Name: Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy (Video) | JoVE Journal
Uploaded: 2014-03-24T20:00:00.000+00:00
Duration: 10 min
Description: Discover dual-color STED nanoscopy for visualizing the NK cell lytic synapse and cytoskeleton structures. This protocol achieves sub-100 nm resolution to study immune cell interactions.

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5 minutes

We thank Geoff Daniels for technical assistance. This work was funded by R01 AI067946 to J.S.O.

1. Coat Coverslips with Antibody
<ol>
	<li>Prewarm (at 37 &#176;C) 30 ml of RPMI 10% FCS media and 1 ml of BD Cytofix/Cytoperm.</li>
	<li>Prepare a solution of 5 &#956;g/ml of purified antibody in phosphate buffered saline (PBS). For activation of the NK92 cell line, use of anti-CD18&#160; and anti-NKp30 is recommended.
	<ol>
		<li>Mark one approximately dime sized circle for each condition on a #1.5 coverslip using a PAP pen. For a dual color experiment, there should be four conditions: unstained, dual stained, and two single stained conditions. Dispense 200 &#181;l of antibody solution in each region and incubate at 37 &#176;C for 30 min.</li>
		<li>Wash coverslips by gently immersing each one in 50 ml of PBS in a 50 ml conical tube at room temperature. Washing should occur immediately prior to the addition of cells and care should be taken to avoid antibody drying on the coverslip.</li>
	</ol>
	</li>
</ol>
2. Activate NK Cells on Coverslips; Fix and Permeabilize
<ol>
	<li>Isolate 5 x 105 NK92 cells per condition. Centrifuge and decant supernatant. Wash once with 10 ml prewarmed media from step 1.1. Centrifuge and decant supernatant.
	<ol>
		<li>Resuspend cells in prewarmed media from step 1.1 at a concentration of 2.5 x 106/ml.</li>
		<li>Gently decant 200 &#181;l to the center of the region created in section&#160;1.2.1. Incubate at 37 &#176;C for 20 min at 5% CO2. (Note: this time can be extended or decreased depending on the biological function of interest. For NK cell granule polarization, 20 min is sufficient).</li>
	</ol>
	</li>
	<li>Following incubation of cells, gently wash coverslips by immersing each in 50 ml of room temperature PBS in a 50 ml conical tube.</li>
	<li>Add 1 &#181;l of Triton X-100 to 1 ml of prewarmed Fix/Perm solution from step 1.1 and vortex thoroughly. Fix and permeabilize by adding 200 &#181;l of Fix/Perm buffer (step 2.3) to cells. Incubate for 10 min in the dark at room temperature.</li>
</ol>
3. Stain Cells
<ol>
	<li>Prepare staining buffer: Phosphate buffered saline (PBS), 1% BSA, 0.1% Saponin.
	<ol>
		<li>Prepare solution of primary antibody in 200 &#181;l staining buffer (see step&#160;3.1). (Note: antibody should be titrated prior to use). Avoid the use of primary antibody that is raised in the same species used to coat the coverslip (step 1.2). Also avoid Strepatividin-biotin linkages for STED imaging.</li>
		<li>Following section 2.3.1, gently wash coverslips in 50 ml staining buffer. Dab edges of PAP-pen region with cotton swab to remove excess buffer. Apply antibody solution created in section 3.1.1. Incubate 30 min in the dark at room temperature. (Recommended: incubate coverslips in slide box with a moist paper towel to maintain humidity).</li>
	</ol>
	</li>
	<li>Prepare solution of secondary antibody in 200 &#181;l staining buffer. Recommended fluorophores are Alexa Fluor 488, Pacific Orange, and V500. Generally, a 1:200 dilution is suitable for STED imaging.
	<ol>
		<li>Gently wash coverslips in 50 ml staining buffer. Dab edges of PAP-pen region with cotton swab to remove excess buffer. Apply secondary antibody solution. Incubate 30 min in the dark at room temperature.</li>
	</ol>
	</li>
	<li>Repeat washing and staining for additional proteins of interest. If detecting F-actin with Phalloidin, this can be included with secondary antibody, generally at a 1:200 dilution.</li>
</ol>
4. Mount Coverslips on Slides
<ol>
	<li>Prepare mounting medium. Note: Prolong or Prolong Gold are preferable. VECTASHIELD must be avoided, as it is not compatible with STED. 2,2-thiodioethanol must be avoided if Phalloidin is used. Mowiol is acceptable.</li>
	<li>Place approximately 10-20 &#181;l of mounting medium on a slide. Invert coverslip (cell-side down) and mount coverslip gently, taking care to avoid introduction of air bubbles. Incubate slides for 24 hr (coverslip up) prior to imaging.</li>
	<li>Seal edges of coverslip with nail polish.</li>
</ol>
5. Experimental Setup
<ol>
	<li>Initiate required lasers and software. Initiate STED depletion laser at 100% power. Align STED laser, which in the case of commercial systems is often an automated procedure.</li>
	<li>Focus the sample, beginning with single stained control, on the microscope using eyepieces.</li>
</ol>
6. Optimization of Settings
<ol>
	<li>Scan the first channel and optimize laser power, excitation beam position and detector range. If possible, avoid a gain of &#62;100. Capture the image in confocal to optimize settings. Line and/or frame averaging will increase resolution. Check for pixel saturation. Note: Some saturation is acceptable in confocal as application of STED will reduce the intensity of emission. For STED, an optimal pixel size will be below 30 nm however better resolution will be obtained with smaller pixel sizes. The size of the region of interest being imaged will dictate the lower limit of pixel size. Smaller pixel sizes may increase photobleaching.
	<ol>
		<li>Apply STED depletion beam and capture image, starting with 50% depletion laser power. If an improvement in resolution is seen, more depletion laser power can be applied. At this stage, it may be necessary to adjust excitation laser power, line average, and/or gain.</li>
		<li>Apply time gating to reduce background (minimum 0.3 nsec). Adjust settings until an improvement in resolution over confocal can be seen. Resolution can be approximated by estimating full width half maximum (FWHM). &#160;This represents the distance at the half maximal intensity of a Gaussian peak created by drawing a line profile across the structure of interest, and is a widely used method of estimating resolution.</li>
		<li>Once the first channel is satisfactory, initiate a second sequence for sequential scanning. In general, it is best to scan the longer wavelength fluorophore first. Repeat step 6.1 on second channel.</li>
	</ol>
	</li>
	<li>Confirm lack of spectral overlap by imaging single stained controls with both scan sequences. Mild spectral overlap can be corrected using spectral un-mixing features in the software, however should be avoided wherever possible.</li>
</ol>
7. Image Acquisition
<ol>
	<li>Acquire images. For quantitative imaging, it is recommended to obtain at least 20 images/condition.&#160; The exact number, however, should be defined according to the experimental question in concert with a statistical approach such as sample size calculation. Save experiment.</li>
</ol>
8. Deconvolution
<ol>
	<li>Open file with deconvolution software or batch processor. Check parameters for each channel using software. Confirm each channel&#39;s excitation and emission spectra, STED depletion emission, and imaging direction (up or down, if the image is 3-dimensional) in particular.</li>
	<li>Deconvolve using the deconvolution wizard. Default settings are generally adequate, however signal to noise ratio (SNTR) will vary from fluorophore to fluorophore and will need to be determined for each channel and each experiment individually.</li>
</ol>

