Discussion Paragraph Bio 477H

The rhodamine labeled f-actin did not appear in the composite image of the three different fluorophores due to the intensity of the fluorescence being too low to register. This could be due to photobleaching that had previously occurred in this specific area of the sample, or if the sample was not labeled adequately with enough fluorescent dye. Time-lapse data for all three fluorophores under the same condition revealed discrepancies in rate of decay and initial intensity for each fluorophore. A relative high initial intensity for DAPI labeled dsDNA can be explained by the relative high net local concentration of bright fluorophores. Each nucleus contains a high concentration of dsDNA, which when stained with DAPI, creates a large solid fluorescent region with overlapping fluorophores. This differs from both tubulin and f-actin, which are of tube like nature, and appear as porous regions of interest where background light can seep through and be analyzed, making the initial brightness in the region of interest inherently darker. This difference is also reflected in the rates of decay, where in samples where there is less light to be diminished, the rate of decay appears much slower as it approaches the plateau of photobleached darkness. Time-lapse data for fluorescein under stained tubules under different conditions demonstrated discrepancies in rate of decay and initial intensity for all three conditions. The relative low rate of decay and low initial intensity for the images taken with a neutral density filters and auto shutter can be explained by the effect of the neutral density filter. The collective effect of the neutral density filters decreases the intensity of the epi-illumination light path by a factor of 32. This in turn lowers the ability for the fluorophores to be excited, causing a low initial intensity and low excitation, which leads to a lower rate of decay. The auto shutter condition displays a lower rate of decay compared to the open shutter condition due to the lower exposure to light over time. When the shutter is left on automatic, the sample is only exposed to light during the time an image is snapped as opposed to the entire duration of the time lapse. This lessens the time window that fluorophores can covalently bond to oxygen and other elements, and less brightness is lost over time. The open shutter condition leaves the sample exposed to the epi-illumination light path during the entirety of the time-lapse, allowing more time for covalent bonds to form with fluorophores and thus lose more brightness intensity over time. This explains the high relative rate of decay.