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

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.