<ol><li>Stinchcombe, J. C., Bossi, G., Booth, S., Griffiths, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+immunological+synapse+of+CTL+contains+a+secretory+domain+and+membrane+bridges%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">The immunological synapse of CTL contains a secretory domain and membrane bridges.</a> Immunity. 15 (5), 751-761 (2001).</li>
<li>Grakoui, A., Bromley, S. K., Sumen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+immunological+synapse%3A+a+molecular+machine+controlling+T+cell+activation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The immunological synapse: a molecular machine controlling T cell activation.</a> Science. 285 (5425), 221-227 (1999).</li>
<li>Monks, C. R., Freiberg, B. A., Kupfer, H., Sciaky, N., Kupfer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+segregation+of+supramolecular+activation+clusters+in+T+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Three-dimensional segregation of supramolecular activation clusters in T cells.</a> Nature. 395 (6697), 82-86 (1998).</li>
<li>Orange, J. S., Harris, K. E., Andzelm, M. M., Valter, M. M., Geha, R. S., Strominger, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mature+activating+natural+killer+cell+immunologic+synapse+is+formed+in+distinct+stages%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">The mature activating natural killer cell immunologic synapse is formed in distinct stages.</a> Proc. Natl. Acad. Sci. U.S.A. 100 (24), 14151-14156 (2003).</li>
<li>Treanor, B., Lanigan, P. M., Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microclusters+of+inhibitory+killer+immunoglobulin-like+receptor+signaling+at+natural+killer+cell+immunological+synapses%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Microclusters of inhibitory killer immunoglobulin-like receptor signaling at natural killer cell immunological synapses.</a> J. Cell Biol. 174 (1), 153-161 (2006).</li>
<li>Abeyweera, T. P., Merino, E., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibitory+signaling+blocks+activating+receptor+clustering+and+induces+cytoskeletal+retraction+in+natural+killer+cells%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells.</a> J. Cell Biol. 192 (4), 675-690 (2011).</li>
<li>Beemiller, P., Jacobelli, J., Krummel, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integration+of+the+movement+of+signaling+microclusters+with+cellular+motility+in+immunological+synapses%5BTitle%5D)+AND+%22Nat.+Immunol%22%5BJournal%5D)">Integration of the movement of signaling microclusters with cellular motility in immunological synapses.</a> Nat. Immunol. 13 (8), 787-795 (2012).</li>
<li>Campi, G., Varma, R., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Actin+and+agonist+MHC-peptide+complex-dependent+T+cell+receptor+microclusters+as+scaffolds+for+signaling%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling.</a> J. Exp. Med. 202 (8), 1031-1036 (2005).</li>
<li>Mossman, K. D., Campi, G., Groves, J. T., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+TCR+signaling+from+geometrically+repatterned+immunological+synapses%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Altered TCR signaling from geometrically repatterned immunological synapses.</a> Science. 310 (5751), 1191-1193 (2005).</li>
<li>Varma, R., Campi, G., Yokosuka, T., Saito, T., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T+cell+receptor-proximal+signals+are+sustained+in+peripheral+microclusters+and+terminated+in+the+central+supramolecular+activation+cluster%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster.</a> Immunity. 25 (1), 117-127 (2006).</li>
<li>Yokosuka, T., Sakata-Sogawa, K., Kobayashi, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Newly+generated+T+cell+receptor+microclusters+initiate+and+sustain+T+cell+activation+by+recruitment+of+Zap70+and+SLP-76%5BTitle%5D)+AND+%22Nat.+Immunol%22%5BJournal%5D)">Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.</a> Nat. Immunol. 6 (12), 1253-1262 (2005).</li>
<li>Toomre, D., Bewersdorf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+wave+of+cellular+imaging%5BTitle%5D)+AND+%22Annu.+Rev.+Cell.+Dev.+Biol%22%5BJournal%5D)">A new wave of cellular imaging.</a> Annu. Rev. Cell. Dev. Biol. 26, 285-314 (2010).</li>
<li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+guide+to+super-resolution+fluorescence+microscopy%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">A guide to super-resolution fluorescence microscopy.</a> J. Cell Biol. 190 (2), 165-175 (2010).</li>
<li>Huang, B., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+fluorescence+microscopy%5BTitle%5D)+AND+%22Annu.+Rev.+Biochem%22%5BJournal%5D)">Super-resolution fluorescence microscopy.</a> Annu. Rev. Biochem. 78, 993-1016 (2009).</li>
<li>Mace, E. M., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+views+of+the+human+NK+cell+immunological+synapse%3A+recent+advances+enabled+by+super-+and+high-resolution+imaging+techniques%5BTitle%5D)+AND+%22Front.+Immunol%22%5BJournal%5D)">New views of the human NK cell immunological synapse: recent advances enabled by super- and high-resolution imaging techniques.</a> Front. Immunol. 3, 421 (2012).</li>
