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. Author manuscript; available in PMC: 2012 Dec 17.
Published in final edited form as: Methods Enzymol. 2012;505:59–80. doi: 10.1016/B978-0-12-388448-0.00012-7

Figure 4.10.

Figure 4.10

Monte Carlo simulation showing the effect of changing the size of the imaging z-step on SNR and precision determination along the z-axis. The calculation was carried out using 3D PSFs (width = 500 nm along the z-axis) and imaging steps of 200, 300, and 500 nm. (A) The SNR drop reflects the effect of photobleaching. (B) Step size has little effect on z-axis precision if the measurements are carried out before photobleaching ensues. The calculation was carried out using 50 PSFs, each simulated by using a Gaussian distribution of fluorescence intensity. The shot noise of each acquisition was obtained from the intensities corresponding to the values on each z-plane replaced with random values generated using the Poisson distribution of the same value. A Gaussian random number generator was added to simulate fluctuations in the background fluorescence. A uniform rate of photobleaching was used for each imaging step.