstruction Microscopy", Science 319, (2008) ) Answer: The definition of resolution is the minimal distance by which two points can be distinguished. In STORM imaging, resolution is equivalent with FWHM of Gaussian fitting to the distribution of single molecule detection for many frames. As shown in the above reference, (Fig. 1c) a single molecule can give many localization spots over many frames. Then for horizontal or vertical distribution, apply a Gaussian fit and measure FWHM. This will be the resolution, since two distributions (from two single molecules) cannot be resolved any further if they are closer than FWHM distance. The picture below demonstrates this concept. (Here Δ is FWHM, and two distributions can be barely distinguishable when they are apart by Δ. Therefore Δ is considered as the resolution.) 1

2 2. Usually one pixel on our EMCCD is corresponding to 100 nm, but STORM imaging claims 20 nm lateral resolution. How can the resolution be better than the size of one pixel sensing? (4 pts) Answer: STORM resolution isn t limited by pixel resolution since by fitting each single molecule peak by FIONA, the center position of each peak achieves sub-pixel accuracy. The final resolution is then determined via the procedure described as in Question 1, which isn t dependent on the pixel size. 3. Within the data acquisition, we ensure that blinking spots don t overlap with each other, by ensuring that spots appear sufficiently sparsely spaced from each other. What is your estimate of the minimal distance between two spots (appearing in a single frame) to be distinguishable by the software? (Assume the excitation laser is 647nm, EMCCD pixel one pixel is corresponding to 100 nm, each peak will be fitted with 19X19 pixels 2 box.) (4pts) Answer: Each fitting box size is (100 nm 19 pixels) (100 nm 19 pixels) = μm 2. Since it is desired that fitting boxes won t overlap with each other, the minimum distance by which two boxes can be separated is 1.9 μm. This can be a simple answer, but also other possible answers can be: We can allow more overlap, and the software will still detect (with less accuracy). 50% overlap case corresponds to 1.9 μm/2 = 0.95 μm. Also we give a little more distance from the 50% overlap. We can give FWHM distance more than 50% overlap, that is, 0.95 μm nm (FWHM of 647nm = 647/(2 1.4), assuming NA = 1.4 ) = 1.18 μm. 4. Exercise 1: In exercise 1, Cy3B-labeled MTs were incubated in NaBH4 solution and the intensity change was observed over time. Show the progression with images and plot the 2

3 average intensity over the course of NaBH4 incubation. Briefly explain the chemical mechanism behind your observation. (5 points) Reference: J. Vaughan, S. Jia, X. Zhuang, "Ultra-bright Photoactivatable Fluorophores Created by Reductive Caging", Nature Methods 9, (2012) Before NaBH4 after NaBH4 The NaBH4 binds to the Cy3B molecules and puts them into a caged dark state. This bond is broken by 405 nm light and the molecule is sent back to the fluorescent state. 5. In Exercise 2, the activation laser (405 nm) power was systematically changed to observe its effect. Plot number of molecules/imaging area, number of photons and localization precision as a function of activation laser power. How did the laser power influence each of these parameters? Explain why each of the parameter changed. (5 pts) Answer: Only the number of molecules/imaging area should increase with increasing activation laser power. In actuality, with increasing activation laser power, the detected number of photons will also increase. Consequently, the localization precision also becomes better. This is an artifact because high activation laser power activates more than one fluorophores within the diffraction limited spot. As a result, the software still treats the SPF as a single molecule, but with a higher number of photons and better localization precision. 3

4 6. In Exercise 3, STORM imaging of MT was done. (10 points) a. Analyze the data and plot drift uncorrected and corrected STORM images. Explain how the drift correction was done and why this is necessary. What is the limitation of this method? (3 pts) b. Sample 5-10 cross sections of MT from the conventional and super-resolution images to determine the average cross sectional diameters. Plot a sample cross section from each type of images. Report the threshold values used to reconstruct the image. Do not forget to add the scale bar. (3 pts) c. Resolution improvement. What is the localization precision from your fitting? Compared to the theoretical resolution limit, how many fold improvement did you achieve? How many fold improvement of the resolution did you achieve from your cross section comparison between the conventional and the super-resolution image? (4 pts) Drift corrected Green Not drift corrected Red Drift correction was done by imaging a non-fluorescent bead during the course of the imaging. A PSF is fit to the bead to get a trace of how the stage drifted. The positions of the Cy3B molecules are then corrected for. Drift correction is done because the microscope stage drifts due to thermal fluctuations. This will cause the super resolution image to blur. 4

5 b.) Super Resolution (pixel size is 10 nm) Normal Resolution (pixel size is 106 nm) c.) The theoretical resolution is ~570 nm/(2*1.45) 200 nm. Most groups achieved a precision of 15 nm, which is about a 13x improvement in resolution. For the microtubule, most groups achieved 70 nm cross section for the super resolution image and 480 nm cross section for the normal resolution, a 7x improvement in resolution. 7. Discuss how each of the following parameters/chemicals influences the photophysical properties of the dye (e.g., on time, off time, number of switching cycles and photons) and ultimately the final STORM image. (10 pts) a. Reducing agent concentration (e.g., BME, MEA) (3 pts) b. Oxygen scavenging system (3 pts) c. Excitation laser power (in MT experiment, 561 nm) (2 pts) d. Exposure time (2 pts) References: Heilemann, M., et al., Super-Resolution Imaging with Small Organic Fluorophores, Angew. Chem. Int. Ed., 2009, 48, doi: /anie

6 Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes Superresolution Microscopy on the Basis of Engineered Dark States A Reducing and Oxidizing System Minimizes Photobleaching andblinking of Fluorescent Dyes Answer. a. Reducing agent concentration (e.g., BME, MEA): This is important in stabilizing the off state (increase τoff) of the dye. With higher concentration of the reducing agent, fluorophores will stay in the dark state longer. This is important in turning off the fluorophores. b. Oxygen scavenging system: This system will remove the oxygen from the solution and prolong the time for the fluorophores to go to the permanent dark state ( photobleaching ). This allows measurement of more cycles of on events. c. Excitation laser power (in MT experiment, 561 nm): In the presence of the reducing agent, higher excitation laser power will increase the rate in which the fluorophore to go to the dark state. Effectively, fluorophores will go to the dark state faster. With higher excitation laser power, the emission fluorescence from the fluorophore will also be greater, leading to better localization precision. d. Exposure time: With shorter exposure time, more frames can be collected in a shorter period of time. better time resolution. With longer exposure time, more photons may be collected per molecule (assuming an on time is relatively long). better localization precision. 8. In one of the experiments we directed labeled the microtubules with the organic dye, what do you expect in terms of difference when measuring the cross section of the microtubule compared to antibody labeling? (4 pts) When we label the microtubules directly with organic dye we expect to measure a cross section of the microtubule smaller than the cross section measured when the microtubule is labeled with antiobody+dye. The broadening of the microtubule measurement by antibody+dye is in the range of 10-20nm while the broadening due direct labeling should be around 5nm. 6

7 9) In the neuron experiment, do AMPA receptors appear to be colocalized with the PSD-95 molecules? Could this experiment be done in live cells as well? What are the advantages/disadvantages of PALM over STORM. (4 pts) Super resolution techniques can be done in live or fixed cells. Palm has the advantage of label specificity, but the fluorophores used (photoactivatable fluorescent proteins) are much dimmer than organic fluorophores. STORM can use brighter probes, but label specificity is a problem. Also, STORM may require reducing agents that are very toxic to the cells. 7