Tracking accuracy. 500-nm fluorescent beads dried onto a coverslip were moved in a sine wave of amplitudes in the range 0.1–10

*μ*m and frequency of 0.02 Hz (

*A*) or in 20 steps of 20–100 nm separated in time by 3 s (

*B*). The tracking routine was run as described in Materials and Methods with

*f*_{data} = 16 kHz,

*f*_{orbit} = 250 Hz,

*n* = 8,

*r*_{x,y} = 0.7

*μ*m, and

*r*_{z} = 2.5

*μ*m. Trajectories recovered for beads moving 1

*μ*m in a sine wave (

*A*) or in 60 nm steps (

*B*). The insets to the figures show the amplitude recovered by fitting the bead trajectories to sine waves plotted as a function of the input amplitude (

*A*) and the average step size recovered plotted as a function of the input step size (

*B*). Dependence of the tracking accuracy on the signal/noise ratio (

*C*). The position of fixed beads with diameters (nm): 552 (□), 100 (○), 24 (▪), and 14 (•) were determined at different laser powers in the range 0.1–15 mW, using the following parameters for the tracking routine:

*f*_{data} = 32 kHz,

*f*_{orbit} = 250 Hz,

*n* = 16,

*r*_{x,y} = 0.7

*μ*m, and

*r*_{z} = 2.5

*μ*m. The accuracy of the tracking was calculated as described in the text. The continuous line represents the fitting of the following function:

with

*α* = 49 ± 5 nm,

*β* = 0.54 ± 0.08. The dashed line represents the theoretical accuracy.

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