Optimizing the excitation laser power density and the data acquisition frame rate for the ultrafast PALM imaging of mEos3.2 in living cells. (A) The mean number of photons emitted from a mEos3.2 molecule during an on-period is maximized (49 ± 0.54 photons) at a laser power density of 30 µW/µm² at the sample plane (c), providing a single-molecule localization precision of 29 ± 0.22 nm (d) with an on-period of ≈ 2 ms (a) and a mean number of on-periods before photobleaching of 1.4 (e). Caveolin-1-mEos3.2 expressed at very low levels (<<1 caveolin-1-mEos3.2 molecule/caveola) in the basal PM of T24 cells was imaged using live cells. (a) Histograms of individual fluorescent on-periods (with a gap closing of 1 frame) obtained at various laser power densities at the sample (indicated on the right of b). 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, which is also the same in b; the numbers of spots observed are the same as those indicated in the boxes in b). (b) Distributions of the numbers of detected photons during an 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. n = number of observed spots. (c) Summary plot for the results in b, showing that the laser power density of 30 µW/µm² at the sample plane provides the maximal number of detected photons during an on-period of a single mEos3.2 molecule (49 ± 0.54 photons). (d) The mean localization precision for mEos3.2 in the basal PM was 29 ± 0.22 (SEM) nm under the optimized laser excitation conditions of 30 µW/µm². The localization precision for each on-period of a single mEos3.2 molecule was estimated using the theoretical equation derived by Mortensen et al. (2010), employing an “excess noise” factor (F) of 1.2 determined for the developed camera system (see Fig. S2 of the companion paper). (e) The distribution of the number of on-events (localizations) for a single mEos3.2 molecule (N) at a laser power density of 30 µW/µm². Each detection was found by examining the proximity of the spots recorded at different frames, with a cutoff time of 3 s (Durisic et al., 2014) and a cutoff distance of 82 nm (⁠$2×2×$ [mean localization precision for mEos3.2 = 29 nm]). The histogram could be fitted well (green curve) 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), yielding the P value (fluorophore bleaching probability) = 0.72 and the mean number of detections (on events)/molecule (1/p) = 1.4. (B) The data acquisition at 1 kHz is nearly an optimal frame rate for PALM imaging using mEos3.2 as a probe. The figure shows typical consecutive single-frame images of caveolin-1-mEos3.2 molecules in the basal PM acquired every 1 ms (1 kHz) using an observation laser power density of 30 µW/µm². Based on the number of detections (localizations), caveolin-1-mEos3.2 was found to be expressed at 42 ± 5.2 copies/caveola, which only includes the fluorescent mEos3.2 and not the non-fluorescent mEos3.2, but was normalized by the overcounting of 1.4; n = 15). While many spots exist in only a single image frame, some spots appear in two or three images (spot colors are changed every 1 ms). Using these spot images, the SD of the Gaussian spot profile was determined to be 129 ± 1.3 nm (n = 50 caveolin-1-mEos3.2 molecules). This value was used for reconstructing the diffraction-limited images.