The Role of Probe Photophysics in Localization-Based Superresolution Microscopy  Francesca Pennacchietti, Travis J. Gould, Samuel T. Hess  Biophysical Journal  Volume 113, Issue 9, Pages 2037-2054 (November 2017) DOI: 10.1016/j.bpj.2017.08.054 Copyright © 2017 Terms and Conditions

Figure 1 Experimental setup for FCS. (A) Light from three laser lines (561 and 488 nm for excitation and 405 nm for activation) is attenuated by ND filters and then coupled at the back-aperture of a high-NA objective lens (OBJ) to produce an ellipsoidal observation volume. The fluorescence emission is collected back through the objective and separated from the excitation by a multiband (405/488/561/635 nm) dichroic mirror. A second dichroic mirror (560-nm long pass) separates the emission of the longer wavelength (e.g., red) from the emission of the shorter wavelength (e.g., green). The tube lens focuses emission onto a pinhole and light passing through the pinhole is detected by an avalanche photodiode (APD). (B) The green and/or orange fluorescence intensity time traces are processed by correlation analysis, yielding an auto- (APD1 × APD1) or cross- (APD1 × APD2) correlation curve. To see this figure in color, go online. Biophysical Journal 2017 113, 2037-2054DOI: (10.1016/j.bpj.2017.08.054) Copyright © 2017 Terms and Conditions

Figure 2 Excitation light-dependence of Dendra2. (A) Autocorrelation curves for iDendra2 (solid blue lines) and aDendra2 (dashed green lines) versus increasing power (direction of the arrow) of 488- and 561-nm laser light, respectively. Apparent diffusion rates for (B) iDendra2 (blue squares) and (C) aDendra2 (green squares) have been obtained as a function of excitation rate after fitting with Eq. 1. For aDendra2, the activation light (at 405 nm) has been kept at 0.31 kW/cm2. The linear relationship between kdAPP and excitation rate, kx, is described as kdAPP = k0 + Φbleach kx. To see this figure in color, go online. Biophysical Journal 2017 113, 2037-2054DOI: (10.1016/j.bpj.2017.08.054) Copyright © 2017 Terms and Conditions

Figure 3 Light-dependent flickering. Given here are transition rate constants (A and B) and fractions (C and D) for the blinking reactions of iDendra2 (blue circles, A and C) and aDendra2 (green circles, B and D). For both states of the chromophore, the description of the autocorrelation time trace requires two separate flickering processes (the slowest flickering component is represented with open symbols, whereas the fastest is represented with solid symbols). To see this figure in color, go online. Biophysical Journal 2017 113, 2037-2054DOI: (10.1016/j.bpj.2017.08.054) Copyright © 2017 Terms and Conditions

Figure 4 Detected count rate per molecule for iDendra2 (open symbols) and aDendra2 (solid symbols) using N, the average number of molecules inside the focal volume (A and C) or Nbright = N(1−F1)(1−F2), the average number of bright molecules (B and D). To see this figure in color, go online. Biophysical Journal 2017 113, 2037-2054DOI: (10.1016/j.bpj.2017.08.054) Copyright © 2017 Terms and Conditions

Figure 5 pH-dependent flickering of iDendra2 (blue) and aDendra2 (green). (A) Shown here are autocorrelation functions for varying pH (from 10 to 5, according to the arrow) for iDendra2 (solid blue lines) and aDendra2 (dashed green lines). (B) Count rate per molecule for the two forms of the chromophore are given. (C and D) Shown here are flicker rates versus pH and related flicker fractions (inset) for the inactive and active form of the chromophore, respectively. (E) Given here is a model of internal and external chromophore protonation processes. (F) Given here is a model of interconversion of states observed. To see this figure in color, go online. Biophysical Journal 2017 113, 2037-2054DOI: (10.1016/j.bpj.2017.08.054) Copyright © 2017 Terms and Conditions

Figure 6 Photoactivation for Dendra2. (A) Given here is an autocorrelation function for increasing level of 405-nm activation light (in the direction of the arrow), in addition to 561-nm illumination at a constant excitation intensity of 75 kW/cm2. (B) Shown here are kinetic processes involved in the photoactivation. The dashed violet and the solid green ellipses represent the focal volume for the two illumination beams. Note that the actual 405-nm illumination volume is significantly larger than that of the 561-nm beam (not drawn to scale). (C) Average number of molecules (using Eqs. 1 and 2) in the focal volume given as a function of the intensity of 405-nm light used to activate molecules in the presence of 561-nm illumination. (D) Shown here is the number of bright molecules in the focal volume calculated from Eq. 3. To see this figure in color, go online. Biophysical Journal 2017 113, 2037-2054DOI: (10.1016/j.bpj.2017.08.054) Copyright © 2017 Terms and Conditions

