The numbers of detected photons/frame from single molecules (N, proportional to the emitted photons during a frame time) are saturated under stronger excitation laser intensities, and thus the improvement of single-molecule localization precision (σ xy ) with an increase of the laser intensity is limited. For experimental details, see the caption to Fig. S2. (A) For various commonly used fluorescent probes, the numbers of detected photons/molecule/frame are plotted against the laser power density at the focal plane (TIR illumination; the additional examinations using oblique illuminations were only performed for Cy3 and 5xCy3-Tf), showing that Cy3 and Alexa555 are less prone to laser-power saturation. With an increase in the excitation laser intensity, the number of detected photons from single dye molecules initially increased proportionally, and then leveled off (saturation occurred), probably due to “triplet bottleneck saturation” (see “Estimation of the number of photons that can be emitted by a single Cy3 molecule during 0.1 ms: Triplet bottleneck saturation” in Materials and methods). This occurred from around 23 µW/µm2 for Cy3 (the results of Cy3 at 10 and 30 kHz shown here are the same as those shown in Fig. 1 E, and are reproduced here for ease of comparison with the results of other dyes). Cy3 and Alexa555 are less prone to saturation, as compared with the other dyes tested here. In the present study, we primarily used Cy3. (B) Distributions of the localization precisions of single molecules of eight fluorophores observed at 10 kHz (an integration time of 0.1 ms), single Cy3 molecules at 30 kHz (0.033 ms), and 5xCy3-Tf at 45 kHz (0.022 ms), evaluated at various laser illumination intensities (provided on the right). Arrowheads indicate the median values. (C) For various commonly used fluorescent probes, single-molecule localization precisions (mean ± SEM) are plotted against the laser intensity (the results for Cy3 at 10 and 30 kHz shown here are the same as those shown in Fig. 1 F, and are reproduced here for ease of comparison with the results of other dyes). These results show that Cy3 and Alexa555 provide better single-molecule localization precisions at higher laser intensities, due to their lower tendency to saturate. Since Cy3 provided slightly better single-molecule localization precision at 10 kHz at saturation than Alexa555, we primarily used Cy3 throughout the remaining part of this report. Based on the results described in A and C, and also due to the versatility of the oblique-angle illumination to enable the observations of single molecules in both the basal and apical PMs, as well as in endomembranes and the cytoplasm in general (see the subsection “TIR and oblique illuminations for ultrafast SFMI” in the main text), we comprehensively tested and performed ultrafast single-molecule imaging-tracking under the oblique-angle illumination conditions, with a laser power density of 23 µW/µm2 at the specimen plane. These are the “standard test conditions,” using Cy3 molecules on the coverslip as the standard sample.
