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 (~20-25 g) according to the approved IACUC protocol (i.e., CO2 chamber followed by cervical dislocation).</li>
	<li>Spray mouse carcass thoroughly with 70% ethanol to soak down the fur and bring into a BSL-2 safety cabinet.</li>
	<li>Turn mouse onto its right side and make a small incision in the body cavity with surgical scissors. Remove the spleen using surgical forceps. Cut away connective tissue as needed with surgical scissors.</li>
	<li>Place the spleen in a 70 &#181;m-pore mesh cell strainer and mash with the back of a 1 mL syringe plunger. Wash through the strainer thoroughly with complete RPMI (RPMI with 10% fetal bovine serum [FBS], 2 mM L-glutamine, 1% penicillin/streptomycin, 50 &#181;M 2-mercaptoethanol).</li>
	<li>Centrifuge the single cell suspension of splenocytes at 300 x g for 5 min. Discard the supernatant. 
	NOTE: The pellet at this point will be red.</li>
	<li>Resuspend the splenocytes in 5 mL RBC lysis buffer. Incubate for 5 min, then quench with 5 mL of complete RPMI.</li>
	<li>Centrifuge the single cell suspension of splenocytes at 300 x g for 5 min. Discard the supernatant. 
	NOTE: The pellet at this point should be white, not red.</li>
	<li>Resuspend the pellet in 5 mL of complete RPMI.</li>
	<li>Transfer to a T-25 flask and add 10 &#181;M moth cytochrome-C (MCC, sequence ANERADLIAYLKQATK). Put the cells in a cell culture incubator at 37 &#176;C, 5% CO2 overnight.</li>
	<li>On the next day, add recombinant mouse IL-2 to a final concentration of 100 U/mL.</li>
	<li>Observe for the following days, adding fresh media as the current media turns yellow. T cells will die if left in yellow media unattended for over 48 h. Cells are ready to use 6-10 days after harvest.</li>
</ol>
2. Prepare Cells
<ol>
	<li>Incubate 5 mm round coverslips with 0.1% poly-L-lysine for 10 min. Aspirate off and let dry naturally.</li>
	<li>Use a density gradient reagent (see the Table of Materials) to separate out dead cells and to obtain 1 x 106 T cells and 1 x 106 APCs (CH27 cells21,22 transduced with cytosolic mCherry) separately. 
	NOTE: Cells are counted using a hemocytometer.
	<ol>
		<li>Add 3 mL of density gradient reagent to a 15 mL conical tube and add cells dropwise to the edge of the tube carefully. Do not mix. Centrifuge at 930 x g for 10 min at 4 &#176;C; use acceleration/deceleration: SLOW/SLOW. Remove the thin middle layer of cells between the complete media and the density gradient reagent carefully, putting each cell type into separate conical tubes. 
		NOTE: It is necessary to prepare more cells than needed for imaging, as we find that 50% is lost on average during the process. The more cells used for the process, the easier it is to harvest them from the density gradient. We use 4-8 mL of both cell types to ensure excess cells. If desired, cells can be counted before this step to ensure volume needed. Any extra cells are put back into their respective flasks.</li>
		<li>Wash both tubes of T cells and CH27 cells three times with 5 mL complete RPMI (300 x g for 5 min). Discard the supernatant each time during the wash. Resuspend each tube in 1 mL complete RPMI and count cells by a hemocytometer.</li>
	</ol>
	</li>
	<li>Resuspend 1 x 106 APCs in 500 &#181;L complete RPMI and add 10 &#181;M MCC. Incubate for 3 h at 37 &#176;C, 5% CO2. Wash cells three times with 500 &#181;L complete RPMI (300 x g for 5 min). Discard the supernatants.</li>
	<li>Resuspend 1 x 106 T cells in 500 &#181;L complete RPMI. Add 2 &#181;g of anti-TCR&#946; Alexa488-labeled Fab (clone H57) to 500 &#181;L of cells. Incubate for 30 min at 37 &#176;C, 5% CO2. Wash cells three times with 500 &#181;L of complete RPMI (300 x g for 5 min). Discard supernatant after each wash. 
	NOTE: The divalent anti-TCR antibody was cut into monovalent Fab using a Fab preparation kit (see the Table of Materials) to avoid crosslinking the T cell receptors by the divalent antibody (this step is optional).</li>
	<li>Resuspend both cell types in 500 &#181;L of imaging media (phenol red-free Leibovitz&#39;s L-15 medium with 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine).</li>
</ol>
3. Conducting LLSM Daily Alignment
NOTE: (Important) This alignment protocol is based on the LLSM instrument used (see the Table of Materials). Each LLSM may be different and require different alignment strategies, especially those that are home-built. Carry out the appropriate routine alignment and continue to section 4.
<ol>
