Assessing Immunological Synapse Topology through Live-Cell Imaging

To live-image the cells, turn on the microscope system, and open the appropriate image capture software. Set the appropriate parameters for automated multichannel time-lapse imaging as well as the interval for acquisition for 10 to 30 seconds and a total duration of approximately 20 to 60 minutes.

Next, add fresh oil to the objective. Mount a microscope dish containing 0.5 milliliters of buffer A onto the heating stage adapter and immediately switch on the adapter, previously set to 37 degrees Celsius. After two to three minutes of equilibration, use a 20-microliter pipette to add the resting Fura-2-loaded lymphocytes into the mounted microscope dish chamber, and turn on the brightfield imaging.

Select the light path to the eyepieces, and use the coarse focus knob to bring the objective into contact with the bottom of the microscope dish. Then, use the eyepiece and the fine focus knob to locate the T cells settled at the bottom of the dish, and use the XY stage controls to select a field containing at least 10 cells.

When the appropriate cells have been located switch from brightfield to the fluorescent light source, and from eyepiece-imaging to the CCD camera. Optimize the acquisition parameters, and then, acquire resting Fura2-340 and Fura2-380 images.

One of the key steps to generating successful results is careful optimization of the fluorescence acquisition parameters, ensuring that sufficient signal is collected and, in the case of Fura-2, that the baseline 340/380 ratio is close to 1.

When the Fura-2 exposure times have been set, replace the dish of T cells with the microscope dish of endothelial cells. Using a disposable transfer pipette, rapidly remove the media, and rinse the cells once with 1 milliliter of pre-warmed buffer A, then, replace the wash with 0.5 milliliters of fresh buffer A.

Using the objective, identify fields in which the brightly fluorescent endothelial cells appear healthy, then, adjust the acquisition parameters for membrane-YFP and DsRed as just demonstrated for Fura-2, taking care that the mean fluorescent signal intensity in each channel falls between 25% and 75% of the dynamic range of the detector.

Before conducting the live-cell imaging experiment, capture several intervals of images of the endothelial cells alone to establish a baseline with the automated software. Then, inserting the tip of a small-volume pipette into the media close to the center of the objective, slowly dispense 5 microliters of concentrated Fura-2-loaded lymphocytes to the center of the microscope dish imaging field.

As the lymphocytes settle, make fine adjustments to the focus to ensure that the T cell/endothelial cell interfaces are maintained in the focal plane. With the 40x and 63x objectives, 10 to 20 cells per field is optimal.