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Fluorescent Microscopy Part 2

Submitted by rmmcdonald on Fri, 10/25/2019 - 10:40

    The impact of photobleaching on a specimen can be detrimental and irreversible. Depending on the fluorescent stained utilized, certain fluorophores photobleach at a more rapid rate than others requiring more care. The time lapse images collected from part I reveal the different properties of each fluorophore. Admittedly, the photobleaching rate constants extracted from the trend lines of Figure 4, Figure 5, and Figure 6 suggested unexpected values. Since each of these rate constants were so similar, I felt that no definitive conclusions may be drawn about comparing each of the fluorophores with this data. In future experiments, I will try to increase the number of significant digits in the data collected and increase the overall quantity of numerical data. Therefore, I utilized the mostly qualitative data to reach the following conclusions.

Visually, DAPI appears in Figure 1 to decrease less in intensity than the fluorophores seen in Figure 2 or Figure 3. Furthermore, the overall trend line of the DAPI stained nuclei in Figure 4 exhibits the smallest rate of photobleaching. In comparison, Figure 6 and Figure 7 suggest that the rhodamine stained F-actin and FITC stained tubulin have the same rapid rate of photobleaching. If more trials were conducted then we could have conclusively stated which fluorophore photobleached the fastest. However from these varying trend lines and visual differences, the data would suggest that different fluorophores bleach at different rates. Due to DAPI’s chemical structure it might be less likely to bind to neutral compounds like oxygen that inhibit photon emission. Conversely, Rhodamine and Fluorescein may act as unstable compounds that prefer to bind to other molecules than undergo periodic photon emission and absorption.