Wonseok Hwang, Sansu Bae, and Sungchul Hohng
Content:
Fig. S1.
The dependence of s and
σ
on SNR
Fig. S2.
The dependence of s and
σ
on the number of single-molecules
Fig. S3.
Representative refocusing time traces
Fig. S4.
B-Z transition dynamics at varying salt conditions

Fig. S1.
The dependence of s and
σ
on SNR. (a-d) FOM at varying objective positions (solid squares) and their linear fit (red line) for four different SNR conditions. The error bars were generated from 30 independent measurements.
The 4
σ positions are indicated by dashed lines. (e) s (obtained from the fitting graphs in (a-d)) at varying SNR. (f)
σ
(standard deviations at z = 0) at varying
SNR.

Fig. S2. The dependence of s and σ on the number of single-molecules. (a) s at varying number of single-molecules. (b)
σ
at varying number of singlemolecules.

Fig. S3.
Representative refocusing time traces. Representative time traces of the objective lens position during refocusing process from defocuses of –0.1
μm (a, left), +0.1 μm (a, right), –0.5 μm (b, left), and +0.5 μm (b, right).
Experiments were performed under four different SNR conditions: 2.0 (black),
2.9 (red), 4.3 (green), and 6.1 (blue). Auto-focusing system was turned on at 0 frame (black dashed lines).

Fig. S4.
B-Z transition dynamics at varying salt conditions. The representative time traces of fluorescence intensities (Cy3: green, Cy5: red), FRET (gray), and HMM [23] (blue) at (a) 3.1 M, (b) 3.4 M, and (c) 3.7 M NaClO
4
.
Distribution of B-Z transition rates at (d) 3.1 M, (e) 3.4 M, (f) 3.7 M, and (g)
4.0 M NaClO
4
. The transition rates were obtained by HMM. To obtain the transition rates in Fig. 4(f), the histograms were fitted to log-normal distribution (red lines) [24].