This work was supported by a grant from the AHA to R.E.D. (15SDG25560035). Authors would like to acknowledge Dr. Fernando Santana for use of his Leica SR GSD 3D microscope, and Dr. Johannes Hell for kindly providing the FP1 antibody.

1. Washing Glass Coverslips
<ol>
	<li>The day before sample processing, clean #1.5 glass coverslips by sonicating in a 1 M solution of KOH for 1 h. This removes any fluorescent contaminants that may be present on the coverslips.
	<ol>
		<li>Thoroughly rinse the KOH from the coverslips with de-ionized water.</li>
		<li>Once cleaned in KOH, store the coverslips in 70% ethanol until ready to use.</li>
	</ol>
	</li>
</ol>
2. Coating Glass Coverslips
NOTE: S...

<ol>
	<li>Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+microscopy+reveals+that+synaptotagmin+remains+clustered+after+synaptic+vesicle+exocytosis.">STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.</a> Nature. 440 (7086), 935-939 (2006).</li><li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+structured-illumination+microscopy:+wide-field+fluorescence+imaging+with+theoretically+unlimited+resolution.">Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.</a> Proc Natl Acad Sci U S A. 102 (37), 13081-13086 (2005).</li><li>Betzig, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313 (5793), 1642-1645 (2006).</li><li>Hell, S. W., Kroug, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ground-state-depletion+fluorscence+microscopy:+A+concept+for+breaking+the+diffraction+resolution+limit.">Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit.</a> JAppl Phys B. 60 (5), 495-497 (1995).</li><li>Bretschneider, S., Eggeling, C., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+the+diffraction+barrier+in+fluorescence+microscopy+by+optical+shelving.">Breaking the diffraction barrier in fluorescence microscopy by optical shelving.</a> Phys Rev Lett. 98 (21), 218103 (2007).</li><li>Flynn, J. M., Santana, L. F., Melov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptional+profiling+of+adult+mouse+cardiomyocytes.">Single cell transcriptional profiling of adult mouse cardiomyocytes.</a> J Vis Exp. (58), e3302 (2011).</li><li>Davare, M. A., Horne, M. C., Hell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+phosphatase+2A+is+associated+with+class+C+L-type+calcium+channels+(Cav1.2)+and+antagonizes+channel+phosphorylation+by+cAMP-dependent+protein+kinase.">Protein phosphatase 2A is associated with class C L-type calcium channels (Cav1.2) and antagonizes channel phosphorylation by cAMP-dependent protein kinase.</a> J Biol Chem. 275 (50), 39710-39717 (2000).</li><li>Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+fluorophores+for+optimal+performance+in+localization-based+super-resolution+imaging.">Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.</a> Nat Methods. 8 (12), 1027-1036 (2011).</li><li>Chozinski, T. J., Gagnon, L. A., Vaughan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twinkle,+twinkle+little+star:+photoswitchable+fluorophores+for+super-resolution+imaging.">Twinkle, twinkle little star: photoswitchable fluorophores for super-resolution imaging.</a> FEBS Lett. 588 (19), 3603-3612 (2014).</li><li>Dempsey, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoswitching+mechanism+of+cyanine+dyes.">Photoswitching mechanism of cyanine dyes.</a> J Am Chem Soc. 131 (51), 18192-18193 (2009).</li><li>Nahidiazar, L., Agronskaia, A. V., Broertjes, J., van den Broek, B., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+Imaging+Conditions+for+Demanding+Multi-Color+Super+Resolution+Localization+Microscopy.">Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy.</a> PLoS One. 11 (7), e0158884 (2016).</li><li>Ovesny, M., Krizek, P., Borkovec, J., Svindrych, Z., Hagen, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ThunderSTORM:+a+comprehensive+ImageJ+plug-in+for+PALM+and+STORM+data+analysis+and+super-resolution+imaging.">ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging.</a> Bioinformatics. 30 (16), 2389-2390 (2014).</li><li>Veeraraghavan, R., Gourdie, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+optical+reconstruction+microscopy-based+relative+localization+analysis+(STORM-RLA)+for+quantitative+nanoscale+assessment+of+spatial+protein+organization.">Stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA) for quantitative nanoscale assessment of spatial protein organization.</a> Mol Biol Cell. 27 (22), 3583-3590 (2016).</li><li>Ries, J., Kaplan, C., Platonova, E., Eghlidi, H., Ewers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+versatile+method+for+GFP-based+super-resolution+microscopy+via+nanobodies.">A simple, versatile method for GFP-based super-resolution microscopy via nanobodies.</a> Nat Methods. 9 (6), 582-584 (2012).</li><li>Dixon, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graded+Ca2+/calmodulin-dependent+coupling+of+voltage-gated+CaV1.2+channels.">Graded Ca2+/calmodulin-dependent coupling of voltage-gated CaV1.2 channels.</a> Elife. 4, (2015).</li><li>Annibale, P., Vanni, S., Scarselli, M., Rothlisberger, U., Radenovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+clustering+artifacts+in+photoactivated+localization+microscopy.">Identification of clustering artifacts in photoactivated localization microscopy.</a> Nat Methods. 8 (7), 527-528 (2011).</li><li>Nan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+superresolution+imaging+allows+quantitative+analysis+of+RAF+multimer+formation+and+signaling.">Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling.</a> Proc Natl Acad Sci U S A. 110 (46), 18519-18524 (2013).</li><li>Fricke, F., Beaudouin, J., Eils, R., Heilemann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One,+two+or+three?+Probing+the+stoichiometry+of+membrane+proteins+by+single-molecule+localization+microscopy.">One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy.</a> Sci Rep. 5, 14072 (2015).</li></ol>

