Name: ground state depletion super-resolution imaging in mammalian cells
Uploaded: 2017-11-05T08:30:00
Description: Watch this Scientific Journal Video about Ground State Depletion Super-resolution Imaging in Mammalian Cells at JoVE.com

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 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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Long-term Imaging Mammalian Cells using Wide-Field Microscopy

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...