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Highly Sensitive Photon Counting Imaging
Avalanche photodiodes (APDs) in the ConfoCor 3 are now available for highly sensitive photon counting imaging in addition to fluorescence correlation spectroscopy. Benefits in APD imaging include
1. Higher sensitivity down to the single molecule level to image cells with fluorescent tagged proteins that are expressed at extreme low levels.
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| APD | PMT |
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Sample:
HeLa cells expressing a CFP-caxbox protein. Caxbox is targeted to the membrane. Laser powers for both, the APD and the PMT taken images were the same. Detector gain setting for the PMT was at 900.
2. Requirement of less laser power to achieve the same intensities, thus minimizing sample bleaching.
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| APD 1 | PMT 1 |
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| APD 500 | PMT 500 |
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Sample:
HepG2 cells expressing an EGFP-CENP I protein. The protein is localized to centromeric regions. The sample was continuously scanned for approximately 10 minutes using the APD detectors and than for another 10 minutes with 5 fold higher laser powers using the PMT detectors with detector gain set at 900. Shown are the 1st and 500th images of each series. Note that using the higher laser powers with the PMT required to achieve the same intensities as with the APDs substantial bleaching occurred.
3. Low detector dark counts result in images with low background.
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| APD | PMT |
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Sample:
HepG2 cells expressing an EGFP-CENP I protein. The protein is localized to centromeric regions. The laser power was 4 fold higher for the PMT image with detector gain set at 950 compared to the APD image. The shown images are the summed representation of 4 scans. Note the lower background in the APD image.
4. Due to their high quantum efficiency in the longer wavelength range, APDs are especially suited for imaging red shifted fluorescence dyes.
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| APD | PMT |
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Sample:
HeLa cells expressing dsRed1. dsRed1 distributes between the cytosol and nucleoplasm. Note that laser power for the dsRed image was about 8 fold higher for the PMT image, using a detector gain of 900, than for the APD image.
5. Time lapse studies of low expressing cells.
| 0 sec | 90 sec | 1140 sec |
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| Keima-PKC |  | |
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Sample:
HeLa cells expressing Keima-PKC and EGFP-MARCKS proteins. In the unstimulated state (0 s), PKC localizes to the cytosol, whereas MARCKS is found at the membrane. After stimulation with PMA the proteins exchange their sites (90 s and 1140s). PKC redistruibutes to the membrane and MARCKS to the cytosol, respectively.
6. Analysing FRAP with subtle protein redistribution kinetics.
Sample:
HepG2 cells expressing an EGFP-CENP I protein. The protein is localized to centromeric regions. Shown are cells before bleaching and at different times after bleaching along with a recovery curve. Most of the CENP I protein is immobilized in the centromeric regions and only a minor fraction freely redistributes between the nucleoplasm and the centromer.
Keima-PKC, EGFP-MARCKS, CFP-Caxbox and dsRED1 kindly provided by Takako Kogure and Atushi Miyawaki, RIKEN Brain Science Institute, Wako, Saitama, Japan
EGFP-CENP I kindly provided by Stefanie Weidtkamp-Peters and Peter Hemmerich, Fritz Lipman Institute of Age Research, Jena, Germany | |
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