The authors have no competing interests to disclose.

The recent explosion of technologies that allow imaging beyond the diffraction limit have offered new windows into the complexities of mammalian cell signaling in space and time. These technologies include STORM, STED, PALM, GSD, SIM, and their variants (e.g., dSTORM, FPALM). The ingenuity of the scientists behind these techniques has allowed us to circumvent the limitations imposed by laws of physics governing the diffraction of light. In spite of this huge accomplishment, each of these techniques makes some so...

Cellular signaling reactions translate changing internal and external environments to initiate a cellular response. They regulate all aspects of human physiology, serving as the foundation for hormone and neurotransmitter release, the heartbeat, vision, fertilization, and cognitive function. Disruption of these signaling cascades can have severe consequences in the form of pathophysiological conditions including cancer, Parkinson&#39;s, and Alzheimer&#39;s disease. For decades, biological and medical investigators have successfully used fluorescent proteins, probes, and biosensors coupled with fluorescence microscopy as the primary tools to understand the precise spatial and temporal organization of these cellular signals.
The strengths of optical techniques such as epifluorescence, confocal, or total internal reflection fluorescence (TIRF) microscopy are their sensitivity, speed, and compatibility with live cell imaging, while the major limitation is their diffraction-limited resolution, meaning structures or protein complexes smaller than 200-250 nm cannot be resolved. With the theoretical and practical development of deterministic super-resolution (e.g., stimulated emission depletion microscopy (STED1), structured illumination microscopy (SIM2) or stochastic super-resolution (e.g., photoactivated localization microscopy (PALM3), or ground state depletion (GSD4,5)), lateral and axial resolution in fluorescence microscopy has been extended beyond the diffraction barrier, to the order of tens of nanometers. Thus, investigators now have the unparalleled ability to visualize and understand how protein dynamics and organization translates to function at the near-molecular level.
Ground state depletion microscopy followed by individual molecule return (GSDIM), or simply GSD as it is known, circumvents the diffraction limit by reducing the number of simultaneously emitting fluorophores4,5. High energy laser light is used to excite the fluorophore-labelled sample, bombarding electrons with photons and increasing the probability they will undergo a &#39;spin-flip&#39; and enter the triplet or &#39;dark-state&#39; from the excited state4. This effectively depletes the ground state, hence the name &#39;ground state depletion&#39;. In the triplet state, fluorophores do not emit photons and the sample appears dimmer. However, these fluorophores stochastically return to the ground state and can go through several photon emitting excited-to-ground state transitions before returning to the triplet state. With less fluorophores emitting at any given time, photon bursts emitted from individual fluorophores become spatially and temporally distinct from neighboring fluorophores. The burst of photons can be fit with a gaussian function, the calculated centroid of which corresponds to the position of the fluorophore with a localization precision that is dependent on the numerical aperture (NA) of the lens, the wavelength of light used for excitation and crucially, the number of photons emitted per fluorophore. One limitation of GSD is that, since only a subset of fluorophores actively emits at any time, thousands of images must be collected over several minutes to build up a complete localization map. The long acquisition time combined with the high laser power requirement, means that GSD is better suited to fixed rather than live samples.
This article, describes the preparation of fixed samples for super-resolution microscopy imaging of membrane and endoplasmic reticulum (ER)-resident proteins&#160;(for a list of necessary consumables and reagents see the Table of Materials). Examples of how this protocol can be easily adapted to quantify the size and degree of clustering of L-type voltage-gated Ca2+ channels (Cav1.2) in the sarcolemma of cardiac myocytes, or used to visualize the cellular distribution of the ER, are demonstrated. Understanding the distribution and organization of these cellular components is critically important in understanding the initiation, translation, and ultimately the function of many Ca2+-dependent signaling cascades. For example, Cav1.2 channels are fundamentally important for excitation-contraction coupling, while receptor-mediated Ca2+ release from the ER is perhaps the most ubiquitous signaling cascade in mammalian cells.

