Trackable durations of single Cy3 molecules under two typical laser illumination conditions and minimal photo-induced damage to cells during ultrafast SFMI. (A–C) Single Cy3 molecules immobilized on a coverslip were observed. The trackable durations are limited by photoblinking/photobleaching, stochastic fluctuations of the signal, and photon shot noise. (A a–c) Typical time-dependent fluorescence signal intensities (number of detected photons/0.1-ms-frame) of three single Cy3 molecules (a, b, and c) excited by oblique-angle illumination at 23 µW/µm², and observed at 10 kHz (background signal in gray). See Materials and methods for details. (B a–c) The signal-to-noise ratios (SNRs) for the images of the three molecules (a, b, and c) shown in A fall in the range between 2.4 and 3.2. See Materials and methods. (C) The distributions of the durations of the on-periods and those after neglecting the off-periods (non-emission periods) lasting for 1, 2, or 3 frames (gap closing; see Materials and methods) of single Cy3 molecules immobilized on a coverslip and excited by oblique-angle illumination at 23 µW/µm² (standard conditions; left) or by TIR illumination at 79 µW/µm² (right), observed at 10 kHz (totals of 264 and 593 molecules, respectively). The three-frame gap closing was employed in single-molecule tracking under the standard test conditions (thick orange curve; left). (D and E) Photo-induced damage to the cells during ultrahigh-speed SFMI is very limited under our standard experimental conditions. Cell viability was examined by staining with 1 µM TOTO-3 iodide, which selectively stains dead cells, at 37°C for 5 min, and then observing the stained cells using epi-illumination with a 594-nm laser. (D) Representative images of the nuclei stained with TOTO-3 iodide. Control, a reference image of a living cell (n = 24 images). H₂O₂, a reference image of a dead cell after the treatment with 100 µM H₂O₂ at 37°C for 1 h (n = 48 images). (E) Histograms showing the fluorescence intensity of TOTO-3 in the 5.5 × 5.5-µm area inside the nucleus (see the square box in D; n = the number of examined cells). Top: Live cells without laser illumination (negative control). Second: Dead cells after the H₂O₂ treatment (positive control). Based on the results of the negative and positive controls (top and second boxes, respectively), a threshold fluorescence intensity of 4.0 × 10⁵ (arbitrary unit = AU) was selected to categorize the live and dead cells (96% [90%] of the negative [positive] control cells were categorized as alive [dead]). Third and Fourth: Cells were subjected to illumination under our typical 10-kHz single Cy3 molecule imaging conditions (oblique-angle 532-nm laser illumination at 23 µW/µm²) for 1 and 10 min, respectively, which are longer by factors of 12 and 120 than our longest illumination duration of 5 s/cell (10 500-ms image sequences = 5 × 10⁴ frames). We concluded that the light-induced damage to the cells is insignificant under our standard experimental conditions.