We would like to acknowledge the advice and guidance from Dr. Vytas Bindokas at the University of Chicago. We thank the Integrated Light Microscopy Core Facility at the University of Chicago for supporting and maintaining the lattice light-sheet microscope. This work was supported by NIH New Innovator Award 1DP2AI144245 and NSF Career Award 1653782 (To J.H.). J.R. is supported by the NSF Graduate Research Fellowships Program.

5C.C7 TCR-transgenic RAG2 knockout mice in B10.A background were used in this study according to a protocol approved by the Institutional Animal Care and Use Committee of the University of Chicago.
1. Harvest and Activate T Cells
NOTE: This part of the protocol is based on previous protocols. See citations for further detail20,21.
<ol>
	<li>Euthanize a 10-12 week-old 5C.C7 transgenic mouse of either sex (~...

<ol>
	<li>Poulter, N. S., Pitkeathly, W. T. E., Smith, P. J., Rappoport, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Physical+Basis+of+Total+Internal+Reflection+Fluorescence+(TIRF)+Microscopy+and+Its+Cellular+Applications.">The Physical Basis of Total Internal Reflection Fluorescence (TIRF) Microscopy and Its Cellular Applications.</a> Methods in Molecular Biology. 1251, 1-23 (2015).</li><li>Mattheyses, A. L., Simon, S. M., Rappoport, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+with+total+internal+reflection+fluorescence+microscopy+for+the+cell+biologist.">Imaging with total internal reflection fluorescence microscopy for the cell biologist.</a> Journal of Cell Science. 123, 3621-3628 (2010).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+Internal+Reflection+Fluorescence+Microscopy.">Total Internal Reflection Fluorescence Microscopy.</a> Methods in Cell Biology. 89, 169-221 (2008).</li><li>Rust, M. J., 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=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nature Methods. 3 (10), 793-796 (2006).</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., Wichmann, J. <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+resolution+limit+by+stimulated+emission:+stimulated-emission-depletion+fluorescence+microscopy.">Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.</a> Optics Letters. 19 (11), 780 (1994).</li><li>Shroff, H., White, H., Betzig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivated+Localization+Microscopy+(PALM)+of+Adhesion+Complexes.">Photoactivated Localization Microscopy (PALM) of Adhesion Complexes.</a> Current Protocols in Cell Biology. 58 (1), 1-28 (2013).</li><li>Liu, Z., Lavis, L. D., Betzig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Live-Cell+Dynamics+and+Structure+at+the+Single-Molecule+Level.">Imaging Live-Cell Dynamics and Structure at the Single-Molecule Level.</a> Molecular Cell. 58 (4), 644-659 (2015).</li><li>Ji, N., Shroff, H., Zhong, H., Betzig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+speed+and+resolution+of+light+microscopy.">Advances in the speed and resolution of light microscopy.</a> Current Opinion in Neurobiology. 18 (6), 605-616 (2008).</li><li>. Atomic Resolution Imaging with a sub-50 pm Electron Probe Available from: <a target="_blank" href="https://escholarship.org/uc/item/3cs0m4vr">https://escholarship.org/uc/item/3cs0m4vr</a> (2019)</li><li>Kizilyaprak, C., Daraspe, J., Humbel, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+ion+beam+scanning+electron+microscopy+in+biology.">Focused ion beam scanning electron microscopy in biology.</a> Journal of Microscopy. 254 (3), 109-114 (2014).</li><li>Chen, B. C., 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=Lattice+light-sheet+microscopy:+Imaging+molecules+to+embryos+at+high+spatiotemporal+resolution.">Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution.