<title>Fluorescence-lifetime imaging microscopy with a high-repetition gated camera and a dual-view assembly for real-time measurement</title>

Abstract

A novel method for real-time imaging of fluorescence lifetimes employing a new framing camera will be introduced. Based on a unique operating principle, this framing camera can be gated with a 480 picosecond shutter. The camera consists of an image converter with a pair of deflection electrodes and an aperture. As the photoelectron image passes through the deflection electrodes, an electric field is applied externally. The photoelectron image is thus deflected, and swept on the aperture. High speed gating can hence be accomplished. We are now constructing a fluorescence lifetime imaging microscope system employing this framing camera whereupon the repetition rate has been increased to 4 MHz. Although application of this method requires multiple acquisitions at different delay times, it enables us for the first time to observe real-time intracellular phenomena. This is achieved by combing the system with a dual-view assembly, composed of a pair of beam splitters and mirrors which produce a certain delay, hence enabling us to capture two time-resolved images simultaneously in a single operation of the framing camera. These two images have different delay times, each of which can be set from 300 picoseconds to 2 nanoseconds. The two images are used for the analysis based on the assumption that the decay is single exponential. We will describe an example of application of the system towards the observation of cellular phenomena.