![Establishing the photophysics of various fluorescent probe molecules by independently evaluating the number of detected photons (proportional to the emitted photons) from a single fluorescent molecule during a single frame time (N; x-axis) and single-molecule localization precision (σxy=[σx+σy]/2; y-axis), under various excitation laser powers and single-frame durations. These plots can be fitted well with the equation later in this legend, indicating that these measurements were performed with satisfactory accuracies. With an increase of the excitation laser power (at the sample), some dyes emit more photons than others, showing that they are more suitable for ultrafast SFMI. These curves are useful for determining the fluorescent probes to be used in the experiments and for predicting the single-molecule localization precisions that can be obtained under the given excitation laser powers. The results for 30, 10, and 0.06 kHz; i.e., the frame times of 0.033, 0.1 and 16.7 ms, respectively, are shown (45 kHz/0.022 ms for 5xCy3-Tf is also shown). For the method to evaluate N, see the subsection “Determination of the number of detected photons/molecule/frame (N)” in Materials and methods. In high-speed single fluorescent-molecule imaging, one of the crucial problems is whether single fluorescent molecules emit sufficient numbers of photons (during a single frame time) required for obtaining the desired single-molecule localization precisions. The results shown here demonstrate that a 10-kHz frame rate is applicable for various dye molecules, and Cy3 could even be used at 30 kHz. These plots were fitted well by the theoretical equation derived previously (Mortensen et al., 2010), indicating that the developed camera system functions as planned, even at high frequencies. The excess noise factor (F) of the developed camera system was evaluated by this fitting. Throughout this report (except for the measurements in the plasma membrane (PM), as described in Fig. S4), the localization precision (σxy) is defined as [σx + σy]/2, where σx and σy are the standard deviations of the x and y position determinations, respectively, following the convention of the super-resolution imaging field (Dietrich et al., 2002; Martin et al., 2002). σxy was determined in 15 consecutive frames for n = 50 trajectories for each condition. All of the fluorescent dye molecules were covalently bound to coverslips coated with 3-aminopropylethoxysilane, and 5xCy3-Tf was adsorbed on the coverslip coated with poly-D-lysine (Materials and methods). (A and B) Plots for single Cy3 molecules observed at 10 and 30 kHz and single 5xCy3-Tf molecules observed at 45 kHz (A) and those for various fluorescent molecules observed at 10 kHz (B). Five TIR laser illumination intensities were employed for each dye (50 molecules for each laser intensity), as indicated by the different colors of the data points. Various ranges of the laser power densities were used for different dyes, because the dyes are saturated differently (shown in each box). The plots (σxy vs. N) shown in A and B could be fitted well (non-linear least-squares fitting by the Levenberg–Marquardt algorithm) using the following equation derived previously (Mortensen et al., 2010). σxy=F{16(s2+a2/12)9N+8πb2(s2+a2/12)2a2N2}1/2, where F is the sole fitting parameter, representing the excess noise factor (a coefficient describing the stochastic gain fluctuation in the electron amplification process in the image intensifier; F is shown in each box, but its value is 1.2—1.4 for all cases), s is the standard deviation of the Gaussian spot profile, 123 ± 1.1 nm for Cy3 on the sample plane (determined by the Gaussian fitting of each image for 50 Cy3 molecules immobilized on the glass excited by the TIR illumination at 79 µW/µm2; compared with our standard condition of the oblique illumination at 23 µW/µm2, these observation conditions provided ∼3 times more detected photons; see Fig. 1 E, top; note that s depends on the observed fluorescent molecules), a is the pixel size (55.1 nm), and b is the standard deviation of the background noise. (For example, 0.038 ± 0.059 detected photons/pixel/frame [mean ± SD] for the TIR illumination and 0.035 ± 0.058 detected photons/pixel/frame for the oblique illumination at 10 kHz; n = 76,800 pixels = 32 × 32 pixels × 15 frames x 5 different positions.) The estimated excess noise factor F of the image intensifier shows that it is comparable to or slightly smaller (less noisy) than that of the EM-CCD electron multiplier (F = 1.4). Cy3 exhibited the least tendency to saturate, and thus provided better single-molecule localization precisions, consistent with the analysis results shown in Fig. S3, A and C. Note that s and b were determined for each fluorescent probe (with different illumination and excitation wavelengths and optics). 