Fluorescence lifetime spectrometers

The most relevant difference between TCSPC and the modulation techniques is that TCSPC works at extremely low emission intensity but cannot exploit extremely high intensities, while the modulation technique fails at extremely low intensities but works at extremely high intensities. 

The intensities of classic lifetime experiments are well within the TCSPC range. Furthermore, sensitivity, time resolution, and accuracy are often more important than short acquisition time. Therefore many classic lifetime systems still use the classic NIM-based TCSPC technique. The general principles of fluorescence lifetime experiments are described in [308, 389], and various fluorescence lifetime spectrometers are commercially available. 

The t q)ieal optical setup of a fluorescence-lifetime spectrometer is shown in Fig. 5.5. The sample is exeited by a laser. The excitation intensity is adjusted by a variable neutral density filter. An additional bandpass filter may be required to bloek unwanted emission wavelengths of the laser. 

Unfortunately the monochromator makes the detection light path relatively inefficient. The focal ratio of monochromators is usually not higher than f 3, and the slit width is 0.1 to 1 mm. The focal ratio and the slit width restrict the numerical aperture by which the fluorescence light can be collected from the sample. Either the luminescent spot is magnified too much and does not fit into the monochromator slit, or the light cone becomes wider than the focal ratio of the monochromator (see Sect. 7.2.4, page 279). The slits of most monochromators are perpendicular, but the laser beam usually excites a horizontal line in the cuvette therefore it can be useful to turn the monochromator by 90°. 

The efficiency is further reduced by the efficiency of the grating of the monochromator itself, which is 60 % to 80 % at best. Moreover, the detection bandwidth is usually much smaller than the width of the fluorescence band of the fluorophore. Therefore, the efficiency of a monochromator-based system can be orders of magnitude smaller than that of a filter-based system (see below). 

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Fluorescence lifetimes were measured with a Laser Strobe Fluorescence Lifetime Spectrometer (Photon Technology International) with 337 nm laser pulses... 

Figure 2.24 Decay curve acquired with a PTI TimeMaster TM-3/2003 Ni -dye laser fluorescence lifetime spectrometer. Curtosey from Photon Technology International.

R.W. Wijnaendts van Resandt, R.H. Vogel, S.W. Provencher, Double beam fluorescence lifetime spectrometer with subnanosecond resolution Application to aqueous tryptophan, Rev. Sci. Instrum. 53, 1392-1397 (1982)... 

Boxcar averagers and their use in signal recovery are described in (39). PTI (Photon Technology International) manu ctures a series of fluorescence lifetime spectrometers (models C-70 to C-73) that use a stroboscopic detection technique (i.e. a gated PMT, as described in the previous paragraph) and either nanosecond fiashlamp or nitrogen/ e laser excitation. 

Dyads (1 - 4) were synthesized by the method described previously. Fluorescence spectra were measured in degassed acetonitrile solutions at 2S °C with a Hitachi 8S0 spectrofluorometer. The excitation wavelength was the Soret maximum. Fluorescence lifetimes were measured at 25 °C using a Horiba NAES-500 ns-fluorometer interfaced to an NEC PC-9801 RX personal computer. The excitation light below 420 nm was cut off with a glass filter. The fluorescence was detected by a single-photon counting system and analyzed as the sum of two exponential components after deconvolution of the instrument response function. NMR spectra were recorded on a JEOL JNM GX-270 NMR spectrometer. 

A low cost fluorescence lifetime apparatus, based on the phase-modulafion technique, has been described" " while a new design has been suggested for a photon-counting fluorescence spectrometer that allows rapid accumulation of decay data. A variety of improvements have been suggested for fluores-... 

Figure 12.1 shows the classic L-format of the most commonly used fluorescence spectrometer configuration which is topologically the same for the measurement of both steady-state spectra and lifetimes. The source and detector options of relevance to IR fluorescence measurements are discussed in Sections 12.3 and 12.4, respectively. The other optical components comprised of the lenses for focusing and collection and monochromators for wavelength selection contain few peculiarities in the near-IR as... 

The fluorescence quenching of chlorophyll a in acetone has been examined by single beam photoacoustic spectrometer ". A Langmiur film has been used to observe the effect of molecular organisation on the lifetime and steady state fluorescence of chlorophyll a in... 

Spectroscopic techniques based on the optical microscope are being used with increasing success in photophysics. Microscopic fluorescence decay measurements have been made on both thin liquid films and droplets of concentrated dye solutions.Illustrative data are given for rhodamine B in 20 pm films. A luminescence lifetime microscope spectrometer based on time-correlated single photon counting with an avalanche diode detector has measured... 

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