This study demonstrates the use of differential interference contrast (DIC) microscopy to visualize axonal branching in mouse embryonic cortical neurons. By treating neurons with netrin-1, researchers can observe the dynamic process of axonal development and branching over time.
Take a glass-bottom imaging dish containing mouse embryonic cortical neurons.
Place the dish inside the environmental chamber of a differential interference contrast, or DIC, microscope to maintain cell viability during imaging.
DIC microscopy splits polarized light into two beams that traverse the specimen.
Variations in the refractive index across the specimen introduce phase shifts between these beams.
When the beams recombine, their interference generates a high-contrast image, making finer cellular structures visible.
Identify neurons with unbranched axons and use time-lapse imaging to visualize axonal branching.
Treat with netrin-1, an extracellular guidance cue that binds to axonal receptors and triggers localized exocytosis.
Exocytic vesicles deliver membrane components to the sites while the cytoskeleton is reorganized to support membrane expansion.
Under the microscope, these changes appear as small protrusions, marking the onset of axonal branching.
Over time, these protrusions develop into mature axonal branches, illustrating the dynamic process of neuronal development.
At two days in vitro, place the glass bottom imaging dish containing untransfected neurons in a pre-warmed humid environmental chamber. Utilize a microscope equipped with a 60x plan apochromat, 1.4 NA, DIC objective lens, and a high numerical aperture condenser for the best image quality and resolution. Next, with transmitted light illumination, adjust the focal plane to find neurons through the oculars.
For DIC imaging of axon branching, proper setup of cooler illumination will be integral for image optimization and is critical to producing analyzable results. Then, with the neurons of interest within the field of view, use the multi-area acquisition function in the imaging software to find and save the XYZ locations of at least six cells. Now, add to a final concentration of 250 nanograms per milliliter of netrin-1 or other factor of interest to stimulate the cells, and press Start Multi-Area Acquisition.
Sequentially, acquire images at each position every 20 seconds for 24 hours, pausing acquisition and refocusing as necessary. Within the imaging software, review images by choosing App, Review Multi-Dimensional Data, and open the desired file. Identify stable axon branches that form during the imaging session. Use the trace region tool to measure a stable axon branch from the base of the axon to the tip.
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