Single-photon fluorescence time-resolved detection

The optieal systems used for both techniques are essentially the same. A small sample volume is obtained by confocal deteetion or two-photon excitation in a microscope. Several detectors are used to deteet the fluorescence in different spectral ranges or under different polarisation angles. Therefore correlation techniques ean be combined with fluorescence lifetime deteetion, and the typical time-resolved single-molecule techniques may use eorrelation of the photon data. The paragraphs below focus on single-molecule experiments that not only use, but are primarily based on pulsed excitation and time-resolved detection. 

Brand L, Eggeling C, Zander C, Drexhage K FI and Seidel CAM 1997 Single-molecule identification of coumarin-120 by time-resolved fluorescence detection comparison of one- and two-photon excitation in solution J. Chem. Phys. A 101 4313-21... 

Mosconi, D., Stoppa, D., Pancheri, L., Gonzo, L. and Simoni, A. (2006). CMOS single-photon avalanche diode array for time-resolved fluorescence detection. IEEE ESSCIRC. 564-67. 

Volkmer, A., Hatrick, D. A. and Birch, D. J. S. (1997). Time-resolved nonlinear fluorescence spectroscopy using femtosecond multiphoton excitation and single-photon timing detection. Meas. Sci. Technol. 8, 1339 19. 

The time-resolved techniques that are usually used for FLIM are based on electronic-basis detection methods such as the time-correlated single photon counting or streak camera. Therefore, the time resolution of the FLIM system has been limited by several tens of picoseconds. However, fluorescence microscopy has the potential to provide much more information if we can observe the fluorescence dynamics in a microscopic region with higher time resolution. Given this background, we developed two types of ultrafast time-resolved fluorescence microscopes, i.e., the femtosecond fluorescence up-conversion microscope and the... 

When deciding to study the dynamics of electronic excitation energy transfer in molecular systems by conventional spectroscopic techniques (in contrast to those based on non-linear properties such as photon echo spectroscopy) one has the choice between time-resolved fluorescence and transient absorption. This choice is not inconsequential because the two techniques do not necessarily monitor the same populations. Fluorescence is a very sensitive technique, in the sense that single photons can be detected. In contrast to transient absorption, it monitors solely excited state populations this is the reason for our choice. But, when dealing with DNA components whose quantum yield is as low as 10-4, [3,30] such experiments are far from trivial. 

The great sensitivity of fluorescence spectral, intensity, decay and anisotropy measurements has led to their widespread use in synthetic polymer systems, where interpretations of results are based upon order, molecular motion, and electronic energy migration (1). Time-resolved methods down to picosecond time-resolution using a variety of detection methods but principally that of time-correlated single photon counting, can in principle, probe these processes in much finer detail than steady-state techniques, but the complexity of most synthetic polymers poses severe problems in interpretation of results. 

The bimolecular reaction dynamics of geminate recombination or acid-base neutralization have until recently been studied with time-resolved techniques probing electronic transitions. Time-resolved fluorescence using time-correlated single photon counting detection is limited to a time resolution of a few picoseconds. UV/vis pump-probe experiments, in principle, may have a time resolution of a few tens of femtoseconds, but may be hampered by overlapping contributions of... 

Spectroscopy of single molecules is based on fluorescence correlation, photoncounting histograms, or burst-integrated-lifetime techniques. Each case requires recording not only the times of the photons in the laser period, but also their absolute time. Modem time-resolved single molecule techniques therefore use almost exclusively the FIFO (time-tag) mode of TCSPC. The FIFO mode records all information about each individual photon, i.e. the time in the laser pulse sequence (micro time), the time from the start of the experiment (macro time), and the number of the detector that detected the photon (see Sect. 3.6, page 43). 

The main luminescence parameters traditionally measured are the frequency of maximal intensity Vmax, intensity I, the quantum yield < >, the hfetime of the exited state T, polarization, parameters of Raman spectroscopy, and excited-state energy migration. The usefulness of the fluorescence methods has been greatly enhanced with the development of new experimental techniques such as nano-, pico-, and femtosecond time-resolved spectroscopy, single-molecule detection, confocal microscopy, and two-photon correlation spectroscopy. 

Note that the application of the convolution scheme in the simple form (44) requires that the nonlinear polarization contains only a single interaction with the probe laser field. Apart from the transient transmittance spectrum considered above, this condition is also fulfilled for related detection schemes such as time-resolved fluorescence, ionization, and excited-state absorption. Coherent spectroscopic signals such as the photon-echo, on the other hand, contain two interactions with the probe laser field, thus requiring the calculation of the full three-time response function, followed by a double convolution. 

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