</a> Science. , (2014).</li><li>Ni, J., 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=Adoptively+transferred+natural+killer+cells+maintain+long-term+antitumor+activity+by+epigenetic+imprinting+and+CD4++T+cell+help.">Adoptively transferred natural killer cells maintain long-term antitumor activity by epigenetic imprinting and CD4+ T cell help.</a> Oncoimmunology. 5 (9), 1219009 (2016).</li><li>Chaudhri, A., Xiao, Y., Klee, A. N., Wang, X., Zhu, B., Freeman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-L1+Binds+to+B7-1+Only+In+Cis+on+the+Same+Cell+Surface.">PD-L1 Binds to B7-1 Only In Cis on the Same Cell Surface.</a> Cancer Immunology Research. 6 (8), 921-929 (2018).</li><li>Forbes, C. A., Scalzo, A. A., Degli-Esposti, M. A., Coudert, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ly49C-dependent+control+of+MCMV+Infection+by+NK+cells+is+cis-regulated+by+MHC+Class+I+molecules.">Ly49C-dependent control of MCMV Infection by NK cells is cis-regulated by MHC Class I molecules.</a> PLoS pathogens. 10 (5), 1004161 (2014).</li><li>Sch&#246;neberg, J., 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=4D+cell+biology:+big+data+image+analytics+and+lattice+light-sheet+imaging+reveal+dynamics+of+clathrin-mediated+endocytosis+in+stem+cell-derived+intestinal+organoids.">4D cell biology: big data image analytics and lattice light-sheet imaging reveal dynamics of clathrin-mediated endocytosis in stem cell-derived intestinal organoids.</a> Molecular biology of the cell. 29 (24), 2959-2968 (2018).</li><li>Gao, R., 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=Cortical+column+and+whole-brain+imaging+with+molecular+contrast+and+nanoscale+resolution.">Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution.</a> Science. 363 (6424), 8302 (2019).</li><li>Cai, 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=Visualizing+dynamic+microvillar+search+and+stabilization+during+ligand+detection+by+T+cells.">Visualizing dynamic microvillar search and stabilization during ligand detection by T cells.</a> Science. 356 (6338), 3118 (2017).</li><li>Ritter, A. 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=Actin+Depletion+Initiates+Events+Leading+to+Granule+Secretion+at+the+Immunological+Synapse.">Actin Depletion Initiates Events Leading to Granule Secretion at the Immunological Synapse.</a> Immunity. , (2015).</li><li>Lim, J. F., Berger, H., Su, I. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Activation+of+Murine+Lymphocytes.">Isolation and Activation of Murine Lymphocytes.</a> Journal of visualized experiments: JoVE. (116), e54596 (2016).</li><li>Huang, J., 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=A+Single+Peptide-Major+Histocompatibility+Complex+Ligand+Triggers+Digital+Cytokine+Secretion+in+CD4++T+Cells.">A Single Peptide-Major Histocompatibility Complex Ligand Triggers Digital Cytokine Secretion in CD4+ T Cells.</a> Immunity. 39 (5), 846-857 (2013).</li><li>Irvine, D. J., Purbhoo, M. A., Krogsgaard, M., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+ligand+recognition+by+T+cells.">Direct observation of ligand recognition by T cells.</a> Nature. 419 (6909), 845-849 (2002).</li><li>Jansson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mathematical+framework+for+analyzing+T+cell+receptor+scanning+of+peptides.">A mathematical framework for analyzing T cell receptor scanning of peptides.</a> Biophysical journal. 99 (9), 2717-2725 (2010).</li><li>Mickoleit, M., 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=High-resolution+reconstruction+of+the+beating+zebrafish+heart.">High-resolution reconstruction of the beating zebrafish heart.</a> Nature Methods. 11 (9), 919-922 (2014).</li></ol>

