The long term goal of this proposal is the development of tools enabling high throughput single-molecule fluorescence spectroscopy.
The specific aims of this proposal are: (1) Develop an optical setup for 3 color, 48 spot excitation and detection. (2) Develop a 48 pixel custom single-photon avalanche photodiode array (48p-SPADA). (3) Develop field-programmable-gate-array (FPGA) data reduction algorithms for up to 144 channels single-molecule burst analysis. (4) Develop high-throughput fluorescence (cross)-correlation spectroscopy data analysis algorithms in graphical processing unit (GPU). (5) Study fast and transient events in transcription initiation and elongation, focusing on the conformation and interactions of ?70 with the bacterial RNAP holoenzyme. The expected outcome of this proposal is an increase in throughput for single molecule fluorescence techniques of a factor 48. This will include most single-molecule fluorescence techniques such as single-molecule fluorescence resonant energy transfer (smFRET) and microsecond alternating laser excitation (?s-ALEX) as well as fluorescence correlation spectroscopy (FCS). The impact of these developments will be twofold. First, single-molecule fluorescence spectroscopy methods will become attractive to a larger research community, which will be able to use is as an efficient discovery and analytical tool. Additionally, it will give access to new temporal regimes that are unattainable with the current technology. Second, the leap in throughput (by a factor 48) will render this technology attractive for point-of-care diagnostics and large scale screening assays used by the biotechnology and pharmaceutical industry. For these reasons, this proposal is very likely to significantly impact the progresses of many areas of biological research including drug discovery and development as well as diagnostics, thus helping increase our understanding of life processes and lays the foundation for advances in disease diagnosis, treatment, and prevention.
The development of new detectors for high-throughput single-fluorescence molecule spectroscopy will result in accelerated measurements and enable highly parallel studies. This will allow mainstream researchers to take advantage of this powerful technology, thus accelerating the pace of discovery. It will also make high-throughput diagnostics and drug screening applications of single-molecule a practical reality, therefore permitting advances in disease diagnosis, treatment, and prevention.
|Gong, S; Labanca, I; Rech, I et al. (2014) A 32-channel photon counting module with embedded auto/cross-correlators for real-time parallel fluorescence correlation spectroscopy. Rev Sci Instrum 85:103101|
|Michalet, Xavier; Ingargiola, Antonino; Colyer, Ryan A et al. (2014) Silicon photon-counting avalanche diodes for single-molecule fluorescence spectroscopy. IEEE J Sel Top Quantum Electron 20:38044201-380442020|
|Burri, Samuel; Maruyama, Yuki; Michalet, Xavier et al. (2014) Architecture and applications of a high resolution gated SPAD image sensor. Opt Express 22:17573-89|
|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|
|Gulinatti, Angelo; Rech, Ivan; Maccagnani, Piera et al. (2013) New silicon technologies enable high-performance arrays of Single Photon Avalanche Diodes. Proc SPIE Int Soc Opt Eng 8727:|
|Gulinatti, Angelo; Rech, Ivan; Maccagnani, Piera et al. (2013) A 48-pixel array of Single Photon Avalanche Diodes for multispot Single Molecule analysis. Proc SPIE Int Soc Opt Eng 8631:|
|Panzeri, Francesco; Ingargiola, Antonino; Lin, Ron R et al. (2013) Single-molecule FRET experiments with a red-enhanced custom technology SPAD. Proc SPIE Int Soc Opt Eng 8590:|