Photo physical characterization of red emissive fluorophores at cryogenic temperature

Abstract

Single molecule localization microscopy (SMLM) is one of the popular and widely used method of super resolution optical fluorescence imaging techniques. It is a common tool for certain life science researches such as performing high resolution characterization of proteins structure or their distribution over cell plasma membrane. However, fast photo-bleaching rates and lesser blinking events of standard fluorophores at room temperature heavily limits the localization precision and resolution of such methods. In this regard, fluorescence measurements at cryogenic conditions have been observed to greatly improve the photostability of fluorophores, resulting in high photon collection from single molecules which in-turns improves the localization precision. However, insu_cient details about the photophysics of fluorophores at cryogenic conditions may limit its application in super resolution imaging experiments. So, in this thesis we attempted to study certain photophysical properties of two important red emissive fluorophores at cryogenic conditions using our home-build cryostat and Widefield fluorescence microscope. A series of excitation power, exposure time, and excitation interval dependent measurements are performed to find an impact on a few fluorescence properties of these dyes such as the photobleaching, blinking and obtainable localization precision in SMLM. Combining the cryogenic treatment, our cryostat provided enhanced thermal and mechanical stability to the sample which can significantly prolong the life time of fluorescent specimen for several hours. On cooling down to 110K, we observed a large increases in the number of detected photons from single molecules of studied red dyes; Alexa fluor 647 and Atto 647N, which directly improves the attainable localization precision closer to the size of fluorophore.