Enhanced Dynamic Range Proteomic Analysis

描述（由申请人提供）：提出了一种新的增强动态范围相机（EDRaC），用于大大改善蛋白质组学中的荧光检测。从长远来看，EDRaC可以提高使用荧光微孔板阅读和/或显微镜的广泛生物医学应用的性能。最初的重点将是开发，演示和应用该技术的荧光2D凝胶成像。该系统将使用数字微镜器件（DMD）和数字反馈，在成像到CCD焦平面阵列之前选择性地衰减亮像素，从而大大减少相机中的杂散光，并能够同时测量样品中非常明亮和非常暗淡的区域。在完美成像和无散射光的限制下，对于大约25-27位的最大系统动态范围，该系统的所得动态范围将是DMD动态范围（~13位）和CCD动态范围（由于杂散光，实际上通常为12-14位）的乘积。在实践中，我们预计这种方法将增加两到四个数量级（100-10，000 x）的动态范围到荧光检测。这一改进对于荧光2D凝胶成像将是极其有价值的，荧光2D凝胶成像目前受到动态范围的限制，具有最先进的荧光检测染料。已经制定了一项工作计划来设计、组装和演示该技术。设计工作将包括详细的光线跟踪光学建模，杂散光分析和机械设计。实验室规模系统的原型测试将包括系统调制传递函数的测量、杂散光测量以及各种场景的动态范围测量。系统表征后，将在凝胶上证明系统动态范围。高动态范围血浆蛋白质模式将在2D变焦凝胶上进行研究，使用来自所选疾病状态的样本，这些疾病状态耗尽了最丰富的蛋白质，使用EDRaC原型寻找与健康和疾病相关的候选蛋白质模式。来自生物医学样本的光信号强度可以变化超过十亿倍（动态范围），而传统相机可以测量大约一万倍的信号。这是蛋白质组学和其他生物医学应用的常见差异，它极大地限制了可以获得的信息。所提出的系统将大大减少这种差异，从而使许多生物医学应用更准确，更容易测量。

DESCRIPTION (provided by applicant): A novel enhanced dynamic range camera (EDRaC) is proposed for greatly improved fluorescence detection in proteomics. In the longer term the EDRaC can improve the performance of a wide range of biomedical applications using fluorescent microwell plate reading and/or microscopy. The initial emphasis will be to develop, demonstrate, and apply the technology to multicolor fluorescent 2D gel imaging. The system will use a Digital Micro-mirror Device (DMD) and digital feedback to selectively attenuate bright pixels before imaging onto a CCD focal plane array, thereby greatly reducing stray light in the camera and enabling simultaneous measurements of very bright and very dim regions in samples. In the limit of perfect imaging and no scattered light, the resulting dynamic range of this system would be the product of the DMD dynamic range (~13 bits) and the CCD dynamic range (typically 12-14 bits in practice due to stray light) for a maximum system dynamic range of approximately 25-27 bits. In practice, we expect that this approach will add between two and four orders of magnitude (100-10,000x) of dynamic range to fluorescent detection. This improvement will be extremely valuable for fluorescent 2D gel imaging, which currently suffers from dynamic range limitations, with the most advanced multicolor fluorescent detection dyes. A work plan has been developed to design, assemble, and demonstrate the technology. The design effort will include detailed ray-trace optical modeling, stray-light analysis, and mechanical design. Prototype testing of a bench scale system will include measurements of the system Modulation Transfer Function, stray-light measurements, and measurements of the dynamic range for various scenes. Following system characterization the system dynamic range will be demonstrated on gels. High dynamic range plasma protein patterns will be investigated on 2D zoom gels, using samples from selected disease states that are depleted of the most abundant proteins, using the EDRaC prototype to seek candidate protein pattern correlations with health and disease. Optical signal strengths from biomedical samples can vary by factors of over a billion (the dynamic range), whereas conventional cameras can measure signals that vary by factors of about ten-thousand. This is a common disparity for proteomics and other biomedical applications and it greatly limits the information that can be obtained. The proposed system will greatly decrease this disparity, thereby enabling more accurate and easier measurements for numerous biomedical applications.