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Development of a Motionless 3D Fluorescence Microscope

静止 3D 荧光显微镜的开发

基本信息

批准号：
7611265
负责人：
Gary Brooker
金额：
$ 14.42万
依托单位：
CELLOPTIC, INC.
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-01 至 2009-09-30
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：全息术是一种有吸引力的成像技术，因为它能够从一张图像中查看完整的 3D 体积，甚至实现超出罗利极限的分辨率。然而，全息术并未广泛应用于 3D 荧光显微成像，因为荧光是不相干的，并且创建全息图需要相干干涉仪系统。已经提出将干涉仪图案的一束扫描穿过物镜的后孔径以激发样本中的荧光来克服相干性限制，但是它仅限于低数值孔径物镜并且机械上很复杂。我们开发了一种新的简单非相干全息技术，我们将其称为 FINCH（菲涅尔非相干相关全息术）。最近，我们将 FINCH 技术应用于荧光显微镜，创建了第一个静止 3D 显微镜系统，我们称之为“FINCHSCOPE”。它可以使用高数值孔径物镜记录生物样本的高分辨率 3D 荧光图像，只需空间光调制器 (SLM)、CCD 相机和一些简单的滤光片。 FINCHSCOPE 无需扫描或任何显微镜移动即可采集 3D 显微图像。 FINCHSCOPE 有潜力大大简化 3D 荧光显微成像，并实现比目前其他方法更快的 3D 成像，因为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探针、颗粒和蛋白质。该 SBIR 项目第一阶段和第二阶段的目的是支持 FINCHSCOPE 发展成为商业上可行的仪器。在该项目的第一阶段，我们将修改当前的工作原型，以证明其性能能够产生与标准 3D 显微成像技术相同的分辨率。这将通过提高系统中速率限制组件的位深度来实现，空间光调制器 (SLM) 从当前的 8 位操作提高到 10 位性能。此外，该软件将从解释型 MATLAB 语言更改为编译型语言，以实现更快的性能和更好地控制数码相机，从而实现其完整的 2Kx2K 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术和高内涵筛选中的成像应用。我们公司最近发明了一种新的全息概念，称为 FINCH，即菲涅尔非相干相关全息术，它极大地简化了全息图像的采集，并且不需要像经典全息术那样使用激光。这种用数码相机从任何场景捕捉全息图像的方法已应用于显微镜，由此产生的显微镜称为 FINCHSCOPES。该项目旨在改进和开发这些显微镜的商业版本，以适应各种应用。