Development of a Motionless 3D Fluorescence Microscope

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.