Fluorescence modulation measurement

Practically, the phase delay and the modulation ratio mR of the light emitted by the scattering solution (solution of glycogen or suspension of colloidal silica) are measured with respect to the signal detected by the reference photomultiplier. Then, after rotation of the turret, the phase delay r/ F and the modulation ratio mF for the sample fluorescence are measured with respect to the signal detected by the reference photomultiplier. The absolute phase shift and modulation ratio of the sample are then — [Pg.179]

Prior to describing the possible applications of laser-diode fluorometry, it is important to understand the two methods now used to measure fluorescence lifetimes these being the time-domain (Tl)/4 5 24 and frequency-domain (FD) or phase-modulation methods.(25) In TD fluorometry, the sample is excited by a pulse of light followed by measurement of the time-dependent intensity. In FD fluorometry, the sample is excited with amplitude-modulated light. The lifetime can be found from the phase angle delay and demodulation of the emission relative to the modulated incident light. We do not wish to fuel the debate of TD versus FD methods, but it is clear that phase and modulation measurements can be performed with simple and low cost instrumentation, and can provide excellent accuracy with short data acquisition times. 

P. Yuan and D. R.Walt, Calculation for fluorescence modulation by absorbing species and its applications to measurements using optical fibers, Anal. Chem. 59, 2391-2394 (1987). 

Figure 11.9. Phase and modulation measurement of fluorescence lifetime, tan = 2 jr/r = phase difference.

Immunoassays based on phase-modulation spectroscopy have been implemented by two distinctly different approaches. Phase-resolved immunoassays rely on fluorescence intensity measurements, in which the emission of one fluorescent species in a mixture is suppressed, and the remainder is quantitated. Phase fluorescence immunoassays utilize measurements of the phase angle and modulation, which change in response to fluorescence lifetime changes. Common aspects of the theory and instrumentation are discussed in this section, followed by individual discussions of the different approaches. 

PFIAs and fluorescence lifetime immunoassays (FLIAs) are uniquely based on measurement of probe emission properties other than the intensity. The phase and modulation are measured, and they directly reflect the fluorescence lifetime of the fluorophore. This provides a major advantage, since the intensity can vary over a broad range, with only minor effects on the results. Phase-modulation measurements can be... 

Fluorescence Correlation Spectroscopy and Fluorescence Burst Analysis. Several nanoscopic chemical imaging approaches work very well for measurements of chemical kinetics, interactions, and mobility in solution. Fluorescence correlation spectroscopy (FCS) measures the temporal fluctuations of fluorescent markers as molecules diffuse or flow in solution through a femtoliter focal volume.54 Their average diffusive dwell times reveal their diffusion coefficients, and additional faster fluctuations can reveal chemical reactions and their kinetics if the reaction provides fluorescence modulation. Cross-correlation of the fluorescence of two distinguishable fluorophore types can very effectively reveal chemical binding kinetics and equilibria at nanomolar concentrations. 

Chlorophyll a fluorescence was measured with a dual-modulated fluorometer (Photosystem Instruments, Trtilek et al. 1997). Minimum (Fa) and maximum fluorescence (Fm) was recorded after dark adaptation (5 min at 4°C, sufficient to attain stabilization of the fluorescence signal for all light regimes). The maximum quantum yield of photosynthesis FvIFm was calculated as (Fm-F0)/Fm (Krause and Weis 1991). 

Munro, I. H., Sabersky, A. P., Synchrotron Radiation as a Modulated Source for Fluorescence Lifetime Measurements and for Time Resolved Spectroscopy in Ref 10, p. 323... 

Fluorescence lifetime measurements are an important aspect of photophysical research. In the past few months the phase-shift measurement technique has become more widely used. This is largely due to the successful achievement of a multifrequency modulation apparatus. An apparatus made from commercially available components has been described and shown to have an accuracy of 10 ps. The performance was checked using mixtures of acridine and quinine sulphate and least-squares-ht procedures. A series of papers from the Illinois group give very detailed account of the state of the art and show the power of the method. The colour delay error arising from the wavelength error in photodetectors can be determined and fluorescence decay times can be obtained with an accuracy of a few picoseconds. ... 

Weber. G. Resolution of the fluorescence lifetimes in a heterogeneous system by pha% and modulation measurements. J. Chem. Phys. In press, communicated privately... 

Tvvo vidcl used approaches are used for lifetime measurcnienis. ilie lime-domain approach and the frt i/iu niy-domain approach. In tinte-domain measurements. a pulsed source is employed and the time-depcndcnr decay of fluorescence is measured. In the frequency-domain method, a sinusoidallv modulated source is used to excite the sample. The phase shift and demodulation of the fluorescence emission relative lo the excitation waveform provide the lifetime information. ( onimercial instrumentation is available to implement both techniques. ... 

Hovenden, M.J., Seppelt, R.D., 1995. Utility of modulated fluorescence in measuring photosynthetic activity of antarctic plants, field and laboratory studies. Aust. J Plant Physiol. 22, 321-330. 

What is the advantage of the cross-correlation method in fluorescence lifetime measurements Fluorescence lifetimes are in the order of the ns or / and ps. Thus, high frequencies from the MHz to the GHz are needed to perform fluorescence lifetimes measurements. Still, the accuracy of the measured values is not reached at high frequencies. Therefore, one can translate the high frequency and phase modulation information to lower frequency carrier signal, Aw. In this case, the measured fluorescence lifetimes are highly accurate. 

Fig. 7.31 Signal-dependent background. Fluorescence signals and dark counts recorded by a cooled H5773-20 PMT module (left) and a R3809U-50 MCP-PMT. The background of the fluorescence decay measurements is substantially higher than the dark count level...

Chemical Mechanisms for Fluorescence Modulation. While UV/visible signalling almost always results from the ionization of a conjugated substituent, there exists a plethora of mechanisms by which fluorescence signal transduction may be engendered. It is useful to categorize the mechanisms for fluorescence modulation described to date via the type of measurement that is made. These are intensity, intensity-ratio, and lifetime. A pictoral summary of each is found in Figure 1 of the chapter by Szmacinski and Lakowicz. 

Intensity measurement By far the most common mode of fluorescence modulation described to date is the increase or decrease of fluorescence intensity at a single emission wavelength upon analyte binding. Methods for halide ion and molecular oxygen analyses rely on non-chelative (coUisional) quenching by these... 

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