<li>Hell, S. W., Wichmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Breaking+the+diffraction+resolution+limit+by+stimulated+emission%3A+stimulated-emission-depletion+fluorescence+microscopy%5BTitle%5D)+AND+%22Opt.+Lett%22%5BJournal%5D)">Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.</a> Opt. Lett. 19 (11), 780-782 (1994).</li>
<li>Klar, T. A., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subdiffraction+resolution+in+far-field+fluorescence+microscopy%5BTitle%5D)+AND+%22Opt.+Lett%22%5BJournal%5D)">Subdiffraction resolution in far-field fluorescence microscopy.</a> Opt. Lett. 24 (14), 954-956 (1999).</li>
<li>Klar, T. A., Jakobs, S., Dyba, M., Egner, A., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+microscopy+with+diffraction+resolution+barrier+broken+by+stimulated+emission%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission.</a> Proc. Natl. Acad. Sci. U.S.A. 97 (15), 8206-8210 (2000).</li>
<li>Rankin, B. R., Moneron, G., Wurm, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanoscopy+in+a+living+multicellular+organism+expressing+GFP%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Nanoscopy in a living multicellular organism expressing GFP.</a> Biophys. J. 100 (12), 63-65 (2011).</li>
<li>Willig, K. I., Kellner, R. R., Medda, R., Hein, B., Jakobs, S., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanoscale+resolution+in+GFP-based+microscopy%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Nanoscale resolution in GFP-based microscopy.</a> Nat. Methods. 3 (9), 721-723 (2006).</li>
<li>Rak, G. D., Mace, E. M., Banerjee, P. P., Svitkina, T., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Natural+killer+cell+lytic+granule+secretion+occurs+through+a+pervasive+actin+network+at+the+immune+synapse%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">Natural killer cell lytic granule secretion occurs through a pervasive actin network at the immune synapse.</a> PLoS Biol. 9 (9), (2011).</li>
<li>Mace, E. M., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual+channel+STED+nanoscopy+of+lytic+granules+on+actin+filaments+in+natural+killer+cells%5BTitle%5D)+AND+%22Commun.+Integr.+Biol%22%5BJournal%5D)">Dual channel STED nanoscopy of lytic granules on actin filaments in natural killer cells.</a> Commun. Integr. Biol. 5 (2), 184-186 (2012).</li>
<li>Singh, H., Lu, R., Rodriguez, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+and+quantification+of+cardiac+mitochondrial+protein+clusters+with+STED+microscopy%5BTitle%5D)+AND+%22Mitochondrion%22%5BJournal%5D)">Visualization and quantification of cardiac mitochondrial protein clusters with STED microscopy.</a> Mitochondrion. 12 (2), 230-236 (2012).</li>
<li>Tonnesen, J., Nagerl UV, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Superresolution+imaging+for+neuroscience%5BTitle%5D)+AND+%22Exp.+Neurol%22%5BJournal%5D)">Superresolution imaging for neuroscience.</a> Exp. Neurol. 242, 33-40 (2013).</li>
<li>Kempf, C., Staudt, T., Bingen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+multicolor+STED+nanoscopy+of+presynaptic+proteins+in+the+calyx+of+held%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Tissue multicolor STED nanoscopy of presynaptic proteins in the calyx of held.</a> PLoS One. 8 (4), (2013).</li>
<li>Jans, D. C., Wurm, C. A., Riedel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STED+super-resolution+microscopy+reveals+an+array+of+MINOS+clusters+along+human+mitochondria%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (22), 8936-8941 (2013).</li>
<li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surpassing+the+lateral+resolution+limit+by+a+factor+of+two+using+structured+illumination+microscopy%5BTitle%5D)+AND+%22J.+Microsc%22%5BJournal%5D)">Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.</a> J. Microsc. 198 (2), 82-87 (2000).</li>
<li>Gustafsson, M. G., Shao, L., Carlton, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+resolution+doubling+in+wide-field+fluorescence+microscopy+by+structured+illumination%29%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination).</a> Biophys. J. 94 (12), 4957-4970 (2008).</li>
<li>Schermelleh, L., Carlton, P. M., Haase, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subdiffraction+multicolor+imaging+of+the+nuclear+periphery+with+3D+structured+illumination+microscopy%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.</a> Science. 320 (5881), 1332-1336 (2008).</li>
<li>Betzig, E., Patterson, G. H., Sougrat, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 13 (5793), 1642-1645 (2006).</li>
<li>Hess, S. T., Girirajan, T. P., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophys. J. 91 (11), 4258-4272 (2006).</li>
<li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy.</a> Nat. Methods. 3 (10), 793-795 (2006).</li>
<li>Cdc42-interacting protein-4 functionally links actin and microtubule networks at the cytolytic NK cell immunological. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22J.+Exp.+Med%22">J. Exp. Med</a>. Banerjee, P. P., Pandey, R., Zheng, R., Suhoski,, Monaco-Shawver, L., Orange, J. S. 204 (10), 2305-2320 (2007).</li>
<li>Vicidomini, G., Moneron, G., Han, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sharper+low-power+STED+nanoscopy+by+time+gating%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Sharper low-power STED nanoscopy by time gating.</a> Nat. Methods. 8 (7), 571-573 (2011).</li>
</ol>

