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

This method can help answer key questions in the immunology field such as how exactly immune cells behave in a physiologically relevant context like how to search target cells efficiently in a three-dimensional environment. The main advantage of this method is to generate a very thin sheet of light to illuminate only the focal plane without affecting the off plane cells. This enables a very fast acquisition speed with a pronounced reduction of bleaching and photocytotoxicity.

Demonstrating this procedure will be Dr.Renping Zhao, a post-doc from my lab. To begin, under a cell culture hood, transfer 400 microliters of chilled collagen stock solution to a sterile 1.5 milliliter tube. Slowly add 50 microliters of chilled 10X PBS.

Then mix the solution by gently tilting the tube. Add 48 microliters of 0.1 molar sodium hydroxide to the collagen solution to adjust the pH to 7.2 to 7.6. Use pH test strips to determine the pH value of the mixture.

Add two microliters of sterile distilled deionized water to bring the final volume to 500 microliters. Mix well and store the collagen solution on ice or at four degrees Celsius until further use. After fluorescently labeling live cells according to the text protocol, under the cell culture hood, transfer one times 10 to the sixth cells into a sterile 1.5 milliliter tube.

Centrifuge the tube at 200 times g for eight minutes. Then discard the supernatant and use 200 microliters of culture medium to resuspend the pellet. Add 85.9 microliters of the neutralized collagen solution to the cell suspension and mix properly to reach a collagen concentration of 2.5 milligrams per milliliter.

Leave the cell collagen mix on ice in the hood. Next, insert a plunger into the matching capillary until the plunger is sticking one millimeter out of the capillary. Then wet the plunger by dipping into culture medium.

Skipping this step can result in undesirable introduction of air bubbles between the plunger and the collagen matrix. Dip the capillary into the cell collagen mixture and slowly pull the plunger back 10 to 20 millimeters. Then with a spray bottle of 70%ethanol, moisten a paper towel and use it to wipe the outer wall of the capillary to remove the remaining collagen solution.

Now use modeling clay to mount the capillary to the inner wall of a five milliliter tube. Push the cell collagen mix to the edge of the capillary. Then incubate the tube with the capillary at 37 degrees Celsius and 5%CO2 for one hour to polymerize the collagen.

Following the incubation, add one to two milliliters of culture medium to the tube. Then carefully expel the polymerized collagen rod out into the medium until approximately half of the collagen is hanging in the medium. Incubate the capillary for another 30 minutes.

To carry out light-sheet microscopy, assemble the sample chamber according to the manufacturer's instructions. After turning on the microscope and incubator if doing live imaging, place the capillary in the sample chamber, locate the sample, and find the area of interest for image acquisition. Activate the corresponding lasers.

Then set the laser power and exposure time. Also set the step size of the Z stack, the start and end positions of the Z stack, and the time interval for live cell imaging. Then start the image acquisition.

Transfer one milliliter of 4%paraformaldehyde in PBS into a five milliliter tube under a chemical hood. Then dip the capillary with the polymerized collagen into the paraformaldehyde solution and use modeling clay to mount the capillary on the inner wall of the tube. Gently press the plunger until half of the collagen rod is hanging in the paraformaldehyde solution.

Then pull back the plunger to take the collagen rod back up into the capillary. Remove the capillary from the tube and discard the paraformaldehyde. Mount the capillary into a fresh tube and add one milliliter of PBS.

Ensure that the capillary is immersed in the PBS. Gently press the plunger until half of the collagen rod is hanging in the solution and incubate for five minutes. Pull back the plunger to take up the collagen rod in the capillary.

Then replace the PBS with fresh PBS and expel the collagen rod into the solution. Following the third wash, add one to two milliliters of blocking permeabilization buffer into the tube. Expel the collagen rod into the solution and incubate the tube at room temperature for 30 to 60 minutes.

Replace the blocking permeabilization buffer with 200 to 500 microliters of primary antibodies in blocking permeabilization buffer and incubate the submerged collagen rod in the solution for one hour. After using PBST to wash the collagen rod three times, incubate the rod in secondary antibodies in blocking permeabilization buffer at room temperature for one hour. Following three more washes in PBS, pull the collagen rod back into the capillary and keep the sample in PBS until imaging.

As seen in this movie, during migration CTL cells transfected with an EGFP actin fusion protein formed lemon-shaped major protrusions, fringed by fine spindle-like structures. The trajectories of CTLs are illustrated here with velocity and persistence or the displacement divided by the total track length as the measured parameters. The velocities range from 0.01 to 0.19 microns per second with an almost 20-fold difference and the persistence ranges from zero to 0.7.

This figure shows fixed CTL cells in 3D collagen gel stained for endogenous perforin-1 and actin. The sample was illuminated from a single side or from both sides. From different Z positions, the cells are evenly stained indicating good penetration of antibodies into the collagen gels.

Fixed CTL exhibited the same morphology as live cells indicating that the morphology is well-maintained with this protocol. While attempting this procedure, it's very important to remember to avoid air bubbles and to adjust pH value properly. Following this procedure, other methods like live cell staining with particular surface molecules can be performed to answer additional questions like how to correlate cell behavior with differentiation or with different cell subtypes or to investigate cell-cell interaction and cell fate.

After its development, this technique paved the way for researchers in the field of cell biology, immunology, and cancer research to explore cell behavior, cell fate, differentiation, and cell interaction in the most physiologically relevant system in vitro. After watching this video, you should have a good understanding of how to prepare samples with cells embedded in a three-dimensional collagen matrix, how to visualize samples using light-sheet microscopy, either live or fixed, and how to track cell migration in 3D.