	<li>Add 10 mL of water plus 30 &#181;L fluorescein (1 mg/mL stock) to the LLSM bath (~10 mL volume), press Image (Home) to move the objective to image position, and look at a single Bessel laser beam pattern. Align the laser beam using the guides and pre-set region of interest (ROI) to make the beam a thin pattern balanced in all directions.
	<ol>
		<li>The beam should also appear focused in the finder camera. Use two mirror tilt adjustors, top micrometer, focus, and emission objective collar to adjust. See Figure 2A,B for correctly aligned beam.</li>
	</ol>
	</li>
	<li>Wash the bath and objectives with at least 200 mL of water to completely remove fluorescein.</li>
	<li>Image standard fluorescent beads in the imaging media (prepared by adhering beads to a 5 mm coverslip with poly-L-lysine, see the Table of Materials; this can be pre-prepared and re-used) for physical point spread function (PSF) in imaging media. 
	NOTE: There can only be one bead in view for later processing, so try to find a bead that is by itself in the viewer or can easily be cropped to obtain a single bead.
	<ol>
		<li>Turn on dither by setting to 3 in the &#39;X Galvo range&#39; box. Press Live to view the current field. Move along the Z direction to find the cover slip and beads. Find the center of a bead by moving along Z, press Stop to pause the laser. Check 3D, press Center and then press Execute. This will collect the data.</li>
		<li>Manually adjust the tilt mirror, objective collar, and focus micrometer for highest gray values, then adjust as necessary to obtain proper patterns for objective scan, z galvo, z+objective (totPSF), and sample scan (samplePSF) capture modes. See Figure 2C-F for properly adjusted maximum intensity projections (MIPs). 
		NOTE: The various captures modes (objective scan, z galvo, z+objective, and sample scan) change how the light-sheet moves through the sample. All scan modes should be used for alignment.</li>
		<li>The sample scan shows how data will be collected during the experiment. Collect the sample PSF by pressing Execute in sample scan mode for deskewing and deconvolution (see section 5). Change lasers to three color mode (488, 560, 647) and press Execute again. 
		NOTE: Since the LLSM images at an angle (57.2&#176;), images captured in the &#34;sample scan&#34; mode are collected at this angle, and are therefore &#34;skewed&#34;. De-skewing is the process of correcting for this angle and &#34;re-aligning&#34; the image to a true z-stack. These data must be collected in imaging media and in all channels that will be imaged during the experiment. If this is not collected properly, the data will not be properly de-skewed. Similarly, make sure media has been warmed to 37 &#176;C (or desired experimental temperature).</li>
	</ol>
	</li>
</ol>
4. Setting Up Cells with LLSM
<ol>
	<li>Add 100,000 APCs (50 &#181;L) from step 2.5 to a 5 mm-diameter circular coverslip from step 2.1 and allow them to settle for 10 min.</li>
	<li>Grease the sample holder then add the coverslip cell-side-up to it. Add a drop of imaging media to the back of the coverslip to avoid bubbles before placing in the bath. Screw the sample holder onto the piezo, and press Image (Home).</li>
	<li>Find an APC to image to ensure that the LLSM and imaging software (see Table of Materials) are functioning properly. 
	NOTE: We image at 0.4 &#181;m step size with 60 z-steps and 10 ms exposure for two colors with dither set to 3, which results in 1.54 s per frame of 3D image with ~200 nm XY and 400 nm Z resolution. These settings may need to be adjusted based on cell size, desired z-resolution, and strength of signal from the fluorescent labeling technique used. Laser power usage will also vary based on fluorescent labeling technique used.
	<ol>
		<li>Press Live to view the current image. Move along Z to find the cover slip and cells.</li>
		<li>Find the center of an APC by moving in the Z direction, then press Stop to pause the laser. Check 3D and input the desired settings (see step 4.3.1), press Center and then press Execute. This will collect the data.</li>
	</ol>
	</li>
	<li>Lower the stage to load position and add 50 &#181;L of T cells in imaging media (100,000 cells, from step 2.5) dropwise 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 stage back by clicking &#34;Image (Return)&#34;.</li>
	<li>Begin imaging. Be sure to set the desired stack size and time lapse length. For example, image 60 z-stacks at a 0.4 &#181;m step size and input 500 time frames. (Typically) stop recording before 500 frames are reached to avoid photobleaching. Use Live mode to search for cell pairs, and when ready and desired settings have been entered, press Execute to collect data. See Movie 1 and Figure 1 for an example.</li>
</ol>
5. Track Surface Dynamics
<ol>
