Metrolaser, Inc. SBIR Phase I Award, January 2020

New instrumentation and bioimaging devices are required for viewing, understanding, and modeling key metabolic processes in living organisms such as within and among microbial cells and microbial communities in terrestrial environments, and/or multicellular plant tissues. Small, low footprint devices are needed to make in situ, nondestructive, functional imaging and quantitative measurement and capable of operation independently of heavy equipment and large instruments e.g. neutron and light sources, cryo- electron microscopes, high resolution mass spectrometers). These devices must also be easily deployed and affordable in public and private sector to make them accessible to the larger scientific community. We will develop an affordable, low footprint microscope to satisfy the stated measurement requirement by exploiting unique features provided by Digital Holographic Microscopy DHM). Such features can remove key limitations in existing bioimaging systems and enable imaging and measuring key metabolic processes within and among microbial cells and multicellular plant tissues. Holography enables optically freezing a 4D space, which allows microscopic study of dynamic events in a large volume at many instants of time. Thanks to recent advances in electronic imaging systems, computers, and memory, DHM holograms can now be recorded on small, lightweight, CCD or CMOS arrays at very high rates, processed in real time, and offer the many advantages associated with digital media. By transferring much of the overall optical process into electronics and software, DHM electronically stores, in a hologram, all optical information in a light wave scattered from, transmitted through, or reflected from a biological surface of interest, enabling dynamic, microscopic imaging over large fields of view and depths of field with a relatively small instrument, capabilities that cannot be achieved with conventional imaging devices. In addition to using traditional holographic methods for 3D diagnostics, we propose to directly analyze the hologram with machine learning techniques, avoiding the computationally costly step of reconstructing images. Existing software that can learn to analyze an image can, in principle, be employed as well to analyze the raw hologram directly, which provides much more information than a focused reconstructed image. Large FoV high-res microscopes require prohibitively expensive large-scale aberration corrected optics, and commercially available DHM systems combine holography and standard microscope optics and methods that severely limit field of view and require focusing on reconstructed images. We will exploit holographic techniques that enable lens aberration compensation in software, allowing low cost, large- aperture, large-area FoVs. In Phase I we will demonstrate, experimentally, the capability of this system to record and measure typical critical parameters of biological cells, their movements, and changes in time, employing a flexible and controllable simulated biological system to refine, qualify, and characterize the measurement system and identify critical issues. In Phase II we will add a microbiologist to the team and employ live biological samples to shift the focus to making measurement on living biological systems and producing biological data. Because of enhanced speed, this DHM technology should have significant applications in industrial inspection, where speed is at a particularly high premium. For example, the semiconductor industry is a particularly attractive target. Because of the inherent flexibility of machine learning, there are almost innumerable areas of non-destructive testing where a low-cost device like this could be used. Also, the wider availability of DHM to research labs, provided by lower cost, would benefit industry and U.S. government funded research, such as university research. Companies that have commercialized various forms of holographic microscopes have concentrated on replicating the functionality of a conventional microscope using holography, and usually with the goal of maximizing resolution. The proposed system differs from available systems and provides a flexible mix of spatial resolution, angular resolution, throughput, field of view, and phase sensitivity by taking digital microscopy to a new level.