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.
|Ingargiola, Antonino; Segal, Maya; Gulinatti, Angelo et al. (2018) 48-spot single-molecule FRET setup with periodic acceptor excitation. J Chem Phys 148:123304|
|Ceccarelli, Francesco; Gulinatti, Angelo; Labanca, Ivan et al. (2018) Red-Enhanced Photon Detection Module Featuring a 32 × 1 Single-Photon Avalanche Diode Array. IEEE Photonics Technol Lett 30:557-560|
|Lerner, Eitan; Cordes, Thorben; Ingargiola, Antonino et al. (2018) Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer. Science 359:|
|Lerner, Eitan; Ingargiola, Antonino; Weiss, Shimon (2018) Characterizing highly dynamic conformational states: The transcription bubble in RNAP-promoter open complex as an example. J Chem Phys 148:123315|
|Ingargiola, Antonino; Peronio, Pietro; Lerner, Eitan et al. (2017) 16-Ch Time-resolved Single-Molecule Spectroscopy Using Line Excitation. Proc SPIE Int Soc Opt Eng 10071:|
|Ceccarelli, Francesco; Gulinatti, Angelo; Labanca, Ivan et al. (2017) Development and characterization of an 8×8 SPAD-array module for gigacount per second applications. Proc SPIE Int Soc Opt Eng 10229:|
|Ingargiola, Antonino; Lerner, Eitan; Chung, SangYoon et al. (2017) Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules. PLoS One 12:e0175766|
|Lerner, Eitan; Ingargiola, Antonino; Lee, Jookyung J et al. (2017) Different types of pausing modes during transcription initiation. Transcription 8:242-253|
|Gulinatti, Angelo; Ceccarelli, Francesco; Rech, Ivan et al. (2016) Silicon technologies for arrays of Single Photon Avalanche Diodes. Proc SPIE Int Soc Opt Eng 9858:|
|Ingargiola, Antonino; Laurence, Ted; Boutelle, Robert et al. (2016) Photon-HDF5: An Open File Format for Timestamp-Based Single-Molecule Fluorescence Experiments. Biophys J 110:26-33|
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