The authors declare that they have no competing financial interests. Partial publication cost of this article was defrayed by Leica Microsystems, Inc.

The improvement in resolution over confocal will be somewhat dependent upon factors which cannot be controlled. These factors include minor aberrations in cover slip thickness and inconsistencies in mounting media. It is important to keep the temperature and humidity in the imaging room as consistent as possible, and the STED beam should be realigned approximately every 60 min. As mentioned in Procedures, use of VECTASHIELD mounting medium must be avoided, as this is not compatible with STED. In addition to which, one should always use #1.5 cover slips, and if available, use those which have been verified to a specific thickness.
One modification of the approach described here is to image additional channels in confocal, using fluorophores that emit at a longer wavelength than the STED beam. In this way, one can image up to four channels (two in confocal, two in STED). If taking this approach, however, the channels with fluorophores emitting above the STED depletion laser will need to be imaged first, as application of the STED beam will deplete photons in these channels. One advantage to this technique is the application of time gating, which will also improve resolution in confocal by eliminating emission from photons with short lifetimes34. In particular, the use of time gating, the timing of emission detectors to correspond with pulsed excitation STED, will decrease background fluorescence from reflection off coverslip glass when imaging close to it. Even in an experiment not suitable for STED, if using a pulsed excitation source, time gating can be a useful tool for improving resolution in confocal.
There are various modifications that can be utilized to improve resolution in STED. One is to decrease the size of the pinhole from the standard 1 Airy unit, although this will also decrease the amount of light reaching the sample. This can be compensated for by increasing laser power or gain. Another is to increase line average, which will increase the amount of information gathered for each photon, improving resolution. Again, however, this may be at the cost of photobleaching of the sample, so a balance will need to be struck between resolution and bleaching. Similarly, use of fluorescent proteins such as GFP will require careful optimization to avoid bleaching. This may be accomplished by decreasing STED laser power if necessary. Longer time points will also allow for greater photon recovery and reduce bleaching. Correction for photobleaching should be accounted for when analyzing live STED.
Of course, imaging in 3 dimensions in STED is also possible, and will also give an improvement over conventional confocal imaging. This is particularly true if it is done in combination with deconvolution, although care should be taken to correct for drift that occurs during imaging multiple planes in the z-axis. If using Huygens software to deconvolve, this correction is obtained using the "stabilize image" feature. Using this approach, resolution in the z-axis will be improved. This is a great improvement over conventional confocal imaging, which has poor axial resolution, and even over just STED itself, which also has relatively poor z-axis resolution. While acquiring multiple stacks in STED, care must be taken to avoid bleaching of the sample, and if necessary one can reduce line averaging or laser power intensity in order to do so. Again, it should be noted that if imaging other fluorophores that are not suitable for STED, application of the depletion beam in the first sequential scan would prevent emission from these channels. Therefore, a mixed STED/confocal approach (when using confocal scanning in channels that emit at a wavelength greater than 592 nm) will unfortunately not be suitable for 3D.
To summarize, we have chosen STED as an approach due to its relative ease of application and improvement in resolution over standard confocal imaging. For imaging the immune synapse, it has proven an effective and valuable technique that allows us to see details in F-actin architecture not possible at resolution over 200 nm. While many of these details seem subtle, they can have a profound effect on NK cell function. Thus, we are applying the latest nanoscopic imaging technology to deriving information that is critical for maintaining human health.

The immunological synapse is a complex milieu of signaling proteins and cytoskeletal elements. The cytolytic synapse was originally described as having a &#34;bulls-eye&#34; like structure with a ring of actin and adhesion molecules surrounding a central secretory domain1-4. However, we now know that it consists of microscopic domains of active signaling that require continuous dynamic cytoskeletal reorganization for function5-11. Much of the information that we now have about the synapse has been derived from microscopy, and immunologists have been early adopters of cutting-edge imaging technology.
One such novel technology is super-resolution microscopy. Conventional light microscopy is spatially limited by the diffraction barrier of light, which sets the lower limit of resolution for all fluorescence microscopy, including confocal, at approximately 200 nm. In recent years, several techniques have been developed that allow resolution below the diffraction barrier. These include stimulation emission depletion microscopy (STED), structured illumination microscopy (SIM), stochastically resolved microscopy (STORM), and photoactivatable light microscopy (PALM). These techniques have been reviewed in detail elsewhere12-15, but are outlined below. Subdiffraction limited resolution is generated in unique ways in each system. The selection of a super-resolution technique, therefore, should be dictated by the experiment and experimental system of interest.
STED super resolution is achieved using a high-intensity torroidal depletion beam that selectively &#34;silences&#34; fluorescence around each fluorophore of interest following excitation, resulting in subdiffraction limited fluorescence microscopy16-18. One advantage of STED is that image acquisition is rapid and requires relatively little post-processing. While dye selection is dictated by the spectral position of the depletion beam, which in the commercially available system is situated at 592 nm, several commercially available dyes are available that make combinations of two fluorophores possible. In addition, commonly used fluorescent reporters such as GFP can be imaged, making live cell experiments possible19,20.
We have previously used STED to identify and quantify regions of F-actin hypodensity that are utilized by NK cells for degranulation21,22. We propose that STED is a good choice for imaging the immune synapse due to its relatively flexible availability of fluorophores and superior improvement in resolution in the x-y axis. In addition, on the commercially available STED system utilized for these experiments, the use of a high-speed (12,000 Hz) resonance scanner allows for rapid acquisition of images with minimal damage to samples. Limited flexibility in dye selection is considered a disadvantage of STED12, however dual color STED is relatively straightforward with several commercially available fluorophores. The integration of STED with a laser scanning confocal microscope also allows for additional confocal imaging in combination with STED, so while STED is limited to two channels, additional structures can be imaged in confocal with resolution of approximately 200 nm (E. Mace, unpublished observations). While we describe the use of STED for imaging immune cells, this technology is applied to a variety of cell types, including neural cells, and for visualizing a variety of cell structures23-26.
SIM uses a different approach to generate subdiffraction-limited images. By visualizing known periodic excitation patterns, information can then be obtained about the unknown structure being studied following mathematical transformation27. This yields an increase in resolution to ~100 nm laterally28,29. The advantage of SIM is that it is compatible with all standard confocal dyes and probes, however the disadvantage is that it is much slower to acquire images and these require lengthy post-processing12. This also limits its use for live cell imaging.
Finally, super-resolution images can be generated by stochastic photo-switching of fluorophores. This approach is exploited in photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). By scanning multiple camera frames and localizing randomly activated molecules that are turned &#34;on&#34; and &#34;off&#34; over time, images with 20-30 nm resolution are generated from accumulated frames30-32 . The trade-off for this resolution is the time required to acquire images.
Here we show, in detail, the protocol for preparing and imaging dual color samples in STED. In this system, excitation is with a pulsed, tunable, white light laser. Due to the nature of the pulsed excitation beam, time gating of detection is made possible and further increases resolution. In addition, the system is equipped with gadolinium hybrid (HyD) detectors, which are more sensitive than conventional photomultiplier tubes, thus allowing for lower laser power requirements. The depletion beam for STED is applied continuously and is tuned to 592 nm, which will dictate the choice of dyes available for two color STED. Commonly used dye combinations generally include one excitable by 488 nm (such as Alexa Fluor 488, Oregon Green, DyLights green, or Chromeo 488) and one excitable by 458 nm (such as Pacific Orange or Horizon V500). Thus, while detection of the two dyes will be in a similar range (and both are accessible by the depletion laser), excitation will occur with different wavelengths. With a tunable white light laser and tunable detectors, maximizing signal while eliminating spectral overlap is made fairly easy. As such, we have had good success with combinations of commercially available dyes, such as Pacific Orange and Alexa Fluor 488 (used here).&#160; Our protocol is tailored towards and describes the evaluation of human NK cells as that represents the historical focus of our laboratory.&#160;We are specifically utilizing the NK92 cell line in this example as that is one we have regularly applied in our experimental work21,33.

