You are here

Fluorescent Microscopy

Submitted by rmmcdonald on Thu, 10/24/2019 - 17:14

A Nikon Inverted Optical microscope at 40x magnification was utilized throughout all experiments. For studying the rate of photobleaching of DAPI stained nuclei, we set the exposure time 600 ms and a previously untouched area of cells was exposed to constant fluorescent light for 5 minutes. During those 5 minutes, 31 photos were automatically captured at 10 second intervals and the results are visualized by Figure 1. The brightness of each nuclei clearly decreased and the images blurred overtime as evident in Figure 1.

We repeated this process to capture the rate of photobleaching of fluorescein stained tubulin. Once we located an unbleached area of cells, we set the exposure to 2,000 ms and begun the time lapse video. Figure 2 represents the specimen over the course of 5 minutes of constant exposure. The decrease in intensity in Figure 2 seems more evident than in Figure 1, clearly exhibiting effects of photobleaching.

Finally, we captured evidence of photobleaching of rhodamine stained F-actin. We initially set the exposure time to 8,500 ms, but found that it was difficult to visualize those results. Therefore we completed the experiment again with a lower exposure time of 7,500 ms. Figure 3 visualizes the results from the second trial of photobleaching the sample. Figure 3 reveals a more rapid decrease in intensity as picture B and C look relatively similar, unlike those time points from Figure 1 and Figure 2.