<title>Digital deconvolution FRET microscopy: 3D visualization of protein-protein interactions in a single living cell</title>

Abstract

The spatiotemporal dynamics of dimerization of the transcription factor Pit-1 in the living cell nucleus have been visualized and monitored by expression of genetic vectors encoding green (GFP) and blue (BFP) fluorescent protein fusions and fluorescence resonance energy transfer (FRET) imaging techniques. However, in wide-field FRET imaging microscopy the FRET signals are a combination of signals from all focal planes and the fine image details are obscured by 'out-of-focus' light. Digital deconvolution FRET imaging microscopy is used here to remove the 'out-of-focus' light to improve the resolution of the protein localization in the optical axis. Cells expressing fluorescent Pit-1 fusion proteins were imaged with a high speed, high sensitivity CCD camera and a water immersion objective lens. The point spread function (PSF) of the system was used to deconvolve the donor and acceptor images which then were ratioed to obtain the FRET signal at different optical sections. These signals were used to create three-dimensional visualization of the distribution of Pit-1 protein dimers. Digitally deconvolved data with a water immersion lens has a better signal-to-noise ratio than data obtained with an oil immersion lens. The 3-D energy transfer imaging of Pit-1 protein in the nucleus of living cells offers the possibility of studying domains within the nucleus.