Enhanced Dynamic Range Proteomic Analysis

描述(申请人提供)：提出了一种新的增强型动态范围照相机(EDRaC)，用于极大地改进蛋白质组学中的荧光检测。从长远来看，EDRaC可以使用荧光微孔板读数和/或显微镜来改善广泛的生物医学应用的性能。最初的重点将是开发、演示并将该技术应用于多色荧光2D凝胶成像。该系统将使用数字微镜装置(DMD)和数字反馈，在成像到CCD焦平面阵列之前选择性地衰减明亮的像素，从而大大减少相机中的杂散光，并能够同时测量样品中的非常亮和非常暗的区域。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，该系统的最终动态范围将是DMD动态范围(~13比特)和CCD动态范围(实际上由于杂散光通常为12-14比特)的乘积。在实践中，我们预计这种方法将使荧光检测的动态范围增加两到四个数量级(100-10,000倍)。这一改进对于目前受到动态范围限制的荧光2D凝胶成像将是非常有价值的，因为它使用了最先进的多色荧光检测染料。已经制定了一项工作计划来设计、组装和演示这项技术。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、杂散光测量和各种场景的动态范围测量。在系统表征之后，将在凝胶上演示系统的动态范围。高动态范围的血浆蛋白质模式将在2D Zoom凝胶上进行研究，使用从缺乏最丰富蛋白质的选定疾病状态的样本，使用EDRaC原型来寻找与健康和疾病相关的候选蛋白质模式。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。对于蛋白质组学和其他生物医学应用来说，这是一个常见的差异，它极大地限制了可以获得的信息。建议的系统将大大缩小这种差异，从而使更准确和更容易的测量许多生物医学应用。

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.