The presented protocol was optimized for the usage of CD4+ T cells isolated from 5C.C7 transgenic mice on the LLSM instrument used, and therefore other cell systems and LLSMs may need to be optimized differently. However, this protocol shows the power of 4D imaging, as it can be used to quantify the dynamics of a surface receptor on an entire cell with the least distortion in physiological conditions. Therefore, there are many possible future applications of this technique.
A critic...

The precise dynamics of molecules trafficking and diffusing on the three-dimensional cell surface in real time have been an enigma to solve. Microscopy has always been a balance of speed, sensitivity, and resolution; if any one or two are maximized, the third is minimized. Therefore, due to the small size and immense speed with which surface receptors move, tracking their dynamics has remained a major technological challenge to the field of cell biology. For example, many studies have been conducted using total internal reflection fluorescence (TIRF) microscopy1,2,3, which has high temporal resolution, but can only image a very thin slice of the T-cell membrane (~100 nm), and therefore misses events happening farther away in the cell. These TIRF images also only showing a two-dimensional section of the cell. By contrast, super-resolution techniques, such as stochastic optical reconstruction microscopy (STORM)4, photoactivated localization microscopy (PALM)5, and stimulated emission depletion microscopy (STED)6, can overcome the Abbe diffraction limit of light. These techniques have high spatial resolution (~20 nm resolution)4,5,6,7, but they often take many minutes to acquire a full two-dimensional (2D) or three-dimensional (3D) image, and therefore the temporal resolution is lost. In addition, techniques such as STORM and PALM that rely on blinking signals may have inaccuracies in counting8,9. Electron microscopy has by far the highest resolution (up to 50 pm resolution)10; it can even be conducted three-dimensionally with focused ion beam scanning electron microscopy (FIB-SEM), resulting in up to 3 nm XY and 500 nm Z resolution11. However, any form of electron microscopy requires harsh sample preparation and can only be conducted with fixed cells or tissues, eliminating the possibility of imaging live samples over time.
Techniques to obtain the high spatiotemporal resolution required to identify the dynamics of surface and intracellular molecules in live cells in their true physiological 3D nature is only being recently developed. One of these techniques is Lattice Light-Sheet Microscopy (LLSM)12, which utilizes a structured light sheet to drastically lower photobleaching. Developed in 2014 by Nobel Laureate Eric Betzig, the high axial resolution, low photobleaching and background noise, and ability to simultaneously image hundreds of planes per field of view make LLS microscopes superior to widefield, TIRF and confocal microscopes12,13,14,15,16,17,18,19. This four-dimensional (x, y, z and time) imaging technique, while still diffraction limited (~200 nm XYZ resolution), has incredible temporal resolution (we have achieved a frame rate of about 100 fps, resulting in a 3D reconstructed cell image with 0.85 seconds per frame) for 3D spatial acquisition.
LLSM can be generally used to track real-time dynamics of any molecules within any cell at the single-molecule and single-cell level, particularly those in highly motile cells such as immune cells. For example, we show here how to use LLSM to visualize T-cell receptor (TCR) dynamics. T cells are the effector cells of the adaptive immune system. TCRs are responsible for recognizing peptide-MHC (pMHC) ligands displayed on the surface of antigen-presenting cells (APC), which determines the selection, development, differentiation, fate, and function of a T cell. This recognition occurs at the interface between T cells and APCs, resulting in localized receptor clustering to form what is called the immunological synapse. While it is known that TCRs at the immunological synapse are imperative for T-cell effector function, still unknown are the underlying mechanisms of real-time TCR trafficking to the synapse. LLSM has allowed us to visualize in real time the dynamics of TCRs before and after trafficking to the synapse with the resultant pMHC-TCR interaction (Figure 1). LLSM can therefore be used to solve current questions of the formative dynamics of TCRs and provide insights to understand how a cell distinguishes between self and foreign antigens.