<table>
	<tbody>
		<tr>
			<td>KOH</td>
			<td>Thermo Scientific</td>
			<td>P250-500</td>
			<td>To clean coverglass</td>
		</tr>
		<tr>
			<td>#1.5 coverglass 18 x 18 mm</td>
			<td>Marienfeld Superior</td>
			<td>0107032)</td>
			<td>To grow/process/image cells</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Thermo Scientific</td>
			<td>BP3994</td>
			<td>Dilute to 1X with de-ionized water</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P4832</td>
			<td>Aids with cell adhesion to cover glass</td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Sigma</td>
			<td>114956-81-9</td>
			<td>Aids with cell adhesion to cover glass</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Thermo Scientific</td>
			<td>11150-059</td>
			<td>Ventricular myocyte culture media</td>
		</tr>
		<tr>
			<td>DMEM 11995</td>
			<td>Gibco</td>
			<td>11995</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>Fetal bovine Serum (FBS)</td>
			<td>Thermo Scientific</td>
			<td>10437028</td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>0.05% trypsin-EDTA</td>
			<td>Corning</td>
			<td>25-052-CL</td>
			<td>Cell culture solution</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Transient transfection reagent</td>
		</tr>
		<tr>
			<td>Ca2+-free PBS</td>
			<td>Gibco</td>
			<td>1419-144</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>100 % Methanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>A414-4</td>
			<td>Cell Fixation</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Cell Fixation</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma Aldrich</td>
			<td>SLBR6504V</td>
			<td>Cell Fixation</td>
		</tr>
		<tr>
			<td>SEAblock</td>
			<td>Thermo Scientific</td>
			<td>37527</td>
			<td>BSA or other blocking solution alternatives exist</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Detergent to permeabilize cells</td>
		</tr>
		<tr>
			<td>Rabbit anti-CaV1.2 (FP1)</td>
			<td>Gift</td>
			<td>N/A</td>
			<td>Commercial anti-CaV1.2 antibodies exist such as Alomone Labs Rb anti-CaV1.2 (ACC-003)</td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-RFP</td>
			<td>Rockland Inc.</td>
			<td>200-301-379</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 donket anti-rabbit IgG (H+L)</td>
			<td>Invitrogen (Thermo Scientific)</td>
			<td>A31573</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 goat anti-mouse IgG (H+L)</td>
			<td>Invitrogen (Thermo Scientific)</td>
			<td>A11031</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>Prevents microbial growth for long term storage of samples</td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C40</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Sigma</td>
			<td>G2133</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>beta-Mercaptoethylamin hydrochloride</td>
			<td>Fisher</td>
			<td>BP2664100</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma</td>
			<td>63689</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S271-3</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher</td>
			<td>D14-212</td>
			<td>Photoswitching buffer ingredient</td>
		</tr>
		<tr>
			<td>Glass Depression slides</td>
			<td>Neolab</td>
			<td>1 &#8211; 6293</td>
			<td>To mount samples</td>
		</tr>
		<tr>
			<td>Twinsil</td>
			<td>Picodent</td>
			<td>13001000</td>
			<td>To seal coverglass</td>
		</tr>
		<tr>
			<td>sec61&#946;-mCherry plasmid</td>
			<td>Addgene</td>
			<td>49155</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SR GSD 3D microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Washing block solution</td>
			<td></td>
			<td></td>
			<td>20 % SEAblock in PBS</td>
		</tr>
		<tr>
			<td>Primary antibody incubation solution</td>
			<td></td>
			<td></td>
			<td>0.5 % Triton-X100, 20 % SEAblock, in PBS</td>
		</tr>
		<tr>
			<td>Secondary antibody incubation solution</td>
			<td></td>
			<td></td>
			<td>1:1000 in PBS</td>
		</tr>
	</tbody>
</table>

ground state depletion super-resolution imaging in mammalian cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

As documented in the introduction, there are many different super-resolution microscopy imaging modalities. This protocol, focuses on GSD super-resolution imaging. Representative images and localization maps are shown in Figure 2 and Figure 3.
Figure 2 shows a COS-7 cell transfected with the ER protein, mCherry-Sec61&#946;, and processed us...

