Summary of the camera frame rate, maximal data acquisition image size (pixels), position determination precision, and number of observable frames for various fluorescent probes for the developed camera system and other cameras
| Frame Rate (kHz) . | Maximal Data Acquisition Image Size (pixels) . | Probe and Illumination Power Density . | Position Determination Precision (nm; mean ± SEM) . | Reference . | ||
|---|---|---|---|---|---|---|
| 1024PCI-CMOSa | SA1-CMOSa | TIR illumination | Oblique illumination | |||
| 0.002 | 128 × 128 (EM-CCD) | Cy3b | 3.2 molecules lasting 10–100 frames | ND | Pertsinidis et al. (2010) | |
| 0.06 | 1,024 × 1,024 | 1,024 × 1,024 | Cy3c 79 µW/µm2 | 2.6 ± 0.099 (n = 50) molecules lasting ≈1 frame | ND | This work |
| 0.065 | 512 × 512 (EM-CCD) | JF549d | 23 4.5% molecules lasting >100 frames | ND | Banaz et al. (2019) | |
| 0.065 | 512 × 512 (EM-CCD) | TMRd | 24 6.1% molecules lasting >100 frames | ND | Banaz et al. (2019) | |
| 10 | 256 × 256 | 768 × 640 | Cy3e 23 µW/µm2 | 27 ± 1.1 (n = 50) | 38 ± 1.7 (n = 50) 14% molecules lasting >100 frames | This work |
| 10 | 256 × 256 | 768 × 640 | Cy3c 79 µW/µm2 | 20 ± 0.71 (n = 50) 1.6% molecules lasting >100 frames | 27 ± 1.2 (n = 50) | This work |
| 30 | 128 × 112 | 512 × 256 | Cy3c 79 µW/µm2 | 34 ± 1.0 (n = 50) | 55 ± 1.7 (n = 50) | This work |
| 45 | 128 × 64 | 256 × 256 | 5xCy3-Tff 79 µW/µm2 | 29 ± 0.73 (n = 50) | 34 ± 0.96 (n = 50) | This work |
| 110 | 128 × 16 | 128 × 80 | - | ND | ND | This work |
| 0.1–0.15g | 128 × 128 (EM-CCD; PALM) | mEos2h | 11i | ND | Jones et al. (2011) | |
| 0.6 | 2,048 × 256 (sCMOS; PALM) | mEos3.2j | ≈22 | ND | Huang et al. (2013) | |
| 1 | 1,024 × 1,024 PALM | 1,024 × 1,024 PALM | mEos3.2k | 29 ± 0.22 (n = 2,940) | ND | Fujiwara et al. (2023) |
| 0.033 | 512 × 512 (EM-CCD; STORM) | Alexa647l | 7.2i | ND | Dempsey et al. (2011) | |
| 0.8 | 2,048 × 256 (sCMOS; STORM) | Alexa 647m | ≈10 | ND | Lin et al. (2015) | |
| 1.6 | 2,048 × 128 (sCMOS; STORM) | Alexa 647m | ≈12 | ND | Lin et al. (2015) | |
| Frame Rate (kHz) . | Maximal Data Acquisition Image Size (pixels) . | Probe and Illumination Power Density . | Position Determination Precision (nm; mean ± SEM) . | Reference . | ||
|---|---|---|---|---|---|---|
| 1024PCI-CMOSa | SA1-CMOSa | TIR illumination | Oblique illumination | |||
| 0.002 | 128 × 128 (EM-CCD) | Cy3b | 3.2 molecules lasting 10–100 frames | ND | Pertsinidis et al. (2010) | |
| 0.06 | 1,024 × 1,024 | 1,024 × 1,024 | Cy3c 79 µW/µm2 | 2.6 ± 0.099 (n = 50) molecules lasting ≈1 frame | ND | This work |
| 0.065 | 512 × 512 (EM-CCD) | JF549d | 23 4.5% molecules lasting >100 frames | ND | Banaz et al. (2019) | |
| 0.065 | 512 × 512 (EM-CCD) | TMRd | 24 6.1% molecules lasting >100 frames | ND | Banaz et al. (2019) | |
| 10 | 256 × 256 | 768 × 640 | Cy3e 23 µW/µm2 | 27 ± 1.1 (n = 50) | 38 ± 1.7 (n = 50) 14% molecules lasting >100 frames | This work |
| 10 | 256 × 256 | 768 × 640 | Cy3c 79 µW/µm2 | 20 ± 0.71 (n = 50) 1.6% molecules lasting >100 frames | 27 ± 1.2 (n = 50) | This work |
| 30 | 128 × 112 | 512 × 256 | Cy3c 79 µW/µm2 | 34 ± 1.0 (n = 50) | 55 ± 1.7 (n = 50) | This work |
| 45 | 128 × 64 | 256 × 256 | 5xCy3-Tff 79 µW/µm2 | 29 ± 0.73 (n = 50) | 34 ± 0.96 (n = 50) | This work |
| 110 | 128 × 16 | 128 × 80 | - | ND | ND | This work |
| 0.1–0.15g | 128 × 128 (EM-CCD; PALM) | mEos2h | 11i | ND | Jones et al. (2011) | |
| 0.6 | 2,048 × 256 (sCMOS; PALM) | mEos3.2j | ≈22 | ND | Huang et al. (2013) | |
| 1 | 1,024 × 1,024 PALM | 1,024 × 1,024 PALM | mEos3.2k | 29 ± 0.22 (n = 2,940) | ND | Fujiwara et al. (2023) |
| 0.033 | 512 × 512 (EM-CCD; STORM) | Alexa647l | 7.2i | ND | Dempsey et al. (2011) | |
| 0.8 | 2,048 × 256 (sCMOS; STORM) | Alexa 647m | ≈10 | ND | Lin et al. (2015) | |
| 1.6 | 2,048 × 128 (sCMOS; STORM) | Alexa 647m | ≈12 | ND | Lin et al. (2015) | |
Single-molecule localization precisions listed here were obtained with the 1024PCI-CMOS sensor, which was used in most of the experiments in the present research.
Attached to the end of a short DNA duplex and immobilized on the glass surface.
Covalently linked to the glass surface. In the basal PM, Cy3-DOPE observed under the same TIR illumination conditions at 79 µW/µm2 with the camera operated at 10 and 30 kHz, the single-molecule localization precisions were 34 ± 5.1 and 51 ± 10 nm, respectively (Fig. S4 A). Compare these values with the data of Cy3 on the glass shown here (20 and 34 nm, respectively).
Conjugated to MukB-Halo and observed in fixed E. coli.
Covalently linked to the glass surface. When our standard laser illumination conditions (oblique illumination at a power density of 23 µW/µm2 at the sample plane) were employed to observe Cy3-DOPE in the apical PM, with the camera operated at 10 kHz, the single-molecule localization precision was 49 ± 2.1 nm (Fig. S4 B, top). Compare this value with the data of Cy3 on the glass shown here (38 nm).
Single transferrin (Tf) molecules bound by an average of 5.0 Cy3 molecules, immobilized on the glass surface.
From this row to the bottom row, single-molecule localization precisions applicable for PALM and STORM experiments are listed, so under these observation conditions, most single molecules are photobleached within one or a few frames.
Fused to clathrin light chain and observed in live BS-C-1 cells.
Estimated from the reported FWHM value.
Fused to clathrin light chain and observed in live HeLa cells.
Fused to caveolin-1 and observed in live T24 cells.
Conjugated to in vitro-prepared microtubules and immobilized on the glass surface.
Covalently linked to antibodies and immobilized on the glass surface.
ND: Not Determined.