Figure S1.
The PRNU of the developed camera system scarcely affects the single-molecule localization precision, examined at 10 kHz. Two image intensifier amplifications were employed: 8,100× (A) and 253× (B).(A and B) (a) Images used for evaluating the PRNU effects of the developed camera system on the localization precision of a single Cy3 molecule. Top: 256 × 256–pixel images representing the PRNU of the developed camera system, obtained by averaging images over 40,000 consecutive frames recorded at 10 kHz under uniform illumination, so that the mean pixel intensity counts became 515 ± 34 (A) and 513 ± 32 (B; SD for 256 × 256 pixels), which are approximately half of the maximum intensity count of 10 bits. The uniform illumination was generated by Köhler illumination, using the halogen lamp of the microscope and a 572- to 642-nm bandpass filter (FF01-607/70, Semrock). Bottom: Modulation of the image of a single Cy3 molecule by PRNU, evaluated by calculation. Left: The Cy3 image was approximated by an ideal two-dimensional Gaussian point spread function (PSF) in the 15 × 15–pixel region, based on an experimentally determined SD of 2.2 pixels for 50 Cy3 molecules immobilized on the glass, obtained by the TIR illumination at 79 µW/µm2 and a peak intensity count of 511 (half of the maximum intensity count of 10 bits). In the actual imaging experiments, we employed 55.1 nm/pixel: 2.2 pixels = 123 nm. The PSF peak was placed at the center of the 15 × 15–pixel region. Middle: The 15 × 15–pixel yellow regions shown in the top images are magnified. Right: Images on the left and middle were multiplied pixel by pixel and normalized, generating the PRNU-modulated images of a single Cy3 molecule. (b) The effect of PRNU on the single-molecule localization precision is quite limited. Top: Maps of the 2D position deviation from the pixel center (coded on the gray scale). These maps were generated by moving the 15 × 15–pixel image of an ideal Gaussian PSF simulating the Cy3 image (a, bottom left), scanning over the 256 × 256–pixel PRNU images (a, top) pixel-by-pixel, and calculating the 2D position deviation at every position in the scan. Bottom: Distributions of the 2D position deviations, showing that the mean deviations are 0.033 and 0.034 pixels (= 1.8 and 1.9 nm at 55.1 nm/pixel; n = 57,600 pixels; arrowheads) for A and B, respectively, which are comparable to the typical PRNU effect found with the EM-CCD camera (Pertsinidis et al., 2010). Furthermore, the reverse cumulative distributions (shown in red) indicate that 95% of the 2D position deviations are within the range of 0.066 pixels (= 3.6 nm) and 0.068 pixels (= 3.7 nm) for A and B, respectively. These results suggest that the effects of PRNU on the single-molecule localization precision are limited and almost identical at amplifications of 8,100× (A) and 253× (B).

The PRNU of the developed camera system scarcely affects the single-molecule localization precision, examined at 10 kHz. Two image intensifier amplifications were employed: 8,100× (A) and 253× ( B ). (A and B) (a) Images used for evaluating the PRNU effects of the developed camera system on the localization precision of a single Cy3 molecule. Top: 256 × 256–pixel images representing the PRNU of the developed camera system, obtained by averaging images over 40,000 consecutive frames recorded at 10 kHz under uniform illumination, so that the mean pixel intensity counts became 515 ± 34 (A) and 513 ± 32 (B; SD for 256 × 256 pixels), which are approximately half of the maximum intensity count of 10 bits. The uniform illumination was generated by Köhler illumination, using the halogen lamp of the microscope and a 572- to 642-nm bandpass filter (FF01-607/70, Semrock). Bottom: Modulation of the image of a single Cy3 molecule by PRNU, evaluated by calculation. Left: The Cy3 image was approximated by an ideal two-dimensional Gaussian point spread function (PSF) in the 15 × 15–pixel region, based on an experimentally determined SD of 2.2 pixels for 50 Cy3 molecules immobilized on the glass, obtained by the TIR illumination at 79 µW/µm2 and a peak intensity count of 511 (half of the maximum intensity count of 10 bits). In the actual imaging experiments, we employed 55.1 nm/pixel: 2.2 pixels = 123 nm. The PSF peak was placed at the center of the 15 × 15–pixel region. Middle: The 15 × 15–pixel yellow regions shown in the top images are magnified. Right: Images on the left and middle were multiplied pixel by pixel and normalized, generating the PRNU-modulated images of a single Cy3 molecule. (b) The effect of PRNU on the single-molecule localization precision is quite limited. Top: Maps of the 2D position deviation from the pixel center (coded on the gray scale). These maps were generated by moving the 15 × 15–pixel image of an ideal Gaussian PSF simulating the Cy3 image (a, bottom left), scanning over the 256 × 256–pixel PRNU images (a, top) pixel-by-pixel, and calculating the 2D position deviation at every position in the scan. Bottom: Distributions of the 2D position deviations, showing that the mean deviations are 0.033 and 0.034 pixels (= 1.8 and 1.9 nm at 55.1 nm/pixel; n = 57,600 pixels; arrowheads) for A and B, respectively, which are comparable to the typical PRNU effect found with the EM-CCD camera (Pertsinidis et al., 2010). Furthermore, the reverse cumulative distributions (shown in red) indicate that 95% of the 2D position deviations are within the range of 0.066 pixels (= 3.6 nm) and 0.068 pixels (= 3.7 nm) for A and B, respectively. These results suggest that the effects of PRNU on the single-molecule localization precision are limited and almost identical at amplifications of 8,100× (A) and 253× (B).

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