The long-term objective of this project is to provide the biomedical research community with a new device for high-throughput single-molecule fluorescence assays. The domains of applications of these methods range from basic research to high-throughput diagnostics and drug screening. The goal of this research proposal is to develop an integrated system for multiplexed, high-throughput single-molecule assays. This system will be based on a new type of single-photon counting capable mutichannel hybrid photodetector (HPD) recently developed by Hamamatsu Photonics Corporation. We will incorporate this detector into a multiple excitation/multiple detection spot confocal microscope and demonstrate its capabilities in actual single-molecule assays. We expect this detector to find applications not only in fundamental research applications using single-molecule methods, but also in the emerging fields of high-throughput single-molecule diagnostics and drug screening.
The specific aims of this research proposal are: (1) to develop an electronic interface and data acquisition system (including software) for the multichannel hybrid detector developed by Hamamatsu. (2) to integrate this system into a multiplexed single-molecule fluorescence excitation/detection confocal microscope. (3) to demonstrate the capabilities of this system for multiplexed, high-throughput single- molecule assays, by performing multiplexed fluorescence correlation spectroscopy (FCS) measurements on model systems of biological relevance as well as multiplexed single-molecule FRET measurements using an alternate laser excitation (ALEX) scheme.
| Michalet, X; Colyer, R A; Scalia, G et al. (2013) Development of new photon-counting detectors for single-molecule fluorescence microscopy. Philos Trans R Soc Lond B Biol Sci 368:20120035 |
| Ingargiola, A; Colyer, R A; Kim, D et al. (2012) Parallel multispot smFRET analysis using an 8-pixel SPAD array. Proc SPIE Int Soc Opt Eng 8228: |
| Michalet, Xavier; Berglund, Andrew J (2012) Optimal diffusion coefficient estimation in single-particle tracking. Phys Rev E Stat Nonlin Soft Matter Phys 85:061916 |
| Colyer, Ryan A; Siegmund, Oswald H W; Tremsin, Anton S et al. (2012) Phasor imaging with a widefield photon-counting detector. J Biomed Opt 17:016008 |
| Colyer, Ryan A; Scalia, Giuseppe; Villa, Federica A et al. (2011) Ultra high-throughput single molecule spectroscopy with a 1024 pixel SPAD. Proc SPIE Int Soc Opt Eng 7905: |
| Michalet, X; Colyer, R A; Scalia, G et al. (2011) New photon-counting detectors for single-molecule fluorescence spectroscopy and imaging. Proc SPIE Int Soc Opt Eng 8033:803316 |
| Michalet, X; Colyer, R A; Scalia, G et al. (2010) High-throughput single-molecule fluorescence spectroscopy using parallel detection. Proc SPIE Int Soc Opt Eng 7608: |
| Colyer, Ryan A; Scalia, Giuseppe; Rech, Ivan et al. (2010) High-throughput FCS using an LCOS spatial light modulator and an 8 × 1 SPAD array. Biomed Opt Express 1:1408-1431 |
| Michalet, Xavier (2010) Mean square displacement analysis of single-particle trajectories with localization error: Brownian motion in an isotropic medium. Phys Rev E Stat Nonlin Soft Matter Phys 82:041914 |
| Colyer, R; Siegmund, O; Tremsin, A et al. (2009) Phasor-based single-molecule fluorescence lifetime imaging using a wide-field photon-counting detector. Proc SPIE Int Soc Opt Eng 7185: |
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