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td>For T cell harvest</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148-25ML</td>
			<td>For T cell culture</td>
		</tr>
		<tr>
			<td>5 mm round coverslips</td>
			<td>World Precision Instruments</td>
			<td>502040</td>
			<td>For Imaging</td>
		</tr>
		<tr>
			<td>70um Sterile Cell Strainer</td>
			<td>Corning</td>
			<td>7201431</td>
			<td>For T cell harvest</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-mouse TCR &#946; chain Antibody</td>
			<td>BioLegend</td>
			<td>109215</td>
			<td>For Imaging</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>X&#38;Y Cell Culture</td>
			<td>FBS-500</td>
			<td>For T cell culture</td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Denisty gradient reagent for T cell harvest</td>
		</tr>
		<tr>
			<td>Fluorescein sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>F6377</td>
			<td>For microscope alignment</td>
		</tr>
		<tr>
			<td>FluoSpheres Carboxylate-Modified Microspheres</td>
			<td>Thermo Fisher Scientific</td>
			<td>F8810</td>
			<td>For microscope alignment</td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td>N/A</td>
			<td>Tracking Software; Other options for tracking software include Amira or Trackmate (Fiji).</td>
		</tr>
		<tr>
			<td>Lattice Light-Sheet Microscope</td>
			<td>3i</td>
			<td>N/A</td>
			<td>Microscope Used</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>21083027</td>
			<td>For Imaging</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-081</td>
			<td>For T cell culture</td>
		</tr>
		<tr>
			<td>LLSpy</td>
			<td>Janelia Research Campus</td>
			<td>N/A</td>
			<td>LLSpy was used under license from Howard Hughes Medical Institute, Janelia Research Campus. Contact innovation@janelia.hhmi.org for access. Other deconvolution and deksewing methods are available in image processing softwares such as Fiji, Slidebook, Amira, and others. <a href="https://llspy.readthedocs.io/en/latest/" target="_blank">https://llspy.readthedocs.io/en/latest/</a></td>
		</tr>
		<tr>
			<td>Moth Cytochrome C (MCC), sequence ANERADLIAYLKQATK</td>
			<td>Elimbio</td>
			<td>Custom Synthesis</td>
			<td>For T cell harvest</td>
		</tr>
		<tr>
			<td>Penacillin/Streptamycin</td>
			<td>Life Technologies</td>
			<td>15140122_3683884612</td>
			<td>For T cell culture</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Phenix Research Products</td>
			<td>P8920-100ML</td>
			<td>For Imaging</td>
		</tr>
		<tr>
			<td>RBC Lysis Buffer</td>
			<td>eBioscience</td>
			<td>00-4300-54</td>
			<td>For T cell harvest</td>
		</tr>
		<tr>
			<td>Recombinant mouse IL-2</td>
			<td>Sigma-Aldrich</td>
			<td>I0523</td>
			<td>For T cell culture</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Corning</td>
			<td>MT10040CV</td>
			<td>For T cell culture</td>
		</tr>
		<tr>
			<td>Slidebook</td>
			<td>3i</td>
			<td>N/A</td>
			<td>LLSM imaging software</td>
		</tr>
		<tr>
			<td>Surgical Dissection Tools</td>
			<td>Nova-Tech International</td>
			<td>DSET10</td>
			<td>For T cell harvest</td>
		</tr>
		<tr>
			<td>T-25 Flasks</td>
			<td>Eppendorf</td>
			<td>2231710126</td>
			<td>For T cell culture</td>
		</tr>
		<tr>
			<td>Thermo Scientific Pierce Fab Micro Preparation Kits</td>
			<td>Thermo Fisher Scientific</td>
			<td>44685</td>
			<td>For preparing Fab</td>
		</tr>
	</tbody>
</table>

