Contact Fluorescence Imaging

描述（申请人提供）：研究细胞结构和功能的光学荧光探针广泛应用于免疫学、组织化学、细胞凋亡、细胞粘附、趋化、信号转导、电生理学等生物学领域。这些探针包括传统的基于化学的传感器和基因编码的荧光蛋白生物传感器，它们都能够实时测量活细胞和组织中的生理信号。为此，冷却电荷耦合器件（CCD）或光电二极管阵列通常与成像光学一起使用。这些商用系统的采样速度有限，成像区域的大小也有限制，而且通常体积庞大，价格昂贵。一种新的方法——接触荧光成像（CFI），是一种对大面积荧光信号进行时空成像的简单方法。该方法的广泛应用面临的挑战是能否获得快速响应、高灵敏度的大画幅成像仪。在探索性R21阶段，我们将展示一种基于互补金属氧化物半导体（CMOS）技术的新型成像仪的可行性。先进的模拟信号处理技术将使成像仪具备像素级基线减法、对比度模式操作、双波长成像和比例成像等所有商用成像阵列所缺乏的能力。在R33阶段，我们将扩大传感器阵列的尺寸，并生产可以从较小的模块化阵列平铺的大格式CMOS成像仪。这个项目的成果将是一个紧凑的成像系统，能够用荧光生物传感器监测静态和动态细胞事件。它将是微型的，便携的，坚固的，可重复使用的，并适应广泛的应用。我们将演示如何使用新的成像系统来可视化大尺寸心肌细胞培养中钙和电压波的时空动态，以及它作为传统荧光成像仪的一般适用性，可以连接到商业显微镜的相机端口。

DESCRIPTION (provided by applicant): Optical fluorescent probes of cellular structure and function are widely used in biology in fields as diverse as immunology, histochemistry, apoptosis, cell adhesion, chemotaxis, signal transduction, and electrophysiology. These probes include traditional chemically-based sensors and genetically-encoded fluorescent protein biosensors, all of which enable the measurement of physiological signals in real-time in living cells and tissue. For this purpose, cooled charge-coupled device (CCD) or photodiode arrays have typically been used with imaging optics. These commercial systems have limited sampling speed and limitations in the size of the imaging area, and are generally bulky and costly. A new method, Contact Fluorescence Imaging (CFI), is a simple solution for the spatiotemporal imaging of fluorescence signals over large areas. The challenge in terms of the widespread application of this method is the availability of a fast response, large format imager with high sensitivity. In the exploratory R21 phase, we will show feasibility of a new type of imager based on complementary metal oxide semiconductor (CMOS) technology. Advanced analog signal processing techniques will render the imager capable of pixel-level baseline subtraction, contrast mode operation, dual wavelength imaging, and ratiometric imaging, all capabilities that are lacking in commercially available imaging arrays. In the R33 phase, we will scale up the size of the sensor arrays, and produce a large format CMOS imager that can be tiled from smaller modular arrays. The outcome of this project will be a compact imaging system with the ability to monitor static and dynamic cellular events with fluorescent biosensors. It will be miniature, portable, rugged, reusable and adaptable to a wide range of applications. We will demonstrate how the new imaging system can be used to visualize the spatiotemporal dynamics of calcium and voltage waves in large-sized cultures of cardiac cells, as well as its general applicability as a conventional fluorescence imager that can be attached to the camera port of commercial microscopes.