Begin by coating the 35-millimeter coverslip dishes with Poly-D-Lysine, or PDL. To do so, dispense a 0.5 microliter droplet of PDL at the center of a coverslip dish. Using a 10-microliter graduated pipette, smear the PDL droplet across the coverslip.
Allow the coated coverslip dish to dry at 37 degrees Celsius for 10 minutes. For imaging the freshly collected sperm from a mouse model expressing AcroSensE, add 80 microliters of the prepared sperm suspension to the center of the PDL-coated coverslip dish. Then, slowly add three milliliters of supplemented, modified Whitten's base media pre-warmed to 37 degrees Celsius to the dish.
Proceed to perform the imaging using a combination of a microscope with the single cell stimulant delivery setup. First, mount the dish containing the sperm on the microscope. Then, using the micromanipulator, position the capillary tip of the stimulant delivery system about 100 microns to the side and five to 10 microns above the plane of the cell of interest.
Position the capillary within 100 microns from the sperm head. Next, start the imaging sequence on the microscope. Image sperm cells at a high frame rate, preferably greater than 10 frames per second.
10 seconds after initiating the image acquisition, activate the single cell delivery system to deliver a 10-second puff of stimulant. Continue capturing images of cells for a duration of 10 to 15 minutes. In a similar manner, image multiple cells from a single dish according to the locations shown on the screen.