visualizing surface t-cell receptor dynamics four-dimensionally using lattice light-sheet microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

Here, we describe the isolation, preparation, and imaging of primary mouse 5C.C7 T cells using a lattice light-sheet microscope. During section 3, it is imperative to align the microscope correctly, and to collect PSF daily with which to deconvolve the data after collection. In Figure 2, we show the correct alignment images that will be seen when aligning the microscope. Figure 2A and Figure 2B show...

Watch this Scientific Journal Video about Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy at JoVE.com

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Lattice light-sheet microscopy is a powerful imaging technology, but few labs in the world are capable of using it. This protocol gives a detailed overview of how to use the commercially available lattice light-sheet microscope. It's an extremely powerful system capable of four-dimensional imaging of individual cells or embryos that allows for real-time tracking of molecules.The main advantage of this technique is that it can three-dimensionally image single live cells or organs with high spatiotemporal resolution and minimal photo bleaching. The lattice pattern of the light sheet allows us to high-speed image samples and obtain real-time videos of three-dimensional living cells and organs with molecular details that other techniques cannot achieve. This first video protocol showing how to us lattice light-sheet microscopy to image single cell four dimensionally.This is a very complicated technique due to alignment and sample preparation. And we believe that visual demonstration will improve accessibility by allowing more scientists to image many different molecules, cells, and organs in biology. To begin this procedure, incubate five millimeter round coverslips with 0.1%poly-L-lysine at room temperature for 10 minutes.Aspirate off the liquid and let the coverslips air dry naturally. Next, add three milliliters of density gradient to a 15 milliliter conical tube. Carefully add cells drop-wise to the edge of the tube.Centrifuge at 930 times g and at four degrees Celsius for 10 minutes. After this, carefully remove the thin middle layer of cells between the complete media and the density gradient reagent making sure to put each cell type into a separate conical tube. Centrifuge at 300 times g for five minutes to wash the cells.Discard the supernatant and add five milliliters of RPMI to the tubes of T-cells and CH27 cells. Repeat the wash with fresh RPMI until three total washes have been performed. Then, resuspend the cells in each tube with one milliliter of complete RPMI and count the cells with a hemocytometer.Resuspend one million antigen-presenting cells in 500 microliters of complete RPMI and add 10 micromolar MCC. Incubate at 37 degrees Celsius with 5%carbon dioxide for three hours. After this, resuspend one million T-cells in 500 microliters of complete RPMI.Add two micrograms of anti-TCR beta Alexa 488 labeled FAB and incubate at 37 degrees Celsius with 5%carbon dioxide for 30 minutes. Next, centrifuge both cells at 300 times g for five minutes and discard the supernatant. Add 500 microliters of complete RPMI and repeat the centrifuge to wash the cells.Repeat the wash with fresh RPMI until three total washes have been performed discarding the supernatant after each wash. After this, resuspend both cell types in 500 microliters of imaging media. First, add 10 milliliters of water and 30 microliters of fluorescein to the LLSM bath.Press Image Home to move the object to image position and look at a single Bessel laser beam pattern. Align the laser beam using the guide and preset region of interest to make the beam a thin pattern that is balanced in all directions. The beam should also appear focused in the finder camera.Use the two mirror tilt adjusters, the top focus micrometer, and the objective color to adjust the beam. Next, wash the bath and the objectives with at least 200 milliliters of water to completely remove any fluorescein. To image standard fluorescent beads, turn on dither by setting to three in the Xgalvo range box and press Live to view current field.Manually adjust the tilt mirror, objective color, and focus micrometer for highest gray values. Then, adjust as necessary to obtain proper patterns for objective scan, Z galvo, Z plus objective, and sample scan capture modes. After this, press Execute in sample scan mode to collect the sample point spread function for de-skewing and de-convolution.Change the lasers to three color mode and press Execute again. To begin, add 100, 000 anti-presenting cells to the prepared five millimeter diameter circular coverslip and allow them to settle for 10 minutes. Grease the sample holder and add the coverslip to it cell side up.Next, add a drop of imaging media to the back of the coverslip. Screw the sample holder onto the Piezo and press Image Home. Find an antigen-presenting cell to image and ensure that the LLSM and imaging software are functioning properly.Press Live to view the current image. Move along Z to find the coverslip and cells. Find the center of an antigen-presenting cell by moving in the Z direction, then press Stop to pause the laser.Check 3D and input the desired settings. Press Center and then press Execute to collect the data. Lower the stage to the load position and add 50 microliters of T-cells and imaging media drop-wise directly over the coverslip.It is best to let a drop form on the end of the pipette tip and then touch the tip to the bath liquid. Raise the stage back by clicking Image Return. Begin imaging.Be sure to set the desired Z stack and timelapse length. For example, image 60 Z stacks at a 0.4 micrometer step size and input 500 timeframes. Stop recording before 500 frames are reached to avoid photo bleaching.Use live mode to search for cell pairs and when ready and desired settings have been entered, press Execute to collect data. In this study, primary mouse 5CC7 T-cells are isolated and prepared and are then imaged using a lattice light-sheet microscope. Shown here are the correct beam path and beam alignment when imaged with fluorescein.The objective scan should show a large X shape in the XZ and YZ projections that is as symmetrical as possible. They should also be adjusted to be as small of an X as possible. The Z galvo scan should show an oval in XZ and XY with a single dot on either side.Finally, both the Z plus objective scan and the sample scan should show dots that look as round as possible. Using this protocol, the four-dimensional dynamics of the T-cell receptors on a T-cell surface could be seen. The main advantage of this microscope lies in the ability to track the visualized surface units of the T-cell receptors and to obtain quantified data from their size, motion, signal intensity, and excreta.The most important thing to remember is the quality of your alignment. The lattice light sheet must be aligned daily and not doing this properly will result in distorted imaging. Similarly, the quality of the PSF collected directly impacts the quality of the de-convolution which ultimately results in the quality of your final image.One of the reasons this method is so powerful is because each cell and label can be replaced to visualize many cell types and molecules. Therefore, this method can be used to answer any question related to four-dimensional tracking of molecules. We think that this technique will pave the way for researchers to explore new questions within the field of molecular trafficking.It is not yet being widely used due to the complicated system so we're hoping this Jove video will improve accessibility to the technique. The lasers used on the system are class four so absolute caution must be taken to ensure laser safety. Please take necessary laser safety training prior to using this method.

visualizing-surface-t-cell-receptor-dynamics-four-dimensionally-using

Research

JoVE Journal

Biochemistry

7.8K visualizações.  The University of Chicago. A microscopia de folhas de luz reticundo é uma poderosa tecnologia de imagem, mas poucos laboratórios no mundo são capazes de usá-la. Este protocolo fornece uma visão geral detalhada de como usar o microscópio de folha de luz disponível comercialmente. É um sistema extremamente poderoso capaz de imagens quadridimensionais de células individuais ou embriões que permite o rastreamento em tempo real de moléculas.A principal vantagem desta técnica é que ela pode imagem tridime...