Our protocol uses regular fluorophore probes and simple specimen preparation techniques that maintain the imaged cell in physiological condition to study highly dynamic cellular process and detailed sub-cellular structures. This technique allows us to investigate cellular processes at a super resolution and under physiological conditions for an extended period without sacrificing either the special or temporary resolution. Begin by cutting a 12 to 14 kilodalton molecular weight cutoff dialysis membrane into small pieces and soaking the pieces with 100 microliters of live cell medium for about five minutes.
While the membranes are soaking, cut the outer ring of a 50 milliliter tube into small pieces and sterilize the pieces in 70%ethanol. For testes dissection and mounting, transfer ten two to three day old anesthetized male flies into a dissection dish under a dissecting microscope and use fine forceps to remove the testes. When all of the testes have been collected, wash the testes two times in live cell medium and use a pipette tip to spread 100 to 150 microliters of live cell medium to the prepared glass bottom dish.
Use fine forceps to transfer the fly testes to the center of the dish and remove all but about 10 microliters of the medium. Quickly, place the pre-wet membrane onto the testes and place two to three small plastic weights onto the membrane. Immediately, add 100 to 150 microliters of fresh live cell medium and place the ring back onto the elevated side of the dish.
Place the cover slip back onto the ring and swirl a piece of tissue paper in water before placing the paper onto the cover slip. Then, cover the dish with a lid and secure the dish on the stage of a super resolution microscope. For germline stem cell imaging, open the imaging software and turn on the transmitted light.
Use the 63X objective to focus on the testes tissue and turn on the lasers. Click Live"to locate germinal stem cells. Adjust the focus and click Frame Size"to set the frame size to between 512 by 512 and 1024 by 1024 pixels, and Averaging"to set the frame average to one or two.
Click Master Gain"to set the electron multiplying gain to less than 800, and Lasers"to set the laser power to 1%to 2%Zoom in to the region or cell of interest to reduce the image acquisition time and to reduce photo bleaching, and confirm that the image has been optimally configured before clicking Start Experiment"to start the time lapse image capture. If Airyscan Acquisition is not configured optimally"is displayed, click Optimal"in the frame size, Z-Stack, and Scan Area"sections, and optimize the time interval, number of Z slices and duration of the time-lapse imaging, according to the experimental design and type of specimen. After imaging, select Processing, Batch"and Airyscan Processing, and select the images to be processed.
Then click Run Process"to obtain the super resolution images. Live cell imaging of drosophila testes expressing alpha tubulin GFP in early stage germ cells using a spinning disc confocal microscope, allows visualization of the asymmetric intensity of GFP signals at two centrosomes, as a brighter signal at the mother centrosome and a relatively weaker signal at the daughter centrosomes. The difference in brightness is likely reflected by the temporal asymmetry of the microtubule nucleation, but the detailed morphology and quantity of the microtubules can not be resolved using spinning disc confocal microscopy.
In contrast, live cell super resolution imaging allows the visualization and quantification of microtubule morphology and numbers. This improved resolution also reveals patterns of asymmetric microtubule nucleation, elongation and increased interaction with the nuclear membrane. Live cell imaging also allows the visualization of asymmetric nuclear membrane invagination, but not of individual microtubules that directly enter the nucleus.
In contrast, this live cell super resolution technique allows the direct observation of these events by simultaneous imaging of both microtubules and the nuclear lamina. During metaphase, both sister centromeres can be detected as one signal using spinning disc microscopy, although the microtubule central attachment can not be observed. In contrast, super resolution live imaging allows visualization of the microtubule centromere attachment in early prophase.
Be sure to place a membrane over the testes to ensure that they do not move or float during imaging and to optimize the microscope settings to avoid photo bleaching and photo toxicity. There are many ways that this method can be applied, such as to investigate protein dynamics, linear stressing, or cellular defenses and processes.
