Fluorescence Detection: Schemes To Combine Speed, Sensitivity And Spatial Resolution

Time resolved fluorescence spectroscopy is rapidly gaining popularity as a tool for studying the structure and dynamics of macromolecules. The multidimensional nature of luminescence has recently been exploited in a variety of data analysis innovations, all of which are intended to reveal the underlying heterogeneity of the emission from macromolecules. Global analysis (1,2) and association methods (3,4,5,6,7) have used multiple emission and/or excitation wavelengths, differing polarizations, concentration variations (especially global Stern Volmer quenching analysis and QDAS;7,8) and system-specific parameters (pH, temperature, and other conformational effectors, see 9) to spread analysis across more dimensions. When conditions change during collection, kinetic decay analysis (10,11) provides another new global view using the nanosecond and second time axes simultaneously ("kinetics of kinetics", see 9). The Laboratory of Technical Development, NIHB1, is dedicated to innovation in instrumental design, and we have focused attention on new, multidimensional approaches to time-resolved spectrophotofluorometry. This contribution will outline the current status of the LTD laser fluorometer along with plans being used for some unusual prototypes now under construction. Since rapid multidimensional data acquisition is likely to accelerate the growth of biochemical applications, we will judge current and planned methods by four criteria: 1) what is the expected limiting lifetime resolution in psec (fraction of TTS)? 2) what gain/sensitivity can be expected (relative to excellent current state of the art)? 3) what acquisition time will be needed for moderate SNR (and does SNR improv.e like (Tacq)**1/2)? 4) how many spatial, wavelength (etc.) channels can be acquired simultaneously? The current (and/or theoretically expected) answers to these questions are summarized in table I.