Time-Resolved Fluorescence Approaches

Two time-resolved fluorescence techniques, pulse Jluorimetry and phase-modulation fluorimetjy, are commonly employed to recover the lifetimes. The former uses a short exciting pulse (from femtoseconds to nanoseconds) of light, which leads to the pulsed response of the sample, which should then be deconvolved from the instrument response. In phase-modulation fluorimetry, the intensity of light used for excitation is modulated at a frequency whose reciprocal is similar to the fluorescence decay time. The sample response is also modulated, but with a time delay, measured as phase shift, from which the emission decay time can be calculated. Thus, the first technique works in the time domain, while the second one in the frequency domain. The most widely used technique in the time domain is the time-correlated single-photon counting [10, 11]. The merits of both techniques have been extensively discussed [12]. 

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Another powerful tool for examining this issue is the use of time-resolved fluorescence spectra, especially when combined with the technique of Time-Resolved Area Normalized Emission Spectra (TRANES) developed by Periasamy and coworkers [78-80]. In this method, separate decay curves are collected over a wide range of emission wavelengths and reconstructed into time-resolved spectra, which are then normalized to constant area. In this model-free approach, it is possible to deduce the nature of heterogeneity of the fluorescent species from the... 

The time-resolved fluorescence spectroscopic approach is very fruitful in elucidating electronic and molecular aspects of laser ablation of fluorescent polymers. While a plasma-like emission and fluorescence behavior of ablated radicals have been reported (21,... 

In a totally independent approach, highly fluorescent but rigid molecules can be used and their reorientational movement probed in solution, mostly by time-resolved fluorescence anisotropy, (85) For rodlike molecules likep-oligo-phenyls, this movement corresponds to the reorientation of the long molecular axis the length of which can be easily varied. 86 The use of fluorescence probes in liquid and solid media and on surfaces has been treated recently in several reviews and books. 87 ... 

Time-resolved approaches for multi-analyte immunoassays have been described recently. Simultaneous determination of LH, follicle stimulating hormone (FSH), hCG, and prolactin (PRL) in a multisite manual strip format has been reported. 88 Four microtiter wells are attached to a plastic strip, two-by-two and back-to-back, such that the wells can be read on a microtiter plate reader. In a quadruple-label format, the simultaneous quantitative determination of four analytes in dried blood spots can be done using europium, samarium, dysprosium, and terbium. 89 In this approach, thyroid-stimulating hormone, 17-a-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM (CK-MM) isoenzyme are determined from dried blood samples spotted on filter paper in a microtiter well coated with a mixture of antibodies. Dissociative fluorescence enhancement of the four ions using cofluorescence-based enhancement solutions enables the time-resolved fluorescence of each ion to be measured through four narrow-band interference filters. 

The approach described above is the simplest variant of time resolved fluorescence detection for the measurement of collision processes. If we choose to excite state A and detect the fluorescence from a second state B, the fluorescence intensity has the form... 

Subsequently, fluorescent MIPs for cGMP were fabricated [46 18, 66, 67]. For that, 1,3-diphenyl-6-vinyl-1 //-pyrazolo 3.4-/ quinoline (PAQ) was introduced as the fluorescent indicator to interact with cGMP in a thin-layer fluorescent MIP chemosensor. Both steady-state (Fig. 1) and time-resolved fluorescence spectroscopy were used as two independent analytical techniques for investigation of the chemosensor properties in the presence of cGMP. Steady-state fluorescence spectroscopy is a common technique applied to MIP sensing. Nevertheless, the use of time-resolved fluorescence spectroscopy combined with microscopy was a new approach to MIP sensing. 

Time-Resolved Spectroscopy. Steady-state solvatochromic techniques provide a reasonable means to study solvation processes in supercritical media (5,17-32,43-45,59-68). But, unless the interaction rates between the solute species and the supercritical fluid are slow, these "static" methods cannot be used to study solvation kinetics. Investigation of the kinetics requires an approach that offers inherent temporal resolution. Fortunately, time-resolved fluorescence spectroscopy is ideally suited for this task. 

Because of the very stringent reliability requirements for diagnostic applications, the only homogeneous techniques that have survived in this field are the fluorescence polarization (FP) methods originally developed by AbbotE and the homogeneous time resolved fluorescence (HTRF) and TRACE approaches developed later by CIS bio international. 

Kakabakos SE, Khosravi MJ. Direct time-resolved fluorescence inunimoassay of progesterone in serum involving the biotin streptavidin system and the iimnobihzed-antibody approach. Clin. Chem. 1992 38 725-730. 

The cyclodextrin-sandwiched porphyrin 18 of Kuroda [98] effectively recognizes hydrophobic quinones in water, with association constants ranging from A a = 7.4 X 10 M for 1,4-naphthoquinone to Ka > 5 x 10 M for an adamantyl-functionalized benzoquinone. In contrast, the association of p-benzoquinone is negligible. Molecular modeling predicts that the quinone approaches the porphyrin from an out-of-plane direction. Time-resolved fluorescence measurements give an estimate of the intramolecular electron transfer rate constant on the order of 10 s for all quinones studied. 

There are several ways to perform time-resolved fluorescence measurements. Since the time dependence of fluorescence emission is typically on a picosecond to nanosecond time scale it is very difficult to achieve. To overcome this difficulty either a frequency domain method or the single photon counting approach is used. 

Because sensitivity is the principal advantage of IA, optimization of assay sensitivity is paramount. Fundamental requirements to optimize assay sensitivity are assay protocol design and reagent concentrations. Computer optimization techniques to improve sensitivity have been reported (13-16). Because of the complexity and variability of assay protocols, empirical approaches are more widely practiced. Ekins compared the sensitivities achieved by different method designs (17,18). Advances in chemiluminescent and time-resolved fluorescent labels enables IA sensitivity to reach attamole levels (18-20). 

Aladan substitution of internal core amino-acid residues provides an approach to characterise the physical characteristics of protein cores. Steady-state fluorescence alone can provide initial insight to the immediate environment of Aladan in the protein core. However, time-resolved fluorescence spectroscopy can be used to understand variations in protein core composition and structure as a function of time through the characterisation of Aladan fluorescence intensity and /max changes that are caused by small fluctuations in the relative permittivity, e, of the protein interior with time (fs-ps timescale). Such spectroscopy is possible since fluorescence lifetimes, Tr, are typically in the ns range (see Section 4.5). Also, time-resolved fluorescence spectroscopy can be performed with non-covalently linked extrinsic fluorophores such as ethidium bromide (EtBr). This fluorophore intercalates between the bases of DNA or RNA double helix and in so doing acquires a substantial increase in (j) and hence fluorescence intensity at /max (595 nm). Should there be a disruption or collapse in double-helical structure, then intercalation fails and fluorescent intensity drops... 

A.C. Mitchell, J.E. Wall, J.G. Murray, C.G. Morgan, Direct modulation of the effective sensitivity of a CCD detector A new approach to time-resolved fluorescence imaging, J. Microsc. 206, 225-232 (2002)... 

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