<title>Time-resolved imaging fluorescence microscopy</title>

Abstract

The extension of microscope luminescence measurements into the temporal domain provides the possibility of determining time-resolved properties of microscope samples and their surrounding environments, and thereby extends the conventional steady state measurements. `Time resolved imaging microscopy' is a relatively new technique whereby fast kinetic and luminescence decay parameters (decay times and the corresponding time or phase resolved amplitudes) are directly and simultaneously measured throughout an image, pixel by pixel, in an optical microscope. Molecular rotation, solvent and matrix relaxation, quenching mechanisms, reactions, and energy transfer are examples of molecular spectroscopic processes that can be studied best by directly measuring the time dependent properties. Dynamic measurements are generally much more informative than their steady state counterparts. The goal of our work is to develop time-resolved methods that can be applied conveniently and routinely to biological material in the microscope over a wide time domain. In addition to the augmented purely spectroscopic and reaction kinetic information, simultaneous spatial and temporal resolution of an image in a microscope provides significant improvement in image contrast, probe identification and differentiation, background (light scattering and inherent luminescence) reduction, and provides additional parameters for digital image analysis. Time resolution makes it possible to recover structures in an image concealed by background luminescence with a different lifetime. Examples of these procedures are given, and the instrumentation required for the data acquisition and analysis is discussed. The technique employs phase-locked coordination between the modulation of the perturbation and the recording of the luminescence image together with a Photometrics (Tucson, Ariz.) series 200 high resolution slow-scan scientific CCD camera. A normal fluorescence microscope is used.