	<li>Export the data from the imaging software (see the Table of Materials). This will create z-stack TIF files for every time point in each color.</li>
	<li>First deskew and deconvolve the data. 
	NOTE: We use the LLSpy pipeline under license by HHMI&#39;s Janelia Research Campus18, but deskewing and deconvolution are also available within multiple imaging softwares (see Table of Materials).</li>
	<li>Debleach the data. 
	NOTE: We use Fiji&#39;s debleach feature with histogram matching (Fiji Pathway: Image | Adjust | Bleach Correction | Histogram Matching | OK).</li>
	<li>Import into the tracking software (see the Table of Materials). Track clusters according to software specifications. See Movie 2 for an example. 
	NOTE: Tracking results will depend upon the tracking software used, algorithm chosen, desired output parameters selected, etc. For example, in the tracking software we utilized (see the Table of Materials), we chose to allow the software to track irregular shapes, rather than assigning each feature a single spot, since TCR clusters are not organized into perfect spheres. In addition, we chose to collect 35 parameters, including speed, direction, volume, intensity, area, location, and track duration information. However, different methods or parameters will be beneficial to answer different questions.
	<ol>
		<li>If tracking is not desired, use the ClearVolume plugin for Fiji for visualizing and creating movies from hyperstack data.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 critical step is allowing the cells to settle at an appropriate concentration. If too many APCs settle on the coverslip and become too dense, it is hard to find a T cell that is interacting with only a single APC. When a T cell has multiple synapses, tracking and interpretation of data can become very complicated. Similarly, if too few APCs are present, finding a T cell forming a synapse is also difficult. In our hands, allowing 50,000 cells to settle for 10 min achieves an optimal density. However, this problem can be avoided if using a system with adherent cells. Cells can be grown in the incubator with the coverslips to a desired confluency.
Similarly, the number of T cells dropped into the system is dependent upon the size of the bath and the distance they can disperse. In the LLSM system used here, there is a 12 mL bath and a 2.5 mL bath, as opposed to the previous version of the system which only had a 10 mL bath available. We use the 12 mL bath for the original fluorescein imaging then switch to the 2.5 mL bath for imaging the cells. This allows for less thorough washing of the bath following the beam visualization step, and also lowers the number of T cells required for each imaging session. In turn, this would allow users to utilize fewer cells.
Finding cells at the correct point of interaction is also a challenge. In our hands, T cells take about 2 min to settle down to the APCs on the coverslip, so it is important to begin searching the coverslip for dynamic T cells close to APCs. A major improvement of this has been the recent addition of the LED light in the finder camera. If using a home-built system, we highly recommend including this feature in the design.
Finally, fluorescent labeling strategies are another important consideration. Each fluorescent protein or dye has a different quantum yield and rate of photobleaching. Fluorescent dyes are typically brighter, but if the cells have been stably transduced with a fluorescent protein labeled molecule, the label is replenished as the cell continues to produce the molecule. Therefore, labeling strategy is an important factor to consider when designing experiments.
We would like to conclude with a discussion on future directions for the technology. LLSM is also capable of structured illumination microscopy (SIM), which results in 150 nm XY resolution and 280 nm Z resolution, and is at least 10 times faster than widefield SIM12. Therefore, while LLSM provides unprecedented speed of 4D imaging, it cannot achieve the spatial resolution of current super-resolution techniques4,5,6. However, this resolution could be improved if a STED LLSM could be created. Light sheet STED and stimulated emission depletion with selective plane illumination microscopy (STED-SPIM) have been utilized, but lack the temporal resolution of LLSM21,22,23,24. If STED-SPIM were adapted to incorporate a lattice, we could potentially obtain 50 nm axial resolution with far less photobleaching, and image faster than currently available techniques. Nevertheless, with current available technologies, LLSM gives us the fastest temporal resolution with high axial resolution.

