Live-cell dSTORM data acquisition conditions established for HMSiR-labeled FA molecules. (A) The MEF cells used in this study stably express mEos3.2-paxillin at the level of 0.64× of the endogenous paxillin in the parental MEFs. The figure shows the paxillin Western blot membrane and the plots of paxillin band intensities of the parental MEFs and paxillin-null MEFs (Sero et al., 2011) rescued by the stable expression of mEos3.2-paxillin (cloned), showing that mEos3.2-paxillin–rescued paxillin-null cells (mEos3.2-paxillin–rescued MEFs) express 0.64× the endogenous paxillin in the parental MEF cells. For the dSTORM experiments, the mEos3.2-paxillin–rescued MEFs were further transfected with the Halo-paxillin cDNA for transient expression, and the expressed Halo-paxillin was labeled with HMSiR at ≈ 90% efficiency (Morise et al., 2019). dSTORM observations were performed using live cells exhibiting similar levels of the HMSiR-labeled Halo-paxillin signal. These cells were found to express Halo-paxillin at the level of 0.16× the endogenous paxillin in the parental MEF line, based on the following observations and calculations (and thus these cells express a total of 0.8× the endogenous paxillin; i.e., 0.64× for mEos3.2-paxillin and 0.16× for Halo-paxillin). The ratio of detected spot densities of mEos3.2-paxillin vs. those of HMSiR-Halo-paxillin in the FA was ≈1.4:1. Considering that (1) the number of on-events for a single HMSiR molecule is 2.7 (B d) and that for mEos3.2-paxillin is 1.4 (Fig. 2 A-e), and (2) 90% of HMSiR and 60% of mEos3.2 are fluorescent, the copy number ratio of mEos3.2-paxillin vs. Halo-paxillin including non-fluorescent molecules is estimated to be ≈4.1:1 (1.4/1.4/0.6: 1/2.7/0.9 = 1.667:0.411). Therefore, the amount of Halo-paxillin in these MEF cells is 0.64x/4.1 = 0.16x (0.64x is the ratio of mEos3.2-paxillin vs. endogenous paxillin found in parental MEF cells here) of the amount of endogenous paxillin in the parental cell line. (B and C) First, we will give an overall explanation, and the detailed legends will be presented later. For establishing optimal dSTORM data acquisition conditions, we first examined the on-period durations of HMSiR because this will limit the data acquisition frame rate for dSTORM. With an increase of the TIR excitation laser illumination intensity at a wavelength of 660 nm from 2.2 to 43 µW/µm² in the sample plane, the on-period durations gradually decreased and plateaued at 2.1 ms at a laser intensity of 23 µW/µm² (Fig. S3 B-a), showing that further increases of the laser intensity will not improve the data acquisition frame rate. Therefore, we decided to use a camera frame rate of 1 kHz for dSTORM data acquisition, which is the same rate as that employed for PALM data acquisition using mEos3.2. Further increasing of the illumination laser intensity beyond 23 µW/µm² continued to increase the numbers of detected photons during the on-period of a single HMSiR molecule, with a concomitant improvement of the localization precision of a single dye molecule for a single on-event. However, the extent of improvement was quite limited (Fig. S3 B-b and -c), and thus increasing the laser intensity beyond 23 µW/µm² was deemed not worthwhile, due to the increased probability of photo-damage to live cells. Since the illumination by a 561-nm excitation laser intensity at 23 µW/µm² for 1 min had minimal impact on cell viability (Fig. 2, D and E, in the companion paper), and since this laser intensity is about optimal for the 1 kHz data acquisition rate for HMSiR (on-duration of 2.1 ms; Fig. S3 B-a), we chose to use the 660-nm laser intensity of 23 µW/µm² for the dSTORM experiments. At this laser excitation power density, the on-period reached the plateau at 2.1 ms, providing 477 ± 6.4 photons and a single-molecule localization precision of 19 ± 0.15 nm per on-event (Fig. S3 B-b and -c), and the mean number of on-events per