Localization precision as a function of the z position of the emitter. (A) Images of a fluorescent bead with 2000 photons detected per acquisition at (from left to right) −960 nm, −510 nm, −20 nm, 510 nm, and 970 nm (scale bar is 1 μm). The z position when the two lobes are horizontal is considered to be z = 0. (B) Experimental determination of the localization precision in x, y, and z for (from left to right) 500, 1000, and 2000 photons/frame detected. Note that the localization precision stays relatively constant over a wide range of z values for the 2000 photon case. (C) Theoretical lower limit of the localization precision for (from left to right) 500, 1000 and 2000 photons detected per acquisition. The theoretical data has much lower precision values, but follows a very similar trend to that in the experimental curves.