Figure 7 Cross correlation (A) between the active and inactive Dendra2 channel without (solid black circles) and with (magenta open squares) 405-nm activation light. Autocorrelation in the same condition (light color with and dark color without UV-light) for each separate channel on themselves is reported (aDendra2, green dots, B and iDendra2, blue dots, C). To see this figure in color, go online. Biophysical Journal 2017 113, 2037-2054DOI: (10.1016/j.bpj.2017.08.054) Copyright © 2017 Terms and Conditions

Figure 8 Theoretical estimates of resolution in localization microscopy as a function of photophysical parameters and resolution model, all for an aDendra2 fluorophore, imaging No = 2 objects within the DLA, each of size L = 50 nm. Shown are localizations per DLA per frame, ν = 1, Mmin = 16 localizations. (Note: the values presented are 1000/R in nm−1, such that higher values indicate better resolution.) Acquisition parameters given: total acquisition time τtot = 2 s, effective pixel size at sample q = 100 nm, λx = 561 nm, and ϕdet = 0.05. For the label, Dendra2, Isat = 28.3 kW/cm2, ηmax = 32,700 photons/s, ε(λx) = 28,700 M−1 cm−1, and ϕB = 1.35 × 10−5. Background: β = 100 photons cm2/kW.s and B0 = 1 photon, yielding background values consistent with experimental observations (data not shown). Ropt indicates the optimum resolution for the given conditions. (A) Shown here is Eq. 18 with α = 1: Ropt = 45 nm @ I = 5.6 kW/cm2 and 44.7 ms/frame. (B) Shown here is Eq. 30 with M = Mmin: Ropt = 39 nm @ I = 11.2 kW/cm2 and 22.4 ms/frame. (C) Shown here is Eq. 30 with M from Eq. 36: Rmax = 28 nm @ I = 25.1 kW/cm2 and 8.9 ms/frame. (D) Shown here is Eq. 3 from Nieuwenheusen et al. (20): Rmax = 38 nm @ I = 14.1 kW/cm2 and 17.8 ms/frame. Black plus-signs (+) mark the conditions with best resolution for the given algorithm. To see this figure in color, go online. Biophysical Journal 2017 113, 2037-2054DOI: (10.1016/j.bpj.2017.08.054) Copyright © 2017 Terms and Conditions

Figure 9 Resolution as a function of excitation intensity and time per frame for various fluorophores. Shown here are (A) Dendra2; (B) Alexa 488; (C) Alexa 546; and (D) QD565, for a 1 s total acquisition. Resolution was calculated using Eq. 30, with photobleaching yield, maximum η, and I at saturation from experimental data (Figs. 4 and S1; Table 1), and β = 200, q = 0.1 μm, Mmin = 4, γ = 0.1, L = 50 nm, and packing factor (ν = 1). (Note: the values presented are 1000/R in nm−1, such that higher values indicate better resolution.) To see this figure in color, go online. Biophysical Journal 2017 113, 2037-2054DOI: (10.1016/j.bpj.2017.08.054) Copyright © 2017 Terms and Conditions

Figure 10 Predicted dependence of resolution (calculated using Eq. 30) as a function of key experimental parameters for (A) acquisition time, (B) count rate per molecule η at saturation, (C) photobleaching quantum yield ϕB, (D) fraction of sample filled by structure γ, (E) background photons per second per unit intensity β, and (F) saturation intensity, for a probe similar to Dendra2 with the following properties: ϕB = 6 × 10−5, Isat = 30 kW/cm2, ηmax = 25,000 photons/s, β = 50 photons cm2/kW.s, ν = 1, L = 10 nm, τtot = 1 s, No = 2, γ = 1, and q = 100 nm. For the dependence of each variable, the resolution was calculated using all other variables constant and equal to their above stated values. Biophysical Journal 2017 113, 2037-2054DOI: (10.1016/j.bpj.2017.08.054) Copyright © 2017 Terms and Conditions