HMSiR molecule was 2.7 (Fig. S3 B-d). At a laser intensity of 23 µW/µm², after the illumination for 10 s (10,000 frames at 1 kHz), ≈80% of HMSiR was photobleached (Fig. S3 C), indicating that the data acquisition for 10 s is close to the optimal conditions for the photon usage. In order to ensure single-molecule detection conditions for all experiments while employing a 660-nm laser intensity of 23 µW/µm² for all ultrafast dSTORM data acquisitions, we adjusted the expression levels of Halo-tagged proteins and/or HMSiR labeling efficiencies. (B) The spontaneous blinking characteristics of HMSiR bound to Halo-paxillin located on the basal PM of live MEF cells observed at 1 kHz (1-ms frame time) with 660-nm excitation laser intensities of 2.2, 6.0, 14, 23, and 43 µW/µm². (a) Duration of on-periods. The histograms show the distributions of consecutive fluorescent on-periods (with a gap closing of 1 frame). They could be fitted by stretched exponential functions $φ(t)=φ0e−(t/τ)α$⁠, where φ₀ is the prefactor, α is the stretching exponent, and τ is the time constant (Morimatsu et al., 2007; mean ± SEM; SEM was determined as a 68.3% confidence limit for the fitting; the number of on-events observed [n] is given in c). (b) Distributions of the numbers of detected photons from a single molecule during a single on-period. The histograms could be fitted with single exponential decay functions, with the decay constants providing the mean numbers of detected photons during an on-period (the SEM was given as a 68.3% confidence limit for the fitting). (c) Histograms of localization precisions for individual on-events of single molecules, which were estimated from the numbers of detected photons during a single on-period using the theoretical equation derived by (Mortensen et al., 2010) with an “excess noise” factor (F) of 1.2 determined for the developed camera system (see Fig. S2 of the companion paper). (d) The distribution of the number of detections (on events) for a single HMSiR molecule (N) bound to Halo-paxillin and observed in the bottom PM of a chemically fixed MEF at the laser power density of 23 µW/µm². Here, the fixed cell was used to exclude the effect of the continuous paxillin exchange between the FA and the cytoplasm. Each detection was found by examining the proximity of the spots recorded at different frames (with a cutoff time of 10 s, which is a typical dSTORM data acquisition period for the observations at 1 kHz employed for this work) with a cutoff distance of $2×3×$ (mean localization precision for HMSiR [19 nm]) = 81 nm. The histogram could be fitted well with the geometric function $f(N)=p∙(1−p)N−1$ based on the model for a monomeric blinking fluorophore by Hummer et al. (2016) (magenta curve), providing the P value (fluorophore bleaching probability) = 0.37 and the mean number of detections (on events)/molecule (1/p) = 2.7. (C) Time-dependent reductions in the numbers of fluorescent spots of HMSiR plotted against the elapsed time after starting the continuous illumination by the 660-nm laser at 2.2 and 23 µW/µm² (frame rates of every 4 and 1 ms, respectively). HMSiR bound to the Halo-paxillin located on the MEF’s bottom PM was detected. The plots represent the sum of the spot numbers in five cells for each condition, normalized to 100% at time 0. The reduction was slower in live cells (blue) than in chemically fixed cells (green), indicating that paxillin in the FA is continuously exchanging with that in the cytoplasm in the time scale of a few tens of seconds, consistent with the previous FRAP data (Legerstee et al., 2019). For longer observations (like 60 s; Fig. 7) we employed an excitation laser intensity of 2.2 µW/µm², whereas 23 µW/µm² was used for shorter observations (like 10 s; Figs. 5 and 6). The total number of counted spots: 1,733,927 in 15,000 frames (blue), 2,476,668 in 15,000 frames (green), and 701,702 in 10,000 frames (red).