TECHNOLOGY AREA(S): Chem Bio_defense, Bio Medical
OBJECTIVE: Develop an optical method for the determination of cellular structure and function in possible pathogen organisms
DESCRIPTION: Label- and reagent-free techniques for three-dimensional (3D) cell imaging with tens of nanometer spatial resolution are needed to characterize the structure and function of potential pathogen organisms. The approach should provide specific biomolecular functional information which will further aid in discrimination. A real time capability would further enhance the utility for time-resolved observation of the organism as it reacts to environmental modifications or stimuli and therapeutic or antibiotic activity.
Historically, sub-diffraction scale resolution has been obtained through fluorescence saturation techniques, typically through manipulating the excitation intensity through interference. These techniques have been limited to certain fluorescent proteins and do not yield functional or 3D information. Nanometer scale molecular confirmation determination has been performed using a 4Ï configuration and spectral self-interference relative to a reflective surface. This technique on the other hand is not label-free. Recently, high resolution (label-free) phase analysis has become of interest for morphological and composition determination of a specimen in areas of tissue disease diagnosis. Diffraction limited phase measurements are possible using digital holographic microscopy (DHM) which is ideal for complex electromagnetic wave fields scattered by biological tissue. Recent efforts have employed DHM in a quasi-2Ï configuration with violet illumination and reported unprecedented lateral resolution (60 nm). Moreover, by scanning a synthetic aperture and recording a series of projections through the sample, tomographic 3D reconstruction can be accomplished.
There is a need to augment current tomographic nanoscopy methods to develop better detection and diagnostic capabilities. A functional capability, such as Raman or infrared absorption spectroscopy, would support species discrimination based on the biomolecular make-up of the organism. High-speed data generation and analysis would enable time-resolved measurements of the organisms response to environmental changes or to therapeutics such as antibiotics. These enhanced capabilities will promote better discrimination of the organism and a more intrinsic understanding of the organisms behavior as it relates to treatment discovery.
PHASE I: Generate a preliminary design of a tomographic nanoscopic cell imaging system capable of biomolecular functional determination. Perform a system level analysis predicting the overall performance including key parameters such as lateral and axial resolutions, sensitivity, specificity, and baseline computational timing. Generate an opto-mechanical design and identify the key components. Breