Fig. 5: BrightEyes-TTM for circular scanning FLFS on freely diffusing fluorescent beads.
From: The BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy
a Schematic representation of the concept of circular scanning FLFS. A pulsed laser beam is scanned in circles of radius R (top panel) while both the absolute arrival times (center-left panel) and the start–stop times (center-right panel) are registered. The autocorrelation function of the intensity trace is calculated, from which the size of the focal spot ω0 and the diffusion time τD can be simultaneously extracted. b Start–stop time histograms for the different pixels, bin width 48 ps, total measurement time 226 s, central pixel in black. c Exemplary filter functions for the central pixel data. d Autocorrelations and fits for the central pixel, sum 3 × 3, and sum 5 × 5, for the unfiltered (left) and filtered (right) case. e Diffusion time as a function of \({\omega }_{0}^{2}\) (left) and average number of particles in the focal volume as a function of the focal volume (right). The corresponding diffusion coefficients are (14.3 ± 0.5) μm2/s (unfiltered) and (14.0 ± 0.4) μm2/s (filtered). The fitted particle concentrations are (7 ± 3)/μm3 (unfiltered) and (1.70 ± 0.03)/μm3 (filtered). Setup: custom-built single-photon laser-scanning microscope equipped with a 5 × 5 SPAD array detector prototype.
