Pulse fluorometers

Compounds 1,2,3,5,10,11,12,13,14 were dissolved in EPIP (diethyl ether, petroleum ether, isopropanol 5 5 2)whereas compounds 4,6,7,8,9,15 were dissolved in THF-DE (tetrahydrofurane, diethyl ether 1 1). These solvent mixtures can be frozen as glassy samples at 77 K. The absorption spectra were recorded on a standard spectrophotometer SF-10 or Beckman-5270. The measurements of fluorescence excitation and emission spectra were made with the aid of a spectrofluorometer SLM-4800 with automatic correction of spectral response. Fluorescence lifetimes were measured with the aid of a pulse fluorometer PRA-3000. Magnetic circular dichroism (MCD) measurements were carried out in a 8 kG magnetic field using a JASCO J-20 circular dichrometer. Triplet state formation was observed for investigated compounds at the experimental set up, whose detailed description can be found in our paper (27). The optical experiments were carried out with a porphyrin concentration of 4.10- - 4.10 mol.l". In NMR investigations (Bruker WM-360) we used higher concentrations ( 5.10" raol.l ) and dried solvents (CDCl, C 2 and toluene-d0). 

Fig. 6.6. Principles of pulse fluoromet and multi-frequency phase-modulation fluoromet. ...

Luminescence speetra of N in CTA films were recorded on a spectrofluorimeter Jobin and Ivon (France). Fluorescence dec kinetics was recorded on a pulse fluorometer with the pulse lamp flash duration of 3 ns. In the mode of photon counting with the help of a multichannel pulse analyzer Nokia (model LP-4050). 

The first photoelectric fhiorimeter was described by Jette and West in 1928. The instrument, which used two photoemissive cells, was employed for studying the quantitative effects of electrolytes upon the fluorescence of a series of substances, including quinine sulfate [5], In 1935, Cohen provides a review of the first photoelectric fluorimeters developed until then and describes his own apparatus using a very simple scheme. With the latter he obtained a typical analytical calibration curve, thus confirming the findings of Desha [33], The sensitivity of these photoelectric instruments was limited, and as a result utilization of the photomultiplier tube, invented by Zworykin and Rajchman in 1939 [34], was an important step forward in the development of suitable and more sensitive fluorometers. The pulse fhiorimeter, which can be used for direct measurements of fluorescence decay times and polarization, was developed around 1950, and was initiated by the commercialization of an adequate photomultiplier [35]. 

The ability of fluorescence to provide temporal information is of major importance. Great progress has been made since the first determination of an excited-state lifetime by Gaviola in 1926 using a phase fluorometer. A time resolution of a few tens of picosecond can easily be achieved in both pulse and phase fluorometries by using high repetition rate picosecond lasers and microchannel plate photo-... 

Historically, the first instrument for the determination of lifetime was a phase fluorometer (designed by Gaviola in 1926) operating at a single frequency. Progress in instrumentation enabled variable modulation frequency by employing a cw laser (or a lamp) and an electro-optic modulator (0.1-250 MHz), or by using the harmonic content of a pulsed laser source (up to 2 GHz). These two techniques will now be described. 

Phase fluorometers using the harmonic content of a pulsed laser... 

The time resolution of a phase fluorometer using the harmonic content of a pulsed laser and a microchannel plate photomultiplier is comparable to that of a single-photon counting instrument using the same kind of laser and detector. 

Problems with data collection by pulse and phase-modulation fluorometers... 

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. 

There has been a considerable decline in the number of papers which deal with the details of techniques of measurement of fluorescence decay. This is no doubt due to the fact that the alternative methods are now essentially well established. Nevertheless a microcomputerized ultrahigh speed transient digitizer and luminescence lifeline instrument has been described . A very useful multiplexed array fluorometer allows simultaneous fluorescence decay at different emission wavelength using single photon timing array detection . Data collection rates could approach that for a repetitive laser pulse system and the technique could be usefully applied to HPLC or microscopy. The power of this equipment has been exemplified by studies on aminotetraphenylporphyrins at emission wavelengths up to 680 nm. The use and performance of the delta function convolution method for the estimation of fluorescence decay parameters has been... 

Fluorometers and fluorescence spectrophotometers are available that offer a variety of features. These features include ratio referencing, microprocessor-controlled excitation and emission monochromators, pulsed xenon light sources, photon counting, rhodamine cell for corrected spectra, polarizers, flow cells, front-surface viewing adapters, multiple cell holders, and microprocessor-based data reduction systems. 

Phase Fluorometers using the Harmonic Content of a Pulsed Laser... 

P.J. Ralph, R. Gademann, W.C. Dennison (1998). In situ seagrass photosynthesis measured using a submersible, pulse-amplitude modulated fluorometer. Mar. Biol, 132, 367-373. 

Phase fluorometers utilize continuous irradiation by a beam of lighf thaf is sinusoidally modulated. If the frequency of fhe modulation is sef correcfly, there will be a phase difference in the modulation of the fluorescent emission that will depend upon x. Phase fluorometry can yield the same information as does pulse fluorometry.327432,133 gy ysing two or more modulation frequencies the decay rates and fluorescence lifetimes for various chromophores in a protein can be observed. For example, the protein colicin A (Box 8-D) contains three tryptophans W86, W130, and W140. Their fluorescence decays with lifetimes Xj, Xy X3 of -0.6-0.9 ns, 2.0-2.2 ns, and 4.2-4.9 ns at pH 7. While X3 originates mainly from W140, both of the other tryptophans contribute to x and X2. Changes in fluorescence intensify with pH reflect a pfC value of... 

The time decay of fluorescence intensity measurements of a i-acid glycoprotein was performed with an Edinburgh Analytical Instruments CD 900 fluorometer. The technique used was time correlated single photon counting. The sample was excited with series of pulses at a frequency of 20 kHz. The time decay of ai-acid glycoprotein was measured by tlie phase method. 

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