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1. Conducting LLSM Daily Alignment
NOTE: (Important) This alignment protocol is based on the LLSM instrument used (see the Table of Materials). Each LLSM may be different and require different alignment strategies, especially those that are home-built. Carry out the appropriate routine alignment and continue to section 2.
- Add 10 mL of water plus 30 µL fluorescein (1 mg/mL stock) to the LLSM bath (~10 mL volume), press Image (Home) to move the objective to the image position, and look at a single Bessel laser beam pattern. Align the laser beam using the guides and pre-set region of interest (ROI) to make the beam a thin pattern balanced in all directions.
- The beam should also appear focused in the finder camera. Use two mirror tilt adjustors, a top micrometer, focus, and an emission objective collar to adjust. See Figure 1 A, and B for the correctly aligned beam.
- Wash the bath and objectives with at least 200 mL of water to completely remove fluorescein.
- Image standard fluorescent beads in the imaging media (prepared by adhering beads to a 5 mm coverslip with poly-L-lysine, see the Table of Materials; this can be pre-prepared and re-used) for physical point spread function (PSF) in imaging media.
NOTE: There can only be one bead in view for later processing, so try to find a bead that is by itself in the viewer or can easily be cropped to obtain a single bead.
- Turn on dither by setting it to 3 in the 'X Galvo range' box. Press Live to view the current field. Move along the Z direction to find the coverslip and beads. Find the center of a bead by moving along Z, press Stop to pause the laser. Check 3D, press Center and then press Execute. This will collect the data.
- Manually adjust the tilt mirror, objective collar, and focus micrometer for the highest gray values, then adjust as necessary to obtain proper patterns for objective scan, z galvo, z+objective (totPSF), and sample scan (samplePSF) capture modes. See Figure 1 C-F for properly adjusted maximum intensity projections (MIPs).
NOTE: The various capture modes (objective scan, z galvo, z+objective, and sample scan) change how the light sheet moves through the sample. All scan modes should be used for alignment.
- The sample scan shows how data will be collected during the experiment. Collect the sample PSF by pressing Execute in sample scan mode for deskewing and deconvolution. Change lasers to three color modes (488, 560, 647) and press Execute again.
NOTE: Since the LLSM images are at an angle (57.2°), images captured in the "sample scan" mode are collected at this angle, and are therefore "skewed". De-skewing is the process of correcting for this angle and "re-aligning" the image to a true z-stack. These data must be collected in imaging media and in all channels that will be imaged during the experiment. If this is not collected properly, the data will not be properly de-skewed. Similarly, make sure the media has been warmed to 37 °C (or desired experimental temperature).
2. Setting Up Cells with LLSM
- Add 100,000 antigen-presenting cells (APCs) (50 µL) to a 5 mm-diameter circular coverslip and allow them to settle for 10 min.
- Grease the sample holder then add the coverslip cell-side-up to it. Add a drop of imaging media to the back of the coverslip to avoid bubbles before placing it in the bath. Screw the sample holder onto the piezo, and press Image (Home).
- Find an APC to image to ensure that the LLSM and imaging software (see Table of Materials) are functioning properly.
NOTE: We image at 0.4 µm step size with 60 z-steps and 10 ms exposure for two colors with a dither set to 3, which results in 1.54 s per frame of the 3D image with ~200 nm XY and 400 nm Z resolution. These settings may need to be adjusted based on cell size, desired z-resolution, and strength of the signal from the fluorescent labeling technique used. Laser power usage will also vary based on the fluorescent labeling technique used.
- Press Live to view the current image. Move along Z to find the coverslip and cells.
- Find the center of an APC 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. This will collect the data.
- Lower the stage to load position and add 50 µL of T cells in imaging media (100,000 cells) dropwise 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 stack size and time-lapse length. For example, image 60 z-stacks at a 0.4 µm step size and input 500-time frames. (Typically) stop recording before 500 frames are reached to avoid photobleaching. Use Live mode to search for cell pairs, and when ready and desired settings have been entered, press Execute to collect data. See Figure 2 for an example.