5xCy3-Tf data are considered to represent fluorescent spots generated by various numbers of Cy3 molecules placed within a few nanometers, mostly in the range of 3 to 8 molecules (1 and 2 Cy3 molecules/Tf, representing ≈12% of the 5xCy3-Tf spots, gave low signals, inducing extremely large errors in single-molecule localizations; meanwhile, the probability of 9 or more Cy3 molecules being attached to a Tf molecule will be <7%). Due to the photobleaching of multiple Cy3 molecules bound to a Tf molecule, the numbers detected on a Tf molecule decreased quickly upon laser illumination. (C) Plot for single Cy3 molecules observed at 60 Hz, with TIR illumination laser power densities ≤0.16 µW/µm2 (indicated by different colors of the data points; 50 molecules for each laser intensity). The excess noise factor F was estimated to be 1.2, consistent with the results shown in A. (D) Summary plot for single Cy3 molecules observed at 60 Hz, with TIR illumination laser power densities up to 79 µW/µm2: 0.018, 0.029, 0.047, 0.088, 0.16 (employed for the plot in c), 0.48, 1.6, 4.8, 14, 23, 43, and 79 µW/µm2 (note that in this plot, in contrast to the others, the x-axis is in the log scale). The single-molecule localization precisions obtained with the laser power densities equal to and >14 µW/µm2 were calculated using the equation above with F = 1.2, as found in C. This method for obtaining the single-molecule localization precisions employed here is different from that used for evaluating the precisions shown in Fig. 1 F and Fig. S2, A–C; and Fig. S3, B and C. Since most Cy3 molecules were photobleached within a single 16.7 ms frame, the more-prevalent method could not be employed. The x-axis of this figure covers the entire practical scale for the number of detected photons/molecule/frame (N) for a single Cy3 molecule, from 25.0 ± 1.4 at a laser power density of 0.018 µW/µm2 up to 11,400 ± 700 at 79 µW/µm2 (mean ± SEM). This upper limit was given by the photobleaching and excitation power saturation of Cy3, and provided the best single-molecule localization precision of 2.6 ± 0.099 nm (mean ± SEM) for Cy3 (no further improvements could be obtain even by employing higher laser intensities; Fig. 1, G and H).](https://cdn.rupress.org/rup/content_public/journal/jcb/222/8/10.1083_jcb.202110160/3/jcb_202110160_figs2.png?Expires=1771137640&Signature=SJApyuUaUMwLgqiWSi9wyIowiF76w017ruhHQF~eqV07Viu540FKkCh8ZtJNbjAA97tyHWR9KL4YvdqXTQ5Yp~jtWX3J4BfrDUj~vg7c~TT2hJCQ9cWXMRLqxFr9j5iqdJlXqKLAy7uWQuqVhrxcajMI9BdurJeJV0K3Oiaj1T3WVIgxRBicq465Os1d71WEBBe7gV0soBX5QI-T4HX9gHOjUxf~8FD3gstfbT9RFA3R46jp8T26YhvPdvppjVlynUVs~2kZaxFHjGMs1yYLkEahDSr~mJz~I35X8ZhzzOR5FfmBdNtO49BIX4jwyubwmdmf7-pAYxbrUttFizDLtA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Establishing the photophysics of various fluorescent probe molecules by independently evaluating the number of detected photons (proportional to the emitted photons) from a single fluorescent molecule during a single frame time (N; x-axis) and single-molecule localization precision ( ; y-axis), under various excitation laser powers and single-frame durations. These plots can be fitted well with the equation later in this legend, indicating that these measurements were performed with satisfactory accuracies. With an increase of the excitation laser power (at the sample), some dyes emit more photons than others, showing that they are more suitable for ultrafast SFMI. These curves are useful for determining the fluorescent probes to be used in the experiments and for predicting the single-molecule localization precisions that can be obtained under the given excitation laser powers. The results for 30, 10, and 0.06 kHz; i.e., the frame times of 0.033, 0.1 and 16.7 ms, respectively, are shown (45 kHz/0.022 ms for 5xCy3-Tf is also shown). For the method to evaluate N, see the subsection “Determination of the number of detected photons/molecule/frame (N)” in Materials and methods. In high-speed single fluorescent-molecule imaging, one of the crucial problems is whether single fluorescent molecules emit sufficient numbers of photons (during a single frame time) required for obtaining the desired single-molecule localization precisions. The results shown here demonstrate that a 10-kHz frame rate is applicable for various dye molecules, and Cy3 could even be used at 30 kHz. These plots were fitted well by the theoretical equation derived previously (Mortensen et al., 2010), indicating that the developed camera system functions as planned, even at high frequencies. The excess noise factor (F) of the developed camera system was evaluated by this fitting. Throughout this report (except for the measurements in the plasma membrane (PM), as described in Fig. S4), the localization precision (σxy) is defined as [σx + σy]/2, where σx and σy are the standard deviations of the x and y position determinations, respectively, following