Watch this Scientific Journal Video about Ground State Depletion Super-resolution Imaging in Mammalian Cells at JoVE.com

Ground State Depletion Super-resolution Imaging in Mammalian Cells

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

The overall goal of this fluorescent imaging technique is to visualize and quantify cellular proteins in fixed cells at subdiffraction-limited resolution using a form of super-resolution microscopy called ground state depletion microscopy and individual molecule return. This method helps answer key questions in the field of cell biology and membrane biophysics, as it circumvents diffraction-limited resolution, which helps investigators more precisely define, visualize and quantify the distribution of cellular proteins. The main advantage of this protocol is that it enables the acquisition of images with an approximate ten-fold improvement in resolution over conventional fluorescence microscopy imaging approaches, such as confocal microscopy.Demonstrating the procedure will be Oscar Vivas, a post-doc in my laboratory, and Karen Hannigan, a post-doc in Rose Dixon's laboratory. To begin, prepare the cover slips and cells as described in the accompanying text protocol. Then fix the samples and perform immunocytochemistry in order to label the plasma membrane and/or intracellular membrane proteins of interest.Next, prepare the oxygen scavenging Glock solution by first adding 12.5 microliters of a catalase stock solution, 3.5 milligrams of glucose oxidase, and 0.5 microliters of one molar Tris to 49.5 microliters of PBS in a 1.5 milliliter microcentrifuge tube. Close the tube and briefly vortex it to dissolve the components. Then centrifuge the solution for three minutes.Now, prepare the photo switching inducing thial solution by dissolving beta-mercaptoethylamine solution into PBS to a concentration of 100 millimolar and modify the pH to 8.0. Aliquot and store the thial solution in the freezer for up to one week. Immediately before imaging, mix the thial and oxygen scavenging components together with a saline buffer and store the solution on ice.Using 100 microliters of the prepared photo switching buffer, mount cover slips containing stained cells onto the depression slides. Next, seal the edges of the cover slip with a silicone glue to prevent rapid oxidation of the imaging buffer. Wait several minutes until the silicone glue has fully cured, prior to imaging.Otherwise, the cover slip will drift. To begin, select an appropriate laser to illuminate the sample, based on the chosen fluorophore. For an Alexa 647 fluorophore, use a 642 nanometer laser.Next, chose the appropriate dichroic imaging cube and objective lens. Open the imaging software. Select the 2D acquisition mode then set the exposure time to 11 milliseconds.Also, set the EM Gain to 300 and select an appropriate detection threshold. Next, turn on Auto Event Control and set the number of Events Per Images to eight. Enter the Pixel Size as 20 nanometers.In the GSD Tools tab, go to the High Resolution Image Calculation options and ensure that Histogram Mode is selected. To image proteins at the plasma membrane, select TIRF mode and modify the incidence angle to determine the penetration depth. Then, set the laser intensity to 100%in order to send electrons to the dark state.Next, select Single Molecule Detection while pumping and set to acquire automatically when the frame correlation is 0.20. For acquisition, set laser intensity to 50%and the number of acquisition frames to 60, 000. Then begin taking images.To quantify protein cluster size, open the image file of a 10 nanometer histogram single molecule localization map in ImageJ. In the Image menu, go to Adjust and use the Auto option to optimize the brightness and contrast of the image. Then, under the Type menu, choose 8-bit.Next, open the Analyze menu, click Set Scale and enter the values shown here. Then, in the Analyze menu, select Set Measurements and check the box beside the Area option. Back in the Image menu, select Adjust, Threshold, and then select Over/Under.Move the maximum value to 255, the minimum value to one and click Apply. Finally, open the Analyze menu and select Analyze Particles. Place a check mark to select Pixel units, Display results and Summarize.Then set the size to four to infinity, select the Bare Outlines option and click OK.The cell imaged here was transfected with the endoplasmic reticulum protein, mCherry-Sec61 beta, and imaged using both defraction-limited TIRF microscopy and super-resolution GSD microscopy in TIRF mode. These curves represent the diameter of the ER tubules. The image on the right shows an improvement in the lateral resolution and represents a more accurate representation of the structure of ER tubules.The improvement in resolution offered by super-resolution GSD imaging is further demonstrated in these images, showing a labeled cardiac myocyte with an anti-CAV 1.2 antibody. Using GSD mode, the improvement in the lateral resolution is prominent and separate clusters of channels are easier to identify. Once mastered, this protocol can be completed in less than three days.The super-resolution imaging of a single cell takes 10 to 30 minutes, depending on the number of frames required. Following this procedure, other methods like electromicroscopy can be performed in order to answer addition questions like how the fluorescent protein of interest interacts with the complex cellular environment. After watching this video, you should have a good understanding of how to prepare, acquire and analyze samples to visualize one or more cellular proteins for super-resolution microscopy using ground state depletion followed by individual molecule return.

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7.1K Views.  University of California School of Medicine, Davis. The overall goal of this fluorescent imaging technique is to visualize and quantify cellular proteins in fixed cells at subdiffraction-limited resolution using a form of super-resolution microscopy called ground state depletion microscopy and individual molecule return. This method helps answer key questions in the field of cell biology and membrane biophysics, as it circumvents diffraction-limited resolution, which helps investigators more precisely define, visualize and quantify the distrib...