This research was supported by the Division of Intramural Research of the National Institute for Allergy and Infectious Diseases, National Institutes of Health. We are grateful to Johannes Huppa and Mark Davis for providing us with the scFv of H57 used in these studies. RV would like to thank Keir Neumann for helpful discussion during the development of this technique.

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All animals used in these experiments were maintained in a specific pathogen-free environment, and the experiments were approved by the National Institutes of Health Animal Care and Use Committee.

We describe here a system to study signaling in antigen-specific primary mouse T cells using TIRFM and glass supported lipid bilayers as artificial APCs. The technique relies on successfully expressing GFP tagged proteins in these cells. Transfecting T cells is always a challenging task. Typically, electroporation or gene delivery using retroviruses or lentiviruses are used. One technique is not superior over the other; both have their limitations and advantages. We find that electroporation has the following advantages: 1) It is not required that the cells be actively dividing as is required for retroviruses but not lentiviruses, and 2) The expression level can be controlled by varying the time the cells are incubated before imaging. The biggest drawback is that we find it very difficult to express proteins that are large in size in primary cells. We have presented a method that gives us very good cell viability after electroporation. As a result it is applicable to using it to achieve siRNA mediated gene silencing.
We also describe here a customized two channel simultaneous acquisition TIRF microscope that is chromatically corrected and does not require separate alignment for different wavelengths. These capabilities; however, are available commercially. Our system is based on principles of TIRF microscopy published by experts in the field and is cheaper than commercial alternatives. One possible criticism of our design is that we are using the same angle of incidence for the different excitation wavelengths. This would result in a different TIRF penetration depth for different excitation wavelengths. We think that these effects are small because the evanescent wave is an exponentially decaying field and the depth of the TIRF field is a linear function of wavelength. Fluorophores close to the glass surface will experience no difference in laser intensities between the two wavelengths. For fluorophores near the penetration depth, the intensity difference will be 1.3 fold for the two wavelengths 488 and 640 nm and 1.15 fold for the two wavelengths 488 and 561 nm. The same system can be used in conjunction with super resolution techniques that make use of TIRF illumination.
We have also described a two camera system which is custom built. Like the TIRF system, similar apparatuses are also commercially available. Our system offers flexibility to change the filters and dichroics, allowing its use with different combinations of wavelengths. The drawback of our system is the one degree of rotation needed to align the two images. This issue can be resolved by using camera stands that are piezo driven and offer rotational degrees of alignment. We have also successfully implemented a two camera system on this microscope, in which the cameras are different and have different pixel sizes. A system like this would be useful if one needed sensitivity in one channel and a large dynamic range in another, both of which are rarely available in the same camera. We used the Photometrics HQ-2 camera at 2x2 binning giving us an effective pixel size of 12.9 &mu;m with the Photometrics Quant-EM camera which has a pixel size of 16 &mu;m. A 180 mm focal length tube lens was used for the Quant-EM and a 145 mm focal length tube lens was used for the HQ-2 camera. The HQ-2 was controlled using Metamorph software and the Quant-EM was controlled via micro-manager software on a separate computer in external trigger mode. The external trigger was provided to the Quant-EM camera using a digital output of the measurement computing DAC board, which was implemented as an additional shutter in the configure illumination settings of Metamorph. The ratio of the focal lengths of the tube lenses matches the ratio of the effective pixel sizes. A representative alignment is shown in Figure 5.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td> Amaxa Nucleofector Kit for Mouse T cells</td>
 <td> Lonza Inc.</td>
 <td>VPA-1006</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PureLink HiPure Plasmid Midiprep Kit</td>
 <td>Life Technologies</td>
 <td>K2100-05</td>
 <td>For endotoxin free DNA preps</td>
 </tr>
 <tr>
 <td>Amaxa Nucleofector Device</td>
 <td>Lonza Inc.</td>
 <td>AAD-1001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Recombinant Murine IL-2</td>
 <td>Peprotech Inc.</td>
 <td>212-12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluoresbrite Multifluorescent Microspheres 0.20&mu;m</td>
 <td>Polysciences, Inc.</td>
 <td>24050-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lab-Tek II 8-well chambered coverglass</td>
 <td>Thermo Scientific, Nunc</td>
 <td>155409</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins at JoVE.com

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

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 ...

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Immunology and Infection

23.7K Views.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. 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.