<table><tbody><tr><td>#1.5 cover slips</td><td>VWR</td><td>48393-172</td><td></td></tr><tr><td>BD Cytofix/Cytoperm</td><td>BD Biosciences</td><td>554722</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>Cotton tipped applicator</td><td>Fisher Scientific</td><td>S450941</td><td></td></tr><tr><td>Falcon centrifuge tubes (50 ml)</td><td>VWR</td><td>352070</td><td></td></tr><tr><td>Fetal calf serum (FCS) (500 ml)</td><td>Atlantic Biologicals</td><td>S11050</td><td></td></tr><tr><td>Goat anti-rabbit Pacific Orange</td><td>Life Technologies</td><td>P31584</td><td></td></tr><tr><td>Laboratory tissue wipers</td><td>VWR</td><td>82003-820</td><td></td></tr><tr><td>Nail polish</td><td>VWR</td><td>100491-940</td><td></td></tr><tr><td>NK-92 cells</td><td>ATCC</td><td>CRL-2407</td><td></td></tr><tr><td>Phalloidin Alexa Fluor 488</td><td>Life Technologies</td><td>A12379</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>Prolong anti-fade reagent</td><td>Life Technologies</td><td>P7481</td><td></td></tr><tr><td>Purified anti-CD18</td><td>Biolegend</td><td>301202</td><td></td></tr><tr><td>Purified anti-NKp30</td><td>Biolegend</td><td>325202</td><td></td></tr><tr><td>Purified anti-perforin</td><td>Biolegend</td><td>308102</td><td></td></tr><tr><td>RPMI 1640 medium (500 ml)</td><td>Life Technologies</td><td>11875-093</td><td></td></tr><tr><td>Saponin from Quillaja bark</td><td>Sigma</td><td>S4521</td><td></td></tr><tr><td>Super PAP pen</td><td>Life Technologies</td><td>008899</td><td></td></tr><tr><td>Triton X-100</td><td>Electron Microscopy Sciences</td><td>22142</td><td></td></tr><tr><td>Huygens deconvolution software</td><td>SVI</td><td>Contact company</td><td></td></tr><tr><td>Leica SP8 TCS STED microscope</td><td>Leica Microsystems</td><td>contact company</td><td></td></tr></tbody></table>

visualization of the immunological synapse by dual color time-gated stimulated emission depletion (sted) nanoscopy

