Time-resolved fluorescence instrumentation

The above section has outlined the physical parameters that describe the fluorescence process. One can measure the fluorescence spectrum, P(X), the singlet excited state lifetime, xs, and determine the Tliese parameters can be interpreted in terms of the structure, environment and ( mamics of the molecule of interest. In this section, the different optical and electronic components comprising an instrument that can measure Fl( ) and will be described. This instrument is generally known as a steady state fluorescence spectrometer, since it integrates the fluorescence intensity over a given time period. Time-resolved fluorescence instrumentation that is used to measure the excited singlet state decay times is described in Chapter 3. 

Product and services of interest include genomics, proteomics, custom research and services, and instruments, accessories, consumables and software. PerkinElmer proprietary technologies include time-resolved fluorescence which is employed in the sensitive Wallac DELFIA system and Wallac LANCE homogeneous assay system, and fluorescence polarization, [FP]2 , a robust fluorescence-based technique for receptor binding assays that is both fast and easy-to-use. 

Wang X. F., Periasamy A., Wodnicki P., Gordon G. W. and Herman B. (1996) Time-Resolved Fluorescence Lifetime Imaging Microscopy Instrumentation and Biomedical Applications, in Wang X. F. and Herman B. (Eds), Fluorescence Imaging Spectroscopy and Microscopy, Chemical Analysis Series, Vol. 137, John Wiley ... 

Time-resolved fluorescence spectroscopy has resulted in significant advances in our understanding of the structure and dynamics of biological macromolecules.<13) There can be no doubt that such experimentation has contributed immensely to our present understanding of biological macromolecules and their assemblies. At present, time-resolved measurements require relatively complex instrumentation, resulting in a number of monographs on this topic.<4 6)... 

D. J. S. Birch, A. S. Holmes, J. R. Gilchrist, R. E. Imhof, S. M. A1 Alawi and B. Nadolski, A multiplexed single-photon instrument for routine measurement of time-resolved fluorescence anisotropy, J. Phys. E Sci. Instrum. 20, 471-473 (1987). 

Volume 4 is intended to summarize the principles required for these biomedical applications of time-resolved fluorescence spectroscopy. For this reason, many of the chapters describe the development of red/NIR probes and the mechanisms by which analytes interact with the probes and produce spectral changes. Other chapters describe the unique opportunities of red/NIR fluorescence and the types of instruments suitable for such measurements. Also included is a description of the principles of chemical sensing based on lifetimes, and an overview of the ever-important topic of immunoassays. 

In addition to fluorescence intensity and polarization, fluorescence spectroscopy also includes measurement of the lifetime of the excited state. Recent improvements in the design of fluorescence instrumentation for measuring fluorescence lifetime have permitted additional applications of fluorescence techniques to immunoassays. Fluorescence lifetime measurement can be performed by either phase-resolved or time-resolved fluorescence spectroscopy. 

In the past ten years, numerous applications of fluorescence methods for monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted the development of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation for lifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician s office testing, future innovations in fluorescence immunoassay development will be expected to center on the simplification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing. 

Figure 8.9 Time-resolved fluorescent lifetime analysis of Cy3 attached to double-stranded DNA (Iqbal et al., 2008b). Fluorescent decay curve for Cy3 attached to a 16 bp DNA duplex, showing the experimental data and the instrument response function (IRF), and the fit to three exponential functions (line). The decay curve was generated using time-correlated single-photon counting, after excitation by 200 fs pulses from a titanium sapphire laser at 4.7 MHz.

Time-Resolved Fluorescence. This instrumental technique may be applied to most immunoassays that require fluorescence intensity measurements,... 

Fig. 4 Time-resolved fluorescence emission decays of PFO in toluene solution obtained at 295 K, with emission collected at 400 nm and 540 nm. Also shown is the instrument response function (IRF) which is deconvoluted with the decay fits to yield a time resolution of 3 ps for the fitted decays...

The LDH/NADH pyruvate ternary complex concentration is quite low, and it was found that the concentration of LDH/NADH + pyruvate equals approximately that of the LDH/NAD+ lactate. A temperature increase tips the equilibrium from right to left. Figure 15.10 shows the time-resolved fluorescence emission of NADH at 450 nm in response to a T-jump from 10 to 23 °C. There are two instrument response times one near 30 ns, which is the pulse width of the laser irradiation heating the sample, and the second is diffusion of heat out from the laser interaction volume that occurs around 15 ms (the latter response is not shown). Fitting the data (solid line) with a function of multiexponentials yielded four rates, as indicated on Fig. 15.10, in addition to these instrument response functions. 

There are also third order instruments available, e.g., GC-GC-MS, LC-MS/MS or time-resolved fluorescence excitation-emission. Such instruments also have the second-order advantage, but the problems of reproducibility become even more pronounced. 

The measurement of the time-dependent depolarization of the fluorescence from molecules rotating on a time-scale comparable to the fluorescence decay time, enables information to be derived concerning the molecular reorientation motion. A review of these techniques has been published. A method involving an optical delay line has been used to record time-resolved fluorescence depolarization methods using only 1 photodetector, and thus some of the possible instrumental distortions are removed. ... 

In this chapter we have mentioned only a few of the more important future developments which can be foreseen in colloid science. Many of these will depend on the availability of modern instrumentation and of powerful computer facilities. In addition to the techniques dealt with in this chapter, mention should also be made of the contributions from greatly improved electron microscopic techniques, ultracentrifuges, and X-ray equipment. Other techniques that will become of increasing significance include dielectric measurements, electrical birefringence, and time-resolved fluorescence. 

Several recent experimental studies in small hydride molecules have indeed demonstrated that molecular rotation is instrumental in accepting the energy. Thus the relaxation in the 4 11 state of NH and ND was examined using pulsed UV laser excitation and time-resolved fluorescence studies. The data showed that, contrary to the energy-gap law predictions, NH relaxes order of magnitude faster than ND. Very similar behavior was observed in the state of OH in solid Ne. ... 

During the last two decades, there has been an enormous increase in the use of photophysical methods in supra-molecular chemistry. Until recently, photophysical methods, such as transient spectrometry and time-resolved fluorescence spectrometry, were primarily research tools in the arenas of photokinetics of small molecules, materials physics, and biophysics. This situation changed dramatically with the introduction of commercial, user-friendly electro-optical components such as charge-coupled detector (ED)-based spectrometers, solid-state pulsed lasers, and other instrumentation necessary for time-resolved measurements. As a result, time-resolved spectrometry became more available to the community of supramolecular chemists, who now reached the level of sophistication that can benefit from the new horizons offered. 

---