Name: visualizing surface t-cell receptor dynamics four-dimensionally using lattice light-sheet microscopy
Uploaded: 2020-01-30T12:00:00
Description: Watch this Scientific Journal Video about Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy at JoVE.com

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>
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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 ...

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