Radiometric calibration of a video fluorescence microscope for the quantitative imaging of resonance energy transfer

Abstract

A video microscope for radiometric imaging of resonance energy transfer (RET) between donor and acceptor fluorophores is described and calibrated. The donor is excited by epi-illumination with monochromatic light. Light emitted by donor and acceptor fluorophores in the specimen is filtered into separate emission images, which are viewed with a pair of multichannel plate image intensifiers in tandem with charge-coupled device (CCD) cameras. The RET rate constant is calculated at each location in the specimen from the ratio of the donor and acceptor emission intensities. These intensities are computed from the video brightnesses after transformation to irradiances using an experimentally obtained calibration, which accounts for the spectral transmission of the optical pathways and the spectral response of the detectors to determine the energies emitted by the probes in the specimen. We describe procedures for measuring the input irradiance–output brightness relations of the intensifier-CCD cameras. With these calibrations, the RET ratio image enables measurement of molecular-scale (nanometer) distances between probes within membranes of biological and biophysical interest.