Portable Hyperspectral Multiphoton Excitation Fluorescence Lifetime Microscopy Imager
- Physical Sciences Inc.
During this Phase I program, Physical Sciences Inc. (PSI) and Michigan State University (MSU) developed and evaluated an advanced multiphoton fluorescence lifetime microscopy (MPM) imager that can be used for measuring and monitoring the metabolic state and the underlying molecular mechanisms of plant cells in situ. The MPM imaging technology leverages proven technologies, such as the optical tissue sectioning and 3 D imaging capability of multiphoton excitation microscopy, and the molecular specificity, sensitivity, and dynamic information provided by fluorescence spectroscopic and lifetime measurements. Innovative approaches were used to develop this novel technology, including a compact and low-cost coherent supercontinuum (SC) source for MPM imaging of multiple fluorophores, and a novel optical sensor for simultaneous fluorescence spectroscopy and lifetime measurements. These technical innovations allowed us to demonstrate that an affordable and portable MPM instrument can be developed and deployed in the field for functional and molecular imaging of plant cells in vivo. These unique features have clear and significant advantages over existing multiphoton and confocal microscopes, which are normally fixed on large optical tables. The goal of the Phase I work was to evaluate the feasibility of the proposed approach through the development of an alpha prototype instrument and to evaluate its capability for imaging plant morphology and for monitoring plant metabolism. The PSI and MSU team achieved this goal by successfully developing a cart mounted MPM prototype, and testing its performance in several preliminary plant imaging experiments. Important Phase I accomplishments and findings include: Development of a fiber-delivered Supercontinuum (SC) laser excitation source. The wavelength selectivity provided by the SC source is critical for efficiently exciting targeted fluorophores and achieving high SNRs. Demonstrated that it is feasible to develop a high-performance, field-deployable instrument based on low cost components and innovative technologies developed by PSI. During the Phase I effort high-resolution imaging was achieved using a novel sample stabilization technique. Redox imaging based on NADH/FAD was used to monitor metabolic changes in plant cells. The high optical sectioning capability of the MPM was critical in suppressing the interference of the out-of-focus autofluorescence fluorophores. The strong spatial confinement of the multi-photon excitation was critical in reducing laser damage to the samples, demonstrating another important benefit of this technology for plant imaging. High-speed, non-stop beam scanning was effective in reducing the laser fluence and thus sample damage. Preliminary Phase I results indicate that detailed spectroscopy and lifetime information provided by the MPM instrument will support future experiments and provide additional cellular dynamics information, including the potential to identify new metabolites. In summary, the Phase I alpha prototype instrument development and application studies provided an important feasibility demonstration for the field-use MPM instrument. PSI will continue to work to mature the technology and demonstrate the application in the plant biology and other fields.
- Research Organization:
- Physical Sciences, Inc., Andover, MA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC)
- DOE Contract Number:
- SC0018607
- OSTI ID:
- 1509705
- Type / Phase:
- SBIR (Phase I)
- Report Number(s):
- DOE-PSI-18607; PSI-4066/FinalReport
- Country of Publication:
- United States
- Language:
- English
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