Homo-FRET detection in wide-?eld, steady-state microscopy, with mCherry as labeling fluorophore

Abstract

HomoFRET is a method in fluorescence microscopy to measure protein clustering in cells by calculating the anisotropy. In this research Homo-FRET is done with steadystate wide-field microscopy. The mCherry fluorophore is used as labeling fluorophore instead of the common mGFP, because it follows from literature that autofluorescence of cells in red spectrum is lower then in the green part of the spectrum. The method obtained anisotropy values with an error of $5\%$. The main problem of measuring HomoFRET with steady state microscopy turns out to be the camera registration. A depth dependence in registration is possible.