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Clearly, a primary goal of super-resolution imaging will be an improvement over conventional confocal microscopy. However, there are some common pitfalls that may lead to suboptimal resolution. These require that each experiment be optimized individually. In our representative experiment, we are imaging the F-actin network in an NK cell activated by antibody bound to glass. Common causes of (and corrections for) a lack of improved resolution of STED over confocal are as follows:
<ol>
	<li>Under-sampling (Figure 1a). This may lead to graininess and loss of pixel information, as shown by poor resolution of F-actin filaments. Increased line or frame averaging can often correct this.</li>
	<li>Bleaching and/or over-sampling (Figure 1b). This may be caused by lengthy pixel dwell time as a result of excessive line averaging. Alternatively, it may be a result of over-scanning of the image prior to acquisition, including over-use of the depletion laser. This commonly results in hazy or fuzzy images. This can be corrected by scanning the field of interest only minimally before acquiring or, if possible, increasing laser scan speed. If the problem persists, the depletion laser power can be reduced.</li>
</ol>
By achieving the correct balance of pixel dwell time, excitation laser power, and depletion laser power, an image with improved resolution and sufficient information can be generated (Figure 1c). Resolution can be further improved by the use of deconvolution (Figure 1d). When acquisition is optimized, deconvolution will improve resolution both qualitatively and quantitatively and sub-100 nm resolution should be routinely attainable.
<img alt="Fluorescence microscopy images showing cellular structure enhancement through deconvolution." fo:content-width="5in" fo:src="/files/ftp_upload/51100/51100fig1highres.jpg" src="/files/ftp_upload/51100/51100fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Optimization of acquisition and common pitfalls of STED imaging. NK92 cells were activated on anti-CD18 and -NKp30 coated glass for 20 min then fixed, permeabilized and stained for F-actin with Phalloidin Alexa Fluor 488. a) An example of loss of image information due to under-sampling. b) An example of loss of resolution due to bleaching/over-sampling c) conditions optimized d) optimized conditions lead to greater improvement in resolution with deconvolution. Scale bar = 5 μm.

