Development of a Motionless 3D Fluorescence Microscope

描述(申请人提供)：全息术是一种很有吸引力的成像技术，因为它提供了从一张图像中查看完整3D体积的能力，甚至达到了超过罗利限制的分辨率。然而，全息术在三维荧光显微成像中的应用并不广泛，因为荧光是非相干的，制作全息图需要一个相干干涉仪系统。为了克服相干性的限制，人们提出了通过扫描一束干涉仪图案穿过物镜的背面孔径来激发样品中的荧光，但是这种方法仅限于低数值孔径物镜，并且机械上很复杂。我们发展了一种用于菲涅耳非相干全息的简单的非相干全息技术，我们称之为Finch。最近，我们将Finch技术应用于荧光显微镜，创造了第一个静止的3D显微镜系统，我们称之为“FINCHSCOPE”。它只需一个空间光调制器(SLM)、一个CCD相机和一些简单的滤光片，就可以使用高数值孔径物镜记录生物标本的高分辨率3D荧光图像。FINCHSCOPE能够在不需要扫描或任何显微镜移动的情况下获取3D显微图像。FINCHSCOPE有可能极大地简化3D荧光显微成像，并实现比目前其他方法更快的3D成像速度，因为它可以在一次曝光中获得完整的3D体积。因此，可以在单个活细胞中快速监测荧光甚至发光的探针、颗粒和蛋白质。第一阶段和第二阶段的SBIR项目的目的是支持FINCHSCOPE发展成为商业上可行的仪器。在该项目的第一阶段，我们将修改我们目前正在工作的原型，以便它的性能可以被证明产生与标准3D显微成像技术相同的分辨率。这将通过将系统中的速率限制组件、空间光调制器(SLM)的位深度从其当前的8位操作提高到10位性能来实现。此外，软件将从解释的MatLab语言改为编译语言，以便更快地性能和更好地控制数码相机，从而实现其2Kx2K的完全分辨率。在第二阶段项目中，FINCHSCOPE将被开发来解决各种3D显微成像问题，包括高速3D显微成像以及在流式细胞仪和高含量筛选中的成像应用。我们公司最近发明了一种新的全息术概念，称为菲涅耳非相干相关全息术的Finch，它极大地简化了全息图像的获取，并且不像经典全息术那样需要激光。用数码相机从任何场景捕捉全息图像的方法已经应用于显微镜，结果被称为FINCHSCOPES显微镜。该项目旨在为各种应用改进和开发这些显微镜的商业版本。

DESCRIPTION (provided by applicant): Holography is an attractive imaging technique as it offers the ability to view a complete 3D volume from one image and even achieve resolution beyond the Raleigh limit. However, holography is not widely applied to 3D fluorescence microscopic imaging, because fluorescence is incoherent and creating holograms requires a coherent interferometer system. Scanning one beam of an interferometer pattern across the back aperture of an objective to excite fluorescence in a specimen has been proposed to overcome the coherence limitation however it is limited to low numerical aperture objectives and is mechanically complex. We developed a new simple incoherent holography technique which we call FINCH for Fresnel Incoherent Correlation Holography. Recently we have applied the FINCH technique to fluorescence microscopy creating the first motionless 3D microscopy system which we call "FINCHSCOPE". It can record high-resolution 3D fluorescent images of biological specimens using high numerical aperture objectives, with just a spatial light modulator (SLM), a CCD camera, and some simple filters. FINCHSCOPE enables the acquisition of 3D microscopic images without the need for scanning or any microscope movement. FINCHSCOPE has the potential to greatly simplify 3D fluorescence microscopic imaging and to enable higher speed 3D imaging than currently possible by other methods because it is possible to obtain the complete 3D volume in one single exposure. Thus fluorescent or even luminescent probes, particles and proteins could be rapidly monitored in single living cells. The purpose of this SBIR project in Phase 1 and 2 is to support the development of the FINCHSCOPE into a commercially viable instrument. In the phase 1 aspect of the project we will modify our current working prototype such that it performance can be demonstrated to yield the same resolution as standard 3D microscopic imaging techniques. This will be done by improving the bit depth of a rate limiting component in the system, the spatial light modulator (SLM) from its current 8 bit operation to 10 bit performance. Furthermore the software will be changed from the interpreted MATLAB language to a compiled language for faster performance and better control of the digital camera such that its full 2Kx2K resolution can be achieved. In the Phase 2 project, the FINCHSCOPE will be developed to solve a variety of 3D microscopic imaging problems including high speed 3D microscopic imaging and application to imaging in flow cytometry and high content screening. Our company has recently invented a new concept in holography called FINCH for Fresnel Incoherent Correlation Holography which dramatically simplifies the acquisition of holographic images and does not require lasers as in classical holography. The method which captures a holographic image from any scene upon a digital camera has been applied to microscopy, the resultant microscopes called FINCHSCOPES. This project is aimed at improving and developing commercial versions of these microscopes for a wide variety of applications.