Strategies For Spatially Resolved Microspectrofluorometric Analysis

Abstract

Any one of the spectroscopic properties of a fluorescent dye (excitation spectrum, emission spectrum, fluorescence lifetime, or quantum yield) could be used to report on its environment. However, pure fluorescence properties such as quantum yield or absolute spectral properties, while sensitive, require complex measurements and calibration procedures for their evaluation. Instead, it is much more common to use fluorescence flux over some part or all of the emission wavelength band as a monitor of a combination of effects including the composite information monitored by the absorption spectrum, the quantum yield and the emission and excitation spectra. The simplest methods use some ratiometric measurement monitoring either a separate reference dye or the signals at two or more wavelengths to read-out the disproportionation of fluorescent species on a relative scale, but other more complex approaches can be successful including spectral shape analysis and the use of the kinetics of photochemical changes. With the appropriate fluorophore, the fluorescence approach has the advantage that it allows both temporal and spatial resolution on the microscopic scale. However, in practice microspectrofluorometry has been difficult to use for quantitative analysis due to optical, physical and chemical problems inherent to microscopic samples and microspectrofluorometric systems. With the advent of very sensitive image detector systems, computer control and data acquisition, and the advanced optical components described in these proceedings, it has become possible to overcome many of these problems. This paper discusses the measurement strategies needed to effectively use advanced microspectrofluorometer systems to make quantitative measurements using fluorescent probes.