Figure 1.
Basic design of the ultrahigh-speed intensified CMOS camera system and ultrafast single fluorescent-molecule imaging and tracking (SFMI) of Cy3 molecules immobilized on coverslips, performed to establish suitable conditions for ultrafast SFMI. To obtain the data shown here, total internal reflection (TIR) illumination was employed. The mean ± SEM and/or the median values are provided in the figures throughout this report. (A) Schematic diagram of the camera system. See Materials and methods. (B and C) All of the single Cy3 molecules detected at 60 Hz were also detectable at 10 (B) and 30 (C) kHz (the same field of view). Typical snapshots are shown. (D) Typical snapshots of single Cy3 molecules excited at various laser powers, observed at 10 kHz. (E) The number of detected photons/molecule/frame emitted from single Cy3 molecules during frame times of 0.1 ms (10 kHz) and 0.033 ms (30 kHz; n = 50 Cy3 molecules for each measured point; the same for F, G, and H), plotted against the excitation laser power density. See the caption to Fig. S3 A. (F) The localization errors for single Cy3 molecules imaged at 10 and 30 kHz, plotted against the excitation laser intensity. Orange, cyan, and purple data points are color-matched with the arrowheads in B, C, and D. (G and H) The number of detected photons/molecule/frame (G) and the localization error (H) for single Cy3 molecules imaged at 60 Hz, plotted as a function of the excitation laser power density. See the caption to Fig. S2 D. The green data points are color-matched with the arrowheads in B, C, and I. (I) Virtually all of the single 5xCy3-Tf molecules (3–8 molecules of Cy3 bound to Tf) were detectable at 45 kHz (compare with the 60-Hz image); typical snapshots.

Basic design of the ultrahigh-speed intensified CMOS camera system and ultrafast single fluorescent-molecule imaging and tracking (SFMI) of Cy3 molecules immobilized on coverslips, performed to establish suitable conditions for ultrafast SFMI. To obtain the data shown here, total internal reflection (TIR) illumination was employed. The mean ± SEM and/or the median values are provided in the figures throughout this report. (A) Schematic diagram of the camera system. See Materials and methods. (B and C) All of the single Cy3 molecules detected at 60 Hz were also detectable at 10 (B) and 30 (C) kHz (the same field of view). Typical snapshots are shown. (D) Typical snapshots of single Cy3 molecules excited at various laser powers, observed at 10 kHz. (E) The number of detected photons/molecule/frame emitted from single Cy3 molecules during frame times of 0.1 ms (10 kHz) and 0.033 ms (30 kHz; n = 50 Cy3 molecules for each measured point; the same for F, G, and H), plotted against the excitation laser power density. See the caption to Fig. S3 A. (F) The localization errors for single Cy3 molecules imaged at 10 and 30 kHz, plotted against the excitation laser intensity. Orange, cyan, and purple data points are color-matched with the arrowheads in B, C, and D. (G and H) The number of detected photons/molecule/frame (G) and the localization error (H) for single Cy3 molecules imaged at 60 Hz, plotted as a function of the excitation laser power density. See the caption to Fig. S2 D. The green data points are color-matched with the arrowheads in B, C, and I. (I) Virtually all of the single 5xCy3-Tf molecules (3–8 molecules of Cy3 bound to Tf) were detectable at 45 kHz (compare with the 60-Hz image); typical snapshots.

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