ABSTRACT
A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Proteins/analysis , Cell Line , Drug Evaluation, Preclinical , Fluorescent Dyes/chemistry , Green Fluorescent Proteins/chemistry , Humans , Microscopy, Fluorescence , Protein Binding , Rhodamines/chemistry , gag Gene Products, Human Immunodeficiency Virus/analysisABSTRACT
We describe a fluorescence lifetime imaging endomicroscope employing a fibre bundle probe and time correlated single photon counting. Preliminary images of stained pollen grains, eGFP-labelled cells exhibiting Förster resonant energy transfer and tissue autofluorescence are presented.
Subject(s)
Endoscopes , Endoscopy/methods , Microscopy, Confocal/instrumentation , Microscopy, Fluorescence/instrumentation , Animals , COS Cells , Chlorocebus aethiops , Fluorescence , Green Fluorescent Proteins/metabolism , Photons , Pollen , Rats , Tendons/anatomy & histology , Time FactorsABSTRACT
We report the design and implementation of spectroscopic and multicolor imaging capabilities into a fibered confocal fluorescence microscope (FCFM) already capable of in vivo imaging. The real time imaging device and the high resolution fiber probe make this system the first reported capable of performing multi color detection in the field of FCFM. The advantages of the system will allow in vivo morphological and functional imaging. Preliminary experiments were carried out in tissue samples to demonstrate the potential of the technique. The quality of the axial sectioning achieved in the confocal fluorescence spectroscopy mode is demonstrated experimentally, and applications to multicolor imaging are shown.