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><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 &amp;beta; chain Antibody</td><td>BioLegend</td><td>109215</td><td>For Imaging</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>X&amp;amp;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&#039;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. &lt;a href=&quot;https://llspy.readthedocs.io/en/latest/&quot; target=&quot;_blank&quot;&gt;https://llspy.readthedocs.io/en/latest/&lt;/a&gt;</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 the correct beam path and beam alignment, respectively, when imaged in fluorescein. The objective scan should show a large X shape in the XZ and YZ projections that is as symmetrical as possible; this should also be adjusted to be as small of an X as possible (Figure 2C). This is mainly achieved by adjusting the emission objective collar. The z galvo scan should show an oval in XZ and XY with a single dot on either side (above and below for XZ and to the left and right for YZ) (Figure 2D). This is mainly achieved by adjusting the galvo mirror tilt, either manually or with the motorized adjustment in the software. Finally, both the z+objective scan and the sample scan should show dots that look as round as possible (Figure 2E and Figure 2F, respectively). These may have a small X, but this should be as dimished as possible. These should be well aligned if the objective scan and z galvo were set well, but if adjustments are necessary, they will be mostly conducted with the galvo mirror. It is important to note that during this alignment, any time the collar and galvo are adjusted, the focus (micrometer above the emission objective) will need to be adjusted as well.
Using this protocol, we can see the four-dimensional dynamics of the TCRs on a T-cell surface (Figure 1, Movie 1). The main advantage of this microscope lies in the ability to track the visualized surface units of the TCRs, and to obtain quantified data from their size, motion, signal intensity, etc. (see Table of Materials). Movie 2 shows an example of the tracks obtained.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59914/59914fig1.jpg" /> 
Figure 1: 4-dimensional imaging of T cell-APC Synapse. (A) A representative example 3D time-lapse LLSM images showing a T cell interacting with an APC. Shown are the TCR (green, labeled by anti-TCR-AF488) dynamics in recognizing antigens presented on the surface of an APC (red, cytosolic mCherry). Scale bar = 5 &#956;m. Also see Movie 1. (B) Orthogonal XY slice of (A). Inset is a reference frame of a whole cell. Scale bar = 5 &#956;m. (C) Orthogonal YZ slice of (A). Inset is a reference frame of a whole cell. Scale bar = 5 &#956;m. (D) Dual orthogonal slice of (A). Inset is reference frame of whole cell. Scale bar = 5 &#956;m. Also see Movie 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59914/59914fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59914/59914fig2a.jpg" /> 
Figure 2: LLSM alignment. (A) Desired beam pattern for LLSM imaging experiment. (B) Screenshot of the beam alignment process; on the left is the focus window showing the narrowed, focused beam; at the top right is a graph showing that the beam is centered within the window; at the bottom right is the finder camera, which should also be a thin, focused beam. (C) Maximum intensity projections (MIPs) of a bead by objective scan. (D) Maximum intensity projections (MIPs) of a bead by z-galvo scan. (E) Maximum intensity projections (MIPs) of a bead by z+objective scan. (F) Maximum intensity projections (MIPs) of a bead by sample scan. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59914/59914fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/59914/59914movie1a.jpg" /> 
Movie 1: T cell synapse formation. Two-color volume rendering of the interaction of a T cell labeled with &#945;TCR-AF488 (green) with a target APC expressing cytosolic mCherry (red) over 70 time points at 1.54 s interval. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59914/JoVE_Movie_1.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 2" src="/files/ftp_upload/59914/59914movie2a.jpg" /> 
Movie 2: Tracking TCR dynamic motion. Compare with Movie 1. Clusters corresponding to visible TCR structures were tracked in 3D over time with imaging software. Dragon tails showing cluster positions over the previous four frames are color-coded by displacement length. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59914/JoVE_Movie_2.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 3" src="/files/ftp_upload/59914/59914movie3a.jpg" /> 
Movie 3: Orthogonal slices of T cell synapse. Compare with Movie 1 and Figure 1B-D. Dual orthogonal slice of Movie 1, showing that &#945;TCR&#946;-AF488 Fab is indeed labeling the cell surface TCRs. Inset is a reference frame of whole cell. Scale bar = 5 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59914/JoVE_Movie_3.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>

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 ...

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

Nanyang Technological University

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JoVE Journal

Biochemistry

7.9K Views.  The University of Chicago. 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.

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

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Immunology and Infection

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Bioengineering

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

Lattice Light Sheet Microscopy to Visualize Receptor-Ligand Interactions in Live Cells

Source: Rosenberg, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59914">Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy.</a> J. Vis. Exp. (2020).
In this video, we describe the lattice light-sheet microscopy technique to visualize interactions between live T-cells and antigen-presenting cells. The method involves focusing an ultrathin lattice light sheet on an interacting cell pair to obtain a series of two-dimensional images, which are combined to obtain high-resolution four-dimensional images that reveal the spatiotemporal dynamics of the cell-cell interaction.

Source: Rosenberg, J., et al. Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy. J. Vis. Exp. (2020).
...

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

Summary

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