Homo-FRET Detection by Fluorescence Polarization Anisotropy in Wide-field Microscopy
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A fluorescence wide-field microscope setup for simultaneous detection of the parallel and perpendicular intensities was used in order to detect homo-FRET between fluorescently labelled proteins, thereby revealing clustering of the biological constructs of interest. Quantitative anisotropy imaging with sub-cellular resolution using seperate detection channels requires high quality registration of the images regarding the data analysis process. Cells expressing green fluorescent protein(GFP) constructs that contain two FKBP binding proteins were used to verify the effect of oligomerization of the constructs to anisotropy. The results show a significant decrease of the anisotropy of $6,5\pm1,2\%$ due to homo-FRET upon clustering of the GFP molecules. Determination of the autofluoresence turned out to be a critical factor in wide-field anisotropy measurements, as the absolute anisotropies show an increase of roughly $2\%$ after subtraction of a slightly higher autofluorescence. However, due to the fact that both the clustered and non-clustered anisotropies increased with the same relative amount, the relative decrease hardly changed. Furthermore, when the resolution of the images is reduced by binning the pixels upon imaging, the decrease in anisotropy is lower and the uncertainty is roughly twice as big: $4,4\pm2,2\%$. Probably, the quality of the registration diminishes due to the lower resolution of the images and hence the overlay is not perfect, resulting in less reliable anisotropy values.