Encyclopedia of Experiments: Biological Techniques
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Transcript
Add 10 milliliters of water and 30 microliters of fluorescein to the LLSM bath. First, press Image (Home) to move the object to image position, and look at a single vessel 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 3 in the 'X Galvo 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 antigen-presenting cells to the prepared 5-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 Enter and then press Execute to collect the data.
Lower the stage to the Load position, and add 50 microliters of T cells in 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 500th-time frames. 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.
