Fluorescence rate per molecule

Figure 17.4 Principle of fluorescence correlation spectroscopy the fluorescence intensity temporal fluctuations originating from a well-defined volume are recorded and correlated to estimate the average number of molecules observed and the characteristic fluctuation time. This data is used to compute the average detected fluorescence rate per molecule in the observation volume.

Fig. 10.30 LIF spectroscopy of predissociating levels, where the predissociating rate is fast compared to the collisional quenching rates (Api, fluorescence rate per molecule predissociating rate collisional quenching rate)...

Moreover, the studies on nanoaperture-enhanced fluorescence point out that for a properly tailored aperture, count rates per molecule greater than a few hundred thousands photons per second were readily obtained, whereas for a single molecule in open solution, fluorescence saturation prevents the count rate from exceeding a few tens of kilocounts per second. This allows for fast and reliable screening for single molecules. 

Hassler, K. Leutenegger, M. Rigler, P. Rao, R. Rigler, R. G6sch, M. Lasser, T. Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule. Opt. Express 2005,13, 7415-7423. 

Another important property of fluorescing molecules is the lifetime of the lowest excited singlet state (X/). If the mean rate of fluorescence is the number of fluorescence events per unit of time, the mean lifetime of the excited state is the reciprocal rate, or the mean time per fluorescence event. The quantum yield of fluorescence and the lifetime of the excited state are related by... 

We solve the rate equations to find the number of molecules in level 2 as a function of time, N2(t). Integrating rN2(t) over the laser-molecule interaction time x, we find the number of fluorescent photons emitted per molecule to be... 

The quantity Trad expresses the rate of spontaneous emission per molecule per unit of angular frequency between E/h and E + d ) jh [2]. For a single fluorescence band, in the absence of non-radiative processes, the radiative decay rate (iCrad) the maximum lifetime (tq) are given by... 

In order to study the molecular dynamics of the outer segments of a dendrimer, one pyrene moiety was selectively and covalently attached to one dendron of poly(aryl ester) dendrimers by Adams (in total three pyrene molecules per dendrimer) [24]. The fluorescence decay of pyrene in the THF solution of the labeled dendrimers provided details of the pyrene excimer formation, such as the excimer formation rate, the excimer decomposition rate constant and the equilibrium constant of the excimer formation. These parameters were utilized to evaluate the diffusional mobility of the dendrimer branches. 

The point is now to estimate the maximum number of photons that can be detected from a burst. The maximum rate at which a molecule can emit is roughly the reciprocal of the excited-state lifetime. Therefore, the maximum number of photons emitted in a burst is approximately equal to the transit time divided by the excited-state lifetime. For a transit time of 1 ms and a lifetime of 1 ns, the maximum number is 106. However, photobleaching limits this number to about 105 photons for the most stable fluorescent molecules. The detection efficiency of specially designed optical systems with high numerical aperture being about 1%, we cannot expect to detect more than 1000 photons per burst. The background can be minimized by careful dean-up of the solvent and by using small excitation volumes ( 1 pL in hydrodynamically focused sample streams, 1 fL in confocal exdtation and detection with one- and two-photon excitation, and even smaller volumes with near-field excitation). 

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