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Photon Counting Histogram Using Numerical Data of Point Spread Function

Photon counting histogram (PCH) is a fluorescence fluctuation method that can quantify the brightness and concentration of different fluorescent molecules in solution at a single molecule level using confocal optics. The current method assumes that the point spread function (PSF) of confocal optics is Gaussian function. However, this could be problematic when the actual PSF profile is not Gaussian. Here, we propose an improved PCH analysis method using numerical PSF data. This method does not require any analytical equation for the PSF profile, and can handle any arbitrary shape of PSF. We report a proof-of-concept estimation of this method using a model PSF.