Single-photon counting APD arrays for high-through-put single-molecule assays

该提案的长期目标是开发能够实现高吞吐量的工具 单分子荧光光谱法。这项建议的具体目标是： (1)开发了一种用于3色48点激发和检测的光学装置。 (2)开发48像素定制单光子雪崩光电二极管阵列（48 p-SPADA）。 (3)开发现场可编程门阵列（FPGA）数据缩减算法， 144通道单分子爆发分析。 (4)开发高通量荧光（交叉）相关光谱数据分析 图形处理器（GPU）中的算法。 (5)研究转录起始和延伸中的快速和瞬时事件，重点是 的构象和与细菌RNAP全酶的相互作用。 这一提议的预期结果是增加单分子的通量， 荧光技术的一个因素48。这将包括大多数单分子 荧光技术，例如单分子荧光共振能量转移 （smFRET）和微秒交替激光激发（<$s-ALEX）以及 荧光相关光谱（FCS）。 这些事态发展的影响将是双重的。第一，单分子荧光 光谱学方法将对更大的研究团体具有吸引力， 能够将其用作有效的发现和分析工具。 此外，它将使人们能够利用新的时间制度，而这是联合国无法实现的。 当前技术。其次，吞吐量的飞跃（系数48）将使这一点 对即时诊断和大规模筛选测定有吸引力的技术 生物技术和制药行业使用。基于这些原因，本提案 很可能会对生物学研究的许多领域产生重大影响 包括药物发现和开发以及诊断，从而帮助增加 我们对生命过程的理解，并为疾病的发展奠定了基础 诊断、治疗和预防。

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.