Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay
Fast frame rate complementary metal–oxide–semiconductor cameras in combination with photon counting image intensifiers can be used for microsecond resolution wide-field fluorescence lifetime imaging with single photon sensitivity, but the time resolution is limited by the camera exposure time. We sh...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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IOP Publishing
2015-01-01
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| Series: | New Journal of Physics |
| Subjects: | |
| Online Access: | https://doi.org/10.1088/1367-2630/17/2/023032 |
| _version_ | 1797751212678840320 |
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| author | Liisa M Hirvonen Zdeněk Petrášek Andrew Beeby Klaus Suhling |
| author_facet | Liisa M Hirvonen Zdeněk Petrášek Andrew Beeby Klaus Suhling |
| author_sort | Liisa M Hirvonen |
| collection | DOAJ |
| description | Fast frame rate complementary metal–oxide–semiconductor cameras in combination with photon counting image intensifiers can be used for microsecond resolution wide-field fluorescence lifetime imaging with single photon sensitivity, but the time resolution is limited by the camera exposure time. We show here how the image intensifier's P20 phosphor afterglow can be exploited for accurate timing of photon arrival well below the camera exposure time. By taking ratios of the intensity of the photon events in two subsequent frames, photon arrival times were determined with 300 ns precision with 18.5 μ s frame exposure time (54 kHz camera frame rate). Decays of ruthenium and iridium-containing compounds with around 1 μ s lifetimes were mapped with this technique, including in living HeLa cells, using excitation powers below 0.5 μ W. Details of the implementation to calculate the arrival time from the photon event intensity ratio are discussed, and we speculate that by using an image intensifier with a faster phosphor decay to match a higher camera frame rate, photon arrival time measurements on the nanosecond time scale could be possible. |
| first_indexed | 2024-03-12T16:44:13Z |
| format | Article |
| id | doaj.art-1c3ed67ee99e4702be7719d5be428aa8 |
| institution | Directory Open Access Journal |
| issn | 1367-2630 |
| language | English |
| last_indexed | 2024-03-12T16:44:13Z |
| publishDate | 2015-01-01 |
| publisher | IOP Publishing |
| record_format | Article |
| series | New Journal of Physics |
| spelling | doaj.art-1c3ed67ee99e4702be7719d5be428aa82023-08-08T14:17:14ZengIOP PublishingNew Journal of Physics1367-26302015-01-0117202303210.1088/1367-2630/17/2/023032Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decayLiisa M Hirvonen0Zdeněk Petrášek1Andrew Beeby2Klaus Suhling3Department of Physics, King's College London , Strand, London WC2R 2LS, UKMax Planck Institute of Biochemistry, Department of Cellular and Molecular Biophysics , Am Klopferspitz 18, D-82152 Martinsried, GermanyDepartment of Chemistry, University of Durham , Durham DH1 3LE, UKDepartment of Physics, King's College London , Strand, London WC2R 2LS, UKFast frame rate complementary metal–oxide–semiconductor cameras in combination with photon counting image intensifiers can be used for microsecond resolution wide-field fluorescence lifetime imaging with single photon sensitivity, but the time resolution is limited by the camera exposure time. We show here how the image intensifier's P20 phosphor afterglow can be exploited for accurate timing of photon arrival well below the camera exposure time. By taking ratios of the intensity of the photon events in two subsequent frames, photon arrival times were determined with 300 ns precision with 18.5 μ s frame exposure time (54 kHz camera frame rate). Decays of ruthenium and iridium-containing compounds with around 1 μ s lifetimes were mapped with this technique, including in living HeLa cells, using excitation powers below 0.5 μ W. Details of the implementation to calculate the arrival time from the photon event intensity ratio are discussed, and we speculate that by using an image intensifier with a faster phosphor decay to match a higher camera frame rate, photon arrival time measurements on the nanosecond time scale could be possible.https://doi.org/10.1088/1367-2630/17/2/023032fluorescence lifetime imaging (FLIM)image intensifiermicrochannel platephosphortime-correlated single photon counting (TCSPC)phosphorescence |
| spellingShingle | Liisa M Hirvonen Zdeněk Petrášek Andrew Beeby Klaus Suhling Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay New Journal of Physics fluorescence lifetime imaging (FLIM) image intensifier microchannel plate phosphor time-correlated single photon counting (TCSPC) phosphorescence |
| title | Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay |
| title_full | Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay |
| title_fullStr | Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay |
| title_full_unstemmed | Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay |
| title_short | Sub-μs time resolution in wide-field time-correlated single photon counting microscopy obtained from the photon event phosphor decay |
| title_sort | sub μs time resolution in wide field time correlated single photon counting microscopy obtained from the photon event phosphor decay |
| topic | fluorescence lifetime imaging (FLIM) image intensifier microchannel plate phosphor time-correlated single photon counting (TCSPC) phosphorescence |
| url | https://doi.org/10.1088/1367-2630/17/2/023032 |
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