Dronpa-1, Dronpa-2 And Dronpa-3 Separation Using Phase Resolved Optical Lock-in Detection (pholid) Microscopy

Abstract

In living cells, protein complexes are dynamic structures that control many distinct aspects of cell behavior. Addressing the complexity of protein function requires a comprehensive understanding of subcellular localization and protein stoichiometry. Fluorescence microscopy provides a high contrast method to determine specific localizations of individual proteins contained within living cells. Determining localizations of protein pairs is made difficult by inherent photobleaching, cross-talk, and autofluorescence contained within the sample being imaged. Traditionally, to separate fluorescent proteins (FPs) with overlapping spectra, techniques such as linear unmixing and fluorescence lifetime imaging (FLIM) have been used. Such techniques rely on complex microscope configurations and often require a compromise in signal to noise ratios. In these studies we use phase information derived from optical lock in detection (OLID) (Mao et al. Biophys J. 2008 Jun;94(11):4515-2) to separate the photoswitchable FPs Dronpa-1, Dronpa-2, and Dronpa-3 from GFP and autofluorescence with no effect in signal to noise. Additionally, relative stochiometric ratios between each of the Dronpa variants contained within microinjected zebrafish embryos were obtained with a standard confocal microscope.