This article describes a method for imaging neural stem cells (NSCs) using a confocal laser scanning microscope. The NSCs are isolated from a transgenic mouse brain and contain a fluorescently tagged protein that allows visualization during the G1 phase of the cell cycle.
Take a multi-well plate containing adhered neural stem cells or NSCs isolated from a transgenic mouse brain. These NSCs contain a fluorescently tagged protein that is specific to the first growth, or G1, phase.
As the NSC divides, it fluoresces in the G1 phase of the cell cycle while emitting no fluorescence in other phases.
Place the plate in a confocal laser scanning microscope imaging chamber, maintained at a controlled temperature.
Select the desired objective lens and adjust the fluorescence intensity.
Configure the parameters for time-lapse imaging.
Choose the large image option and schedule image acquisition at regular intervals for the desired duration.
Use a high-speed resonant scanner to avoid photobleaching and maintain high resolution.
Use the brightfield setting to visualize the cell shape, and use an appropriate fluorescent filter to capture the fluorescence.
Start time-lapse imaging of the NSC to visualize different cell cycle phases.
Prepare a confocal laser scanning microscope for imaging by preheating the attached temperature control chamber to 37 degrees Celsius under a 5% carbon dioxide atmosphere. Position the 96-well plate inside the prewarmed microscope chamber and bring the first well into focus. Then, open the imaging software and right click in the main window to select Acquire Acquisition Controls and Deacquisition in order to open the TI Pad and select the plan Apo VC 20x DIC objective.
Next, click on Acquire Acquisition Controls, and then open the time lapse options. Set the center of each well as a stage position and select the Large Image option to 7 by 7 millimeters squared. Observe a mosaic image around the center of each well. Set the overlap for the large mosaic image to 5% and set the frequency so that pictures are taken every 20 minutes for 24 hours.
Under A1 plus settings, select the photomultiplier tube voltage level for each fluorescence listed in the menu bar. Select to acquire images using a high-speed resonant scanner at a 512 by 512 pixels format, and use DIC to visualize the cells. Then, select the folder to save the data files. Select the Run Now button on the ND acquisition window to begin acquisition.
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