PIE Number and Brightness

Data obtained for PIE RICS can also be used to get information about the number and brightness (N&B) of the particles in each pixel [1,2]. The N&B analysis calculates the particle density and brightness using the first two moments (mean and variance). Even when the sample consists of molecules with different brightness, the N&B analysis provides an estimate of the average number and brightness. For separation of multiple species, a higher-order moment analysis [3] or the photon-counting histogram analysis [4] are more appropriate. In N&B measurements, it is important to choose a suitable pixel dwell time as to not average out the fluctuations. In addition, when using TCSPC detection, the dead-time of the system needs to be accounted for in the analysis. With cross-N&B it is also possible to determine the stoichiometry of a complex of two different labeled species [4].

[1] Digman, M. A., et al. "Stoichiometry of molecular complexes at adhesions in living cells." PNAS USA 106 (2009): 2170-5.

[2] Hendrix, Jelle, et al. "Pulsed interleaved excitation fluctuation imaging."Biophysical journal 105.4 (2013): 848-861.

[3] Sergeev M., et al. "Measurement of monomer-oligomer distributions via fluorescence moment image analysis." Biophysical Journal 91 (2006): 3884-96.

[4] Müller JD, Chen Y, Gratton E. "Resolving heterogeneity on the single molecular level with the photon-counting histogram." Biophysical Journal 78 (2000): 474-486.

[5] Digman, M. A., et al. "Mapping the number of molecules and brightness in the laser scanning microscope." Biophysical Journal 94 (2008): 2320-2332.