Improved Confocal Microscope: Detection & Functionality

描述(由申请人提供)：将开发一种低成本的改装包，以提高标准共聚焦显微镜的灵敏度，并升级其作为研究生物标本的工具的功能。用光子计数装置取代模拟探测器将提高信噪比。通过共焦口的数字检测将使荧光相关光谱(PCS)能够研究细胞结构的单分子动力学、扩散、聚集、粘度和动力学。在中等光线下获得的初步数据显示，数字检测使灵敏度提高了100%。在非常低的光照水平下，预计会有更大的增强效果。脉冲计数电子设备、新的数据采集硬件和固件将集成到一个紧凑型微控制器模块中，该模块通过USB连接到带有Lab view用户界面和图像分析软件的PC。将设计单独的电路来控制振镜，以改进对扫描过程的控制，并固定振镜进行定点测量。定点测量模式是FCS、单分子光谱分析和药物多井筛选所必需的。改装包中将包括一个定制的带有准直光学元件的侧窗，以提高显微镜收集效率。侧窗上将安装一台低成本的光纤光谱仪，从荧光信号中提取单分子动力学信息(定点模式)，并获得样品的高光谱图像(扫描模式)。拟议的改进措施相对简单、成本低廉，而且不局限于特定品牌的乐器。为了促进未来的商业化，已经确定了对改装包的产品版本的分销感兴趣的显微镜维护/改装公司。

DESCRIPTION (provided by applicant): A low-cost modification package will be developed to enhance the sensitivity of a standard confocal microscope and to upgrade its functionality as a tool for studying biological specimens. Replacing analog detectors with photon counting devices will improve signal-to-noise ratio. Digital detection through a confocal port will enable fluorescence correlation spectroscopy (PCS) to study single-molecule kinetics, diffusion, aggregation, viscosity and dynamics of cellular structures. Preliminary data obtained at moderate light levels show > 100% sensitivity enhancement with a digital detection. A much greater enhancement is expected at very low light levels. The pulse-counting electronics, new data acquisition hardware and firmware will be integrated into a compact microcontroller module with a USB connection to a PC with a Lab VIEW user interface and image analysis software. Separate circuitry to control the galvo mirrors will be devised to improve the control over the scanning process and to immobilize the galvo for the fixed-point measurements. Fixed-point measurement mode is necessary for the FCS, single-molecule spectroscopy and pharmaceutical multi-well screening. A custom-designed sideport with collimation optics will be included in the modification package to enhance the microscope collection efficiency. A low-cost fiberoptic spectrometer will be attached to the sideport to extract single molecule dynamics information from fluorescence signal (fixed-point mode) and to obtain hyperspectral image (scanning mode) of the sample. The proposed improvements are relatively simple, inexpensive and not tied to a specific brand of instrument. To facilitate a future commercialization, a microscope maintenance/modification company that is interested in distribution of the product version of the modification package has been identified.