Fluorometer time-resolved

Soini, E., and Kojola, H. (1983) Time-resolved fluorometer for lanthanide chelates—A new generation of monisotopic immunoassays. Clin. Chem. 29, 65-68. 

The fluorescence lifetime can be measured by time-resolved methods after excitation of the fluorophore with a light pulse of brief duration. The lifetime is then measured as the elapsed time for the fluorescence emission intensity to decay to 1/e of the initial intensity. Commonly used fluorophores have lifetimes of a few nanoseconds, whereas the longer-lived chelates of europium(III) and terbium(III) have lifetimes of about 10-1000 /tsec (Table 14.1). Chapter 10 (this volume) describes the advantages of phase-modulation fluorometers for sensing applications, as a method to measure the fluorescence lifetime. Phase-modulation immunoassays have been reported (see Section 14.5.4.3.), and they are in fact based on lifetime changes. 

Self (S4) first proposed the concept of noncompetitive assay for haptens utilizing an adequate combination of an a-type and a jS-type anti-idiotype antibody, in which he used the term, selective antibody for the a-type antibodies. Then, Barnard and Cohen (Bl) applied this assay principle for the determination of serum E2, naming the assay system an idiometric assay. Figure 12A illustrates the assay procedure of the idiometric assay of E2. The target hapten is captured by excess anti-E2 antibody immobilized on microtiter strips by incubation at room temperature for 1 h (step i). After washing the strips, the /3-type anti-idiotype antibody was added in order to saturate (or block) the unoccupied paratope of the anti-E2 antibody (incubation, room temperature for 30 min) (step ii). The a-type anti-idiotype antibody, which has been labeled with a europium chelate (H4), was then added to the plate and incubated at room temperature for a further 2 h (step iii). Finally, fluorescence intensity due to bound europium was measured with a time-resolved fluorometer. Because of large steric hindrance around the bound jS-type antibody (MW 150,000), the labeled a-type antibody would. 

The time delay between absorption of quanta of energy and fluorescence is used in fluorescence instrumentation called time-resolved fluororaeters. The advantage of a time-resolved fluorometer is the efimination of background fight... 

In addition, to the basic spectrofluorometer discussed earlier (see Figure 3-17), other types of fluorometric instruments include a ratio-referencing spectrofluorometer, time-resolved fluorometer, flow cytometer, and hematofluorometer. 

Soini E, Kojola H. Time-resolved fluorometer for lanthanide chelates A new generation of nonisotopic immunoassays. Clin Chem 1983 29 65-8. 

A small number of methods allow for simultaneous determination of FSH and LH in a single assay tube. Doublelabel RiAs using Co-labeled LH and I-labeled FSH as tracers are available as commercial kits. Following separation, each radioisotope is determined in the bound fraction by dual isotope counting or by repeat counting. A simultaneous immunofluorometric assay of LH and FSH, based on the use of the fluorescent lanthanides Eu " " and Tb, has also been described each is detected with a time-resolved fluorometer. ... 

Lanthanide labelled antibodies or antigens, can be applied to most assays based on solid-phase separation. Reagents immobilized to microtitration plates allow an easy and efficient separation of the unbound fraction by using a plate washer. The bound fraction of the labelled gagent is quantified with a specially designed time-resolved fluorometer (DELFIA Research Fluorometer, Victor Multilabel Counter, Wallac Oy, Turku, Finland). 

As a result of enormous development in the technology and production of pulse lasers, laser diodes, detector systems, and powerful computers in recent decades, steady-state and time-resolved fluorometers now belong to the standard equipment of biochemical and macromolecular laboratories. For example, there are apparatuses combined with microscopes that are suitable for time-resolved fluorescence measurements of individual organelles in living cells. However, the widespread use of fluorescence techniques generates certain danger, which is connected with their routine use. We would like to point out that the fluorescence spectroscopy is an indirect technique and that the interpretation of results needs great care and precaution. It almost always requires additional information on the system. 

Abstract The unique properties of lanthanides as analytical luminophores may only be efficiently exploited when the measuring instrument has been especially designed for this particular purpose. Nowadays, many commercial plate fluorometers can be equipped for time-resolved measurements. However, not all of them perform to the hmits of the luminophores time-gating alone does not crmstitute an optimally sensitive measuring device but other factors such as the performance of optical components need also to be considered. The scope of this chapter is to underline these special, practical design requirements and solutions rather than give generic outlines on how fluorometers need to be bruit. 

Keywords Fluorescence lifetime Fluorometer Optical filters Optical materials Photon counting Spectrofluorometer Time-resolved fluorometry Time-gating... 

After the excitation pulse, the intensity of luminescence emission decays with the rate that is characteristic to the used label. The photons emitted during the delay time, the time between the pulse and activation of the detector, are lost and, thus, a short delay time is preferred in the detection. The background luminescence decays typically very rapidly (within tens of nanoseconds) and long delay times are not required. However, many used light sources exhibit a switch-off delays and afterglow that prevent the use of short delay times. This switch-off time is an important characteristic of a light source and may limit the performance of a time-resolved fluorometer significantly. 

In order to apply the Perrin equation [Eq. (16)] the value for the fluorescence lifetime of the excited state at different temperatures has to be determined. In the absence of a fluorometer with time-resolved measurement capabilities, this is performed by measuring the total fluorescence intensity [Eq. (3)] as a function of temperature. This was performed only for paraffin oil, because the other samples showed high interference. The fluorescence intensity reaches a plateau at 0-10°C. The value for the lifetime of DPH in the absence of dynamic quenching processes (at low temperatures) is 11.4 ns (Xo) and is proportional to the plateau... 

If the signal decay is a single-exponential curve, equations 16 and 17 result in values for X that are in agreement with each other. Dissimilar values indicate multiexponential decay, which usually means that the sample contains more than one fluorophore. Multiexponential decay can be resolved by using a phase fluorometer with phase sensitive detection. A time-independent, direct-current signal is produced that is proportional to the cosine of the difference between the phase angle of the detector ( D) and the phase angle of the fluorescence ( ) ... 

In Chapter 5 we described the use of the FD method to measure lifetimes and to resolve complex intensity decays. In FD fluorometers, the sample is excited with intensity-modulated light, and one measures the phase shift and modulation of the emission, both relative to the excitation. The FD method also allows several other types of measurement which can be useful in special circumstances. One method is measurement of phase-sensitive intensities and/or emission spectra. Another method is to use the measured phase and modulation values to resolve the components of species in a mixture based on known decay times. 

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