the convention of the super-resolution imaging field (Dietrich et al., 2002; Martin et al., 2002). σxy was determined in 15 consecutive frames for n = 50 trajectories for each condition. All of the fluorescent dye molecules were covalently bound to coverslips coated with 3-aminopropylethoxysilane, and 5xCy3-Tf was adsorbed on the coverslip coated with poly-D-lysine (Materials and methods). (A and B) Plots for single Cy3 molecules observed at 10 and 30 kHz and single 5xCy3-Tf molecules observed at 45 kHz (A) and those for various fluorescent molecules observed at 10 kHz (B). Five TIR laser illumination intensities were employed for each dye (50 molecules for each laser intensity), as indicated by the different colors of the data points. Various ranges of the laser power densities were used for different dyes, because the dyes are saturated differently (shown in each box). The plots (σxy vs. N) shown in A and B could be fitted well (non-linear least-squares fitting by the Levenberg–Marquardt algorithm) using the following equation derived previously (Mortensen et al., 2010). where F is the sole fitting parameter, representing the excess noise factor (a coefficient describing the stochastic gain fluctuation in the electron amplification process in the image intensifier; F is shown in each box, but its value is 1.2—1.4 for all cases), s is the standard deviation of the Gaussian spot profile, 123 ± 1.1 nm for Cy3 on the sample plane (determined by the Gaussian fitting of each image for 50 Cy3 molecules immobilized on the glass excited by the TIR illumination at 79 µW/µm2; compared with our standard condition of the oblique illumination at 23 µW/µm2, these observation conditions provided ∼3 times more detected photons; see Fig. 1 E, top; note that s depends on the observed fluorescent molecules), a is the pixel size (55.1 nm), and b is the standard deviation of the background noise. (For example, 0.038 ± 0.059 detected photons/pixel/frame [mean ± SD] for the TIR illumination and 0.035 ± 0.058 detected photons/pixel/frame for the oblique illumination at 10 kHz; n = 76,800 pixels = 32 × 32 pixels × 15 frames x 5 different positions.) The estimated excess noise factor F of the image intensifier shows that it is comparable to or slightly smaller (less noisy) than that of the EM-CCD electron multiplier (F = 1.4). Cy3 exhibited the least tendency to saturate, and thus provided better single-molecule localization precisions, consistent with the analysis results shown in Fig. S3, A and C. Note that s and b were determined for each fluorescent probe (with different illumination and excitation wavelengths and optics). 5xCy3-Tf data are considered to represent fluorescent spots generated by various numbers of Cy3 molecules placed within a few nanometers, mostly in the range of 3 to 8 molecules (1 and 2 Cy3 molecules/Tf, representing ≈12% of the 5xCy3-Tf spots, gave low signals, inducing extremely large errors in single-molecule localizations; meanwhile, the probability of 9 or more Cy3 molecules being attached to a Tf molecule will be <7%). Due to the photobleaching of multiple Cy3 molecules bound to a Tf molecule, the numbers detected on a Tf molecule decreased quickly upon laser illumination. (C) Plot for single Cy3 molecules observed at 60 Hz, with TIR illumination laser power densities ≤0.16 µW/µm2 (indicated by different colors of the data points; 50 molecules for each laser intensity). The excess noise factor F was estimated to be 1.2, consistent with the results shown in A. (D) Summary plot for single Cy3 molecules observed at 60 Hz, with TIR illumination laser power densities up to 79 µW/µm2: 0.018, 0.029, 0.047, 0.088, 0.16 (employed for the plot in c), 0.48, 1.6, 4.8, 14, 23, 43, and 79 µW/µm2 (note that in this plot, in contrast to the others, the x-axis is in the log scale). The single-molecule localization precisions obtained with the laser power densities equal to and >14 µW/µm2 were calculated using the equation above with F = 1.2, as found in C. This method for obtaining the single-molecule localization precisions employed here is different from that used for evaluating the precisions shown in Fig. 1 F and Fig. S2, A–C; and Fig. S3, B and C. Since most Cy3 molecules were photobleached within a single 16.7 ms frame, the more-prevalent method could not be employed. The x-axis of this figure covers the entire practical scale for the number of detected photons/molecule/frame (N) for a single Cy3 molecule, from 25.0 ± 1.4 at a laser power density of 0.018 µW/µm2 up to 11,400 ± 700 at 79 µW/µm2 (mean ± SEM). This upper limit was given by the photobleaching and excitation power saturation of Cy3, and provided the best single-molecule localization precision of 2.6 ± 0.099 nm (mean ± SEM) for Cy3 (no further improvements could be obtain even by employing higher laser intensities; Fig. 1, G and H).
