Fig. 4.

Objective axial displacement, $z-z_{51}$, translates the beam intensity center of mass (COM) at the TIR interface through, $\overrightarrow{\rho }-{\overrightarrow{\rho }}_{51}$, on the aqueous side of the coverslip. Axial objective translation is expressed relative to its position in Frame 51 at the center of the axial saw-tooth trajectory shown in Fig. 2. Beam intensity COM is calculated using Eq. (2) and from the image of the fluorescent nanospheres collected by the objective.