The objective of this project is to investigate novel methods aimed at achieving the highest possible spatial resolution with a new instrument being developed for biological microscopy. The device is referred to as a Laser-Plasma Microfluoroscope. In essence, it is a miniaturized fluoroscope that uses low-energy x-rays to image individual cells and other thin samples. Specimens are placed in direct contact with a grainless fluorescent screen (scintillator) and illuminated with pulses of soft x-rays radiated by an extremely hot laser-produced plasma. The resulting unmagnified luminescent shadowgraph of the sample is viewed in real-time using light microscopy. The technique can be thought of as a hybrid of light microscopy and soft x-ray microscopy. The method produces images having extremely high depth-of-field, with three-dimensional information of overlapping features accessible using stereoscopic imaging methods or possibly tomography. Key advantages of the instrument's design are its relatively low-cost, very compact size, and its ability to be used as an accessory device with ordinary light microscopes. Thus, it is not required to have a separate dedicated soft x-ray microscope. The instrument has the unique capacity to rapidly switch imaging modes between light microscopy and microfluoroscopy. Previous research demonstrated a very compact Laser-Plasma Microfluoroscope that was used in conjunction with a standard inverted light microscope to produce microfluoroscopic images having a point-to-point spatial resolution near 200 nm. This represents the highest resolution ever achieved using fluoroscopic methods. In this project, we are seeking to dramatically improve this resolution to a level routinely below 100 nm, and below 50 nm for very thin samples. Such unprecedented performance levels in microfluoroscopy require the development of innovative new approaches. Phase I work studied the technical feasibility of several approaches intended to extend the resolution performance of the current instrument below 100 nm. The most promising directions for Phase II research were identified, which include applying very recently developed state-of-the-art optics in the deep-ultraviolet spectral range to this instrument. In addition to microfluoroscopy, more conventional microimaging modes will be investigated using the extraordinary high-performance optics being developed in this project. The goal of the Phase II effort is to produce a prototype instrument capable of extremely high-resolution imaging that is appropriately designed for subsequent Phase III commercialization. Maintaining reasonable cost and ease of use will be very high priorities with this microscope accessory. In addition to demonstrating outstanding resolution with simple test objects, we plan to apply the instrument to a real research problem during this project. The instrument being developed in this project is designed to be a relatively inexpensive accessory device that can be used with standard light microscopes in life-science laboratories. With the significant improvement in resolution that is achievable using this microscopy technique, it should find widespread use in such fields as medical research, cell biology, microbiology, and possibly clinical medicine. Due to the ubiquitous nature of optical microscopy, the commercial potential of this work is very large.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
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Special Emphasis Panel (ZRG1-SBMI-T (10))
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Deatherage, James F
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Hirsch Scientific
United States
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