Novel wedge-based approach for simultaneous multichannel microscopy

描述（由申请人提供）：物理科学公司（PSI）和波士顿大学医学院克里斯托弗·加贝尔实验室提出设计、开发、表征和演示一种新型的同步多通道显微镜方法，该方法将克服现有技术的许多限制。同时多通道显微镜技术测量由不同波长的光报告的多个可观察物的变化，随着非侵入性测量变得更加可行和可取，这些技术在许多生物研究领域得到了更大的应用。示例应用包括体内比例神经元钙成像，多种荧光蛋白和标记的共定位，以及非侵入性pH测量。尽管多通道成像无处不在，而且目前有三种商业方法可以同时进行多通道成像，但每种方法的局限性阻碍了该技术的充分利用和开发。该项目的长期目标是开发一种简单、廉价的商用设备，使标准单通道荧光显微镜能够同时进行多通道显微镜检查。这种方法可以很容易地扩展到更高数量的通道，并通过同时成像减少光漂白，提高荧光显微镜的能力。此外，该设备不包含移动部件，不需要校准或专业知识来操作，允许先进的成像，同时保持用户友好性。直接受益于这种新方法的科学项目领域包括分子成像、光学成像和光谱学以及组织工程。在第一阶段计划中，我们建议制造和光学测试设备原型，以确认符合商业成像标准。特别是，原型将在常规显微镜的三个位置进行构建和测试，以确定性能，成本和便利性的最佳位置和配置。设备性能将通过在线虫线虫和细胞培养中成像标准荧光团来评估，并将这些数据与传统多通道成像设备的相应测量结果进行比较。先进工程将在第二阶段进行，以降低设备成本，进一步评估，并开发技术商业化的途径。

DESCRIPTION (provided by applicant): Physical Sciences Inc. (PSI) and the laboratory of Christopher Gabel at Boston University School of Medicine propose to design, develop, characterize, and demonstrate a novel approach to simultaneous multichannel microscopy that will overcome a number of limitations of existing technologies. Simultaneous multichannel microscopy techniques measure changes in multiple observables that are reported by different wavelengths of light, and these techniques are finding greater application in many areas of biological research as noninvasive measurements become more attainable and desirable. Example applications include in vivo ratiometric neuronal calcium imaging, co-localization of multiple fluorescent proteins and labels, and non-invasive pH measurement. Despite the ubiquity of multichannel imaging and the existence of three current commercial approaches to perform simultaneous multichannel imaging, limitations to each approach prevent full utilization and exploitation of the technique. The long-term objective of this project is to develop a simple, inexpensive commercial device that enables standard single-channel fluorescence microscopes to perform simultaneous multichannel microscopy. This approach can easily scale to higher numbers of channels and reduces photobleaching by simultaneous imaging, advancing the capabilities of fluorescence microscopy. Furthermore, the device contains no moving parts and does not require alignment or specialized expertise to operate, allowing advanced imaging while maintaining user-friendliness. Scientific program areas benefitting directly from the novel approach include molecular imaging, optical imaging and spectroscopy, and tissue engineering. During the Phase I program we propose to fabricate and optically test prototypes of the device to confirm adherence to commercial imaging standards. In particular, prototypes will be constructed for and tested in three locations in conventional microscopes to determine the optimal location and configuration for performance, cost, and convenience. Device performance will be evaluated by imaging standard fluorophores in the nematode worm C. elegans and cell culture and comparing these data to corresponding measurements on conventional multichannel imaging devices. Advanced engineering will be performed in Phase II to reduce device cost, further evaluate it, and develop a path for technology commercialization.