Brightness, molecular

The fluorescence fluctuations measured by FCS can be analyzed in several ways. The most common technique, autocorrelation analysis, provides information about characteristic diffusion time of fluorescent molecules through the observation volume. It also reports on the average number of molecules present in the observation volume, and thus the concentration of fluorescent moleculesn (14, 49, 56, 57). Other types of FCS analysis can be used to analyze molecular brightness and the oligomeric state of the fluorescent molecule. Finally, cross-correlation FCS monitors fluctuations jointly from molecules labeled with two or more different fluorophores. This technique provides a powerful approach to assay for intermolecular interactions, because molecules that are bound either directly or indirectly to one another should diffuse as a single unit (8, 59). 

Y. Chen, J.D. Muller, Q.Q. Ruan, E. Gratton, Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy, Biophys. J. 82, 133-144 (2002)... 

K. Palo, U. Mets, S. Jager, P. Kask, K. Gall, Fluorescence intensity multiple distribution analysis concurrent determination of diffusion times and molecular brightness, Biophys. J. 79, 2858-2866 (2000)... 

Figure 2.2 Schematic illustration of the conceptual stages in the development of a model to fit photon counting histograms, (a) The case of a non-fluctuating fluorescent particle fixed at the centre of a closed excitation/detection volume (I/q). (b) The case when fluctuations are created by diffusion of the fluorescent molecule around a closed volume with spatially varying excitation/detection efficiency. (c)The case of multiple diffusing molecules in the closed volume, (d) The case when molecules can enter and leave the volume, (e) The case when molecules with different molecular brightness can enter and leave the volume.

Consider the case of two or more distinct species, defined by differing molecular brightnesses, able to diffuse into and out of the volume (Figure 2.2(e)). 

Figure 2.6 PCH for a mixture of the two fluorescent dyes R110 and F27 in 50 mM sodium phosphate buffer at pH 7.0 (open black circles). The molecular brightness of these two dyes differs by a factor of 3.The fit with a single species PCH function (grey line fit parameters are e = 263000 cpspm and N = 52.0 is poor (see residuals) and = 32.1. The fit to a two species PCH model (black line fit parameters are W, = 43.01, Nj = 33.0, = 59200 cpspm and 2 = 321200 cpspm) describes the data well with = 1.1.

Palo, K, Metz, U, Jager, S, Kask, P, and Gall, K, Fluorescence intensity multiple distributions analysis Concurrent determination of diffusion times and molecular brightness. Biophysical Journal 79 (2000) 2858 2866. 

Whether transfer of the dye pair GFP/DsRed or any analogous combinations to in vivo applications will work successfully depends on intracellular photon yields per molecule and the extent of fluorophore oligomerization. Nearly equal molecular brightness values for in vivo and in vitro situations were reported for EGFP implying that molecular brightness issues are unlikely to impede intracellular applications of the presented DsRed/GFP pair. Tetramerization of a reporter construct like STEV-ST devoid of any physiological role probably won t... 

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