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

The overall goal of this procedure is to visualize structures such as F actin in the NK cell lytic synapse using TED microscopy. This is accomplished by first recapitulating the synapse on glass using activating antibody. The second step of the procedure is to fix perme and stain the cells for structures of interest.Next, the slides are imaged by laser scanning confocal microscopy, followed by application of stead depletion for increased image resolution. The final step is to process the images using a deconvolution algorithm. Ultimately, results can show sub 100 nanometer resolution of cellular structures thanks to dual color stead and endoscopy.This method can help answer key questions in the cell biology field, such as a relationship between the cytoskeleton and exocytosis. In our case, we are interested in the secretion of specialized lysosomes termed lytic granules by natural killer cells. Generally, individuals new to this method will struggle because there are multiple variables that can be adjusted in the application of the TED depletion laser.An inappropriate application of the depletion beam will result in a worsening of instead of improvement in resolution In preparation, warm 30 milliliters of RPMI media with 10%FCS and separately. One milliliter of BD CY effects cyop perm both to 37 degrees Celsius. Then begin the process of coating the cover slips with antibodies.Take number 1.5 cover slips and using a PAP pen. Make dime-sized squares on the cover slips. Prepare one square for each tested condition.Load each circle with 200 microliters of antibody at five micrograms per milliliter in PBS. For NK 92 cell activation, use anti CD 18 and anti NK P 30 antibodies. Incubate the loaded cover slips at 37 degrees Celsius for half an hour, and then wash them off with a dip in room temperature PBS proceed immediately with adding the cells for each tested condition, 500, 000 cells must be ready to use.Wash the cells in 10 milliliters of the prewarm media and then resuspend them in the same warmed media. At 2.5 million cells per milliliter with the cells fully in suspension, decant 200 microliters into each antibody square on the cover Slips incubate the cover slips with cells at 37 degrees Celsius with 5%CO2 in this case. 20 minutes is enough time to polarize the granules in the NK cells after the incubation wash, the cover slips gently in PBS at room temperature.Begin with adding a microliter of TRITTON X 100 to a milliliter of warm BD CY effects cyto perm solution. Then vortex. Apply 200 microliters of this solution to each condition square on the cover slips.Then place the cells in the dark at room temperature and wait 10 minutes. When the incubation is finished, gently wash the cover slips in staining buffer. Then dry the edges of the surrounded regions with a cotton swab.Next, add 200 microliters of the primary antibody in staining buffer to the surrounded regions, and let the cover slips incubate in the dark for half an hour. Some good secondary antibodies for stead imaging include Alexa floor 4 88 Pacific orange and Horizon V 500. Each of these work well at a one to 200 dilution.After the incubation wash, the cover slips in staining, buffer, and dab them dry. As before, apply 200 microliters of the secondary antibody to each condition square. Then let them sit in the dark for another 30 minutes.At this point, it's possible to add additional antibodies after additional washes. For example, Phin can be applied at one to 200 to detect f actin. For a mounting medium, choose prolong over vector shield, which is not compatible with stead.If phin staining is used, avoid using two two thio ethanol while however is okay, now apply 10 to 20 microliters of mounting medium to a slide. With the cover slip inverted, gently lower it into the medium, avoiding air bubbles. Then incubate the slide for 24 hours.With the cover, slip up the next day, seal the cover slip with nail polish and proceed with imaging. Start up the lasers and software. The stead depletion laser should warm up at 100%power and be aligned.Using the eyepiece focus on the sample, then turn to the software. Scan the first channel, the one with the longest wavelength. Floor four, optimize the laser's power, the excitation beam position, and the detector range.Avoid setting above 100. Next, capture a confocal image and check for pixel. Saturation, line, and frame averaging will both enhance pixel resolution.The goal is to be below 30 nanometers per pixel. Some saturation is acceptable for stead. Next, apply the stead depletion beam at 50%power and capture an image.If the resolution improves, more power can be applied. Further, adjusting the excitation laser power or the line average or the gain could also improve the resolution. To reduce the background apply 0.3 nanoseconds of time gating and adjust upward.The new resolution should be estimated using a full half width maximum calculation. Once satisfied, repeat these steps with the second channel when all the channels are tweaked. Check for spectral overlap by imaging single stain controls with all the scan sequences.Mild overlap can be corrected with spectral unmixing by the software but is best avoided or kept to a minimum. Now, acquire images for quantitative imaging. Obtain at least 20 images per condition according to the experimental conditions.To devolve the saved images, open the files in the appropriate software. Check the parameters of each channel, specifically the excitation and admission spectra, the stead depletion emission, and the imaging direction. Then apply the software's deconvolution calculations.Default settings usually suffice, but the signal to noise will vary between floor force, so that parameter needs to be set manually for the experiment. There are a few common pitfalls that may lead to suboptimal resolution with stead. One is under sampling.This leads to graininess and a loss of pixel information such as in these f actin filaments. Increasing the line or frame averaging often remedies this issue. Another issue is bleaching or oversampling caused by lengthy pixel.Dwell time usually due to excessive line averaging over scanning prior to image acquisition may also be the culprit and can be corrected using a smaller scan before acquiring images or by using a higher scan speed. Reducing the laser power can also help with everything working. The image will improve dramatically.Deconvolution will even further improve the image. Both quantitatively and qualitatively. Sub 100 nanometer resolution should be routinely obtained While attempting this procedure.It's important to remember to perform stead beam alignment prior to each experiment and approximately once an hour thereafter following this procedure. Other methods like quantitative analysis can be performed in order to answer additional questions regarding colocalization, subcellular localization and molecular interactions.

visualization-immunological-synapse-dual-color-time-gated-stimulated

Research

JoVE Journal

Immunology and Infection

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Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

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Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

This video demonstrates a technique for visualizing the formation of the immunological synapse in natural killer (NK) cells on a supported lipid bilayer (SLB). The SLB is first labeled with a fluorophore-tagged NK cell activating receptor ligand, followed by incubation with NK cells. Subsequently, the cells are stained for polymerized actin and perforin-positive lytic granules. This approach enables the visualization of synapse formation using stimulated emission depletion (STED) microscopy.

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

This video demonstrates live-cell imaging of immunological synapse topology, investigating the interactions between T lymphocytes and epithelial cells carrying fluorescent antigens. Fluorescence microscopy reveals red cytoplasm displacement and yellow membrane rings in endothelial cells, indicating the formation of immunological synapses.

Assessing Immunological Synapse Topology through Live-Cell Imaging

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals.  Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform  2D dual color dSTORM  images of microtubules and intermediate filaments in fixed cells...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

Imaging the Human Immunological Synapse

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Discover dual-color STED nanoscopy for visualizing the NK cell lytic synapse and cytoskeleton structures. This protocol achieves sub-100 nm resolution to study immune cell interactions.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

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