Fig. 5: Axial position localization accuracy of the 3D localization using the PSF generated through mica substrate.

a A schematic illustration of the experimental configurations. 60 nm fluorescent nanospheres deposited on the glass and mica substrates were embedded in a wedge-shaped thin film of polydimethylsiloxane (PDMS). This experiment was conducted using a 164 µm-thick mica substrate. Fluorescence images were captured through the glass substrate. b Representative images of the fluorescent nanospheres deposited on the mica surface (Particle 2 indicated by white arrowheads) and the glass surface (Particle 1 indicated by yellow arrowheads) captured at varied Z-axis positions. Scale bar = 5 µm. c A schematic illustration of the experimental configurations. The experimental configurations were identical to those in (a), except that fluorescence images were captured through the mica substrate. d Representative images of the fluorescent nanospheres deposited on the glass surface (Particle 1) and the mica surface (Particle 2) captured at varied Z-axis positions. Scale bar = 5 µm. e The gap distances between the nanospheres deposited on the mica and glass surfaces determined at different X positions. The gap distances were determined by capturing the images through the glass (blue) and mica (red) substrates. The solid lines show linear fits. Two sets of samples were prepared and tested to ensure data reproducibility. f A schematic illustration of the configurations for the PSF simulation. A point emitter is placed either on the surface of a 164 µm-thick mica substrate or in the water at a distance from the mica surface of d µm. g Simulated PSF of the point emitter placed on the mica surface (Particle 2) and in the water at a distance from the mica surface of d µm (Particle 1) at varied Z-axis positions.