Fig. 3: Theoretical and experimental considerations reveal the upper limit of trackable diffusion rates.

a Fluorescent lipid analogs (DPPE-PEG2000-STAR RED) tagged with Abberior STAR RED were incorporated in homogeneous DOPC:CHOL 7:3 bilayers. (Created in BioRender. Vogler, B. (2025) https://BioRender.com/tmaaqpx). b Single-molecule MSD curves (gray lines), and the ensemble average MSD curve (black line) calculated for trajectories of the fluorescent lipid analogs diffusing in the SLB illustrated in (a). The shading corresponds to the Lower (orange-red) and Upper (orange) Lag Regimes, with an arbitrary threshold set at 5 lags (\(\approx 670\mu s\)). The corresponding distribution of diffusion coefficients (time-average (red diamond) and ensemble-average (black cross)) extracted for the Upper Lag Regime of the MSD can be found as a boxplot (center line, median; red diamond, mean; box limits, first to third quartile; whiskers, 1.5x interquartile range; notch, height proportionally to interquartile range; points, fliers) to the right. The horizontal purple line marks a system-specific reference value taken from literature²⁷. c Upper theoretical limit of trackable diffusion coefficients \(D\) against the average photon detection rate of the particle within one pattern iteration as calculated from Eq. 3, in the absence of noise. Different line styles and colors represent unique TCP diameters \(L\) and photon limits PL as labeled. d Zoom-in of the graph in c) for values of \(D\) and average photon detection rate more common in experimental practice. The black horizontal line indicates the ensemble (and time) average diffusion coefficient \(D\) for the dataset in panel b), while the vertical one represents the average frequency of photon detection \(\left\langle {\psi }_{{iMFX}}\right\rangle\). The shaded dark orange area represents the standard deviation of the distribution of \(D\) from panel b), whereas the lighter orange represents the full extent of the distribution. The vertical dashed lines represent the standard deviation of the photon detection rate.