This article details a protocol for live-cell imaging of adult neural stem cells using phase-contrast time-lapse microscopy. The method emphasizes the importance of optimal cell seeding density and incubation conditions to ensure cell viability and accurate tracking.
Seed adult neural stem cells on a poly-D-lysine-coated multi-well plate at optimal densities to ensure cell viability and single-cell tracking.
During incubation, cells adhere to the coated surface, providing a stable platform for precise visualization during imaging.
Mark a well without cells to establish a reference point for microscope coordinates.
Transfer and secure the plate inside the microscope's incubation chamber under optimized conditions to maintain cell viability and prevent undesired movement during motorized stage shifts.
Allow the cells to equilibrate to the chamber conditions to ensure consistent image focus.
Select the region containing cells for imaging with respect to the reference point. Then, configure time-lapse microscope parameters.
Perform phase-contrast time-lapse microscopy to image live cells at regular intervals, minimizing phototoxicity.
Using single-cell tracking software, process captured images to track cell proliferation and visualize lineage progression.
Live-cell tracking enables the study of cell division and differentiation.
To begin this procedure, seed the cells directly on poly-D-lysine-coated 24-well plates with 1 milliliter of culture medium per well. Incubate the plates at 37 degrees Celsius and 8% carbon dioxide for adult neural stem cells for two hours prior to live imaging. Turn on the microscope, camera, hardware, and incubation systems.
Set the temperature and air pressure to 37 degrees Celsius and 8% carbon dioxide for adult neural stem cells. Allow the temperature and carbon dioxide levels to stabilize for one to two hours. Once the cells are firmly attached to the plate, use a permanent marker to make a small mark on the bottom of one well that will not be used for tracking.
Place the plate inside the microscope's incubation chamber, and firmly attach the plate to the stage to avoid any undesired movements during the displacement of the microscope's motorized stage. Then, allow the temperature of the cell culture medium to equilibrate in the chamber for approximately 20 minutes.
Start the live imaging software and select the time lapse module to set up the experiment. Set the total duration of the experiment, and the image acquisition cycles, and the time scheduled tab menu. Select the image positions defined by the x and y-coordinates, and the focal distance in the XYZ points tab menu.
Use the mark made previously as position number 1, which will be used as the XYZ reference zero position. Select the type of acquisition and the wavelength selection tab menu, Brightfield Only, or in combination with epifluorescence excitation when required. Then, select the exposure time. Afterward, define the name of the experiment and the folder where the image will be stored. Save the list of positions to reload the experiment at any time.
Once all the conditions have been set, run the experiment by clicking on the Run Now button. Pause the experiment and readjust the focus conditions, clicking the Overwrite Z button once per day until the experiment is completed.
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