Measurements of time-resolved fluorescence are increasingly used for research in biophysics, biochemistry, cell biology and medicine. Advances in the technology of light sources and detectors are resulting in more reliable and/or advanced instrumentation, which is resulting in the expanding applications of fluorescence spectroscopy. Time-resolved measurements are often performed by direct measurements in the time-domain. In this article the authors describe the alternative method of frequency-domain fluorometry. The frequency-response of the emission to intensity-modulated excitation can be used to recover the time-dependent decay. Commercial instrumentation now allows measurements to an upper light modulation frequency limit of 200 MHz. This laboratory has developed second and third generation instruments which allows measurements to 2 GHz and subsequently to 10 GHz. The frequency-domain data from such instrumentation provides excellent resolution of picosecond decays of intensity and anisotropy. Additionally, the frequency-domain method appears to provide remarkable resolution of complex decays which are often observed for biochemical samples. In this article the authors describe this instrumentation and applications of this method. Examples are shown using probes with ps decay and correlation times, the intrinsic fluorescence of proteins, and the measurement of end-to-end diffusion in proteins and/or flexible molecules.
We present data on lifetimes of hemoglobin solutions obtained using a 10 GHz frequency domain fluorometer and a specially designed cuvette which allows front face excitation on a free liquid surface. The cuvette eliminates reflections and stray emissions, which become significant for low intensity fluorophores like hemoglobin. Three lifetimes are detectable in the subnanosecond range. At high frequency it is possible to detect components below 10 ps. The average lifetime of hemoglobin is ligand dependent, opening a new chapter in the investigation of the allosteric behavior of hemoglobin.
We describe the design and performance of a 10 GHz harmonic-content frequency-domain fluorometer. The modulated excitation is provided by the harmonic content of a train of ps pulses. High speed and/or high frequency detection was attained with a triode-type MCP PMT from Hamamatsu, R-2566-6, with 6 micron channels. To minimize the cost of the electronic components, and to minimize the noise due to these components, the detection circuits consist of two ranges, 10 MHz to 2 CHz and 2-10 GHz. The upper frequency limit of 10 GHz is determined by the current MCP PMT, so the usual configuration includes a low noise 2-10 GHz amplifier. This amplifier is easily replaced with a 2-18 GHz amplifier which allows operation to 18 GHz and the use of faster PMTs, should they become available in the future. Measurement of known optical delays demonstrates the accuracy of the instrument. For instance, a 1.69 ps optical delay was measured as 1.7 0.4 ps from 0.5 - 10 GHz, and 1.7 0.2 ps from 2- 8 GHz, where the uncertainty indicates the maximum deviation from the expected value. The data were shown to be free of systematic errors by measurements on fluorophores with single exponential decays.
We report fluorescence studies with the single trp protein, S. nucelase A, and several of its site-directed mutants. One of these mutants, PA56, which has an alanine at position 56 in place of proline, has a much lower structural stability than the wild type. This is demonstrated by the much lower Tm (30 degrees C) for PA56 than for the wild type (52 degrees C) and by a much lower (urea)1/2 for denaturation of the mutant. Also we show that PA56 can be unfolded by relatively low hydrostatic pressure (~700 bar). The free energy for unfolding of PA56 is found to be only 1.3 kcal/mole (at 20 degrees C) by thermal, urea, quanidine and pressure unfolding. Fluorescence lifetime measurements with wild type nuclease and several of its mutants show non-exponential decay kinetics. The fluorescence decay profiles are similar for the native state of each protein and the decay data at various temperatures generally reveal differences in the Tm for the various mutants. Anisotropy decay data are analyzed in terms of two rotational correlation times, a longer one for overall rotation of the protein and a shorter one for rapid, segemental motion of the trp residue. The mutant PA56 can be easily denatured by temperature, pressure or urea, and anisotropy decay data for these various denatured forms are reported.
Conference Committee Involvement (7)
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