Yüksek çözünürlükte ışıma mikroskopisi uygulamaları

Confocal microscopy is a technique used for increasing the resolution of microscopeimages at the expense of the acquisition time. As compared with wide field microscopy,confocal microscopy provides a better resolution by rejecting out of focus fluorescent light.Despite this fact, smaller acquisition times can yield wide field microscopy superior toconfocal microscopy in certain applications.Fluorescence Resonance Energy Transfer (FRET) is the nonradiative energy transferfrom an optically excited molecule (donor, D) to an unexcited nearby molecule (acceptor, A).Measurement of the FRET efficiency is a powerful tool in determining the relativelocalizations of the donor and acceptor molecules with nanometer (<10 nm) accuracy. Wehave used total internal reflection fluorescent microscopy to investigate the energy transferbetween two fluorescent molecules Rhodamine B and Fluorescein Na in PMMA thin films.Following the steps of spectra FRET, the efficiency of FRET was quantified by the decreasein donor intensity after energy is transferred from donor to acceptor molecule.We have used confocal microscopy to determine the exact localization of proteins incells. It was observed that the melanopsin proteins are located in cell membrane, while the cryproteins are distributed in cytoplasm of the cell. Experiments were also performed using cellscontaining both melanopsin and cryptochrome tagged with red fluorescent protein (RFP) andgreen fluorescent protein (GFP), respectively. The ultimate goal of these experiments is toobserve whether there is an interaction between these two proteins by measuring the FRETefficiency between GFP and RFP labels.Finally, we observed the single molecule behavior of Rhodamine B molecules inPMMA thin films by wide field microscopy and confocal microscopy.