While superresolution microscopy has opened the doors to insights into biological phenomena we couldn’t have dreamed of in the last century, its methodology is naturally limited. We aim to push the envelope of its capabilities by testing the effect that Ca2+ and H+ ions have on the fluorescent protein Dendra2. Utilizing a newly designed perfusion chamber, we flow separate solutions containing Ca2+ and H+ ions into a cellular environment, in which the cells in question have been tagged with Dendra2. Utilizing the superresolution technique known as Spectral Fluorescence Photoactivation Localization Microscopy, we are able to obtain information about the emission intensity and emission spectrum of Dendra2 while under the influence of these ions. Preliminary findings suggest that Ca2+ ions have no effect on the emission intensity of Dendra2. Our results testing the effects H+ ions agree with previous findings that acidic environments reduce the bulk emission intensity of Dendra2. Our findings on emission intensity work to increase the imaging capabilities of Dendra2, as increasing emission intensity and prolonging photobleaching are both highly desirable effects when it comes to producing high quality videos of microscopic biological phenomena. Unfortunately, due to a poor calibration between our spectral and space channels, we were unable to observe the emission spectra of Dendra2 under the effect of these ions. Potential shifts in emission spectra were found to be smaller than our localization precision, further suggesting poor calibration data and emphasizing the delicate, precise nature of the FPALM imaging procedure.
Waterman, Benjamin, "Investigating Ions’ Effects on the Fluorescent Protein Dendra2" (2019). Honors College. 563.