Recently, Franke, Sauer and van de Linde introduced a way to estimate the axial position of single molecules (TRABI). To this end they compared the detected photon count from a temporal radial-aperture-based intensity estimation to the estimated count from Gaussian Point Spread Function (PSF) fitting to the data. Empirically they found this photometric ratio to be around 0.7-0.8 close to focus and decreasing away from it. Here, we provide an explanation for this reported but unexplained discrepancy and furthermore show that the photometric ratio as indicator for axial position is susceptible even to small optical aberrations.

1. Rasmus Thorsen
2. Christiaan Hulleman
3. Mathias Hammer
4. Daivd Grunwald (David.Grunwald@umassmed.edu)
5. Sjoerd Stallinga
6. Bernd Rieger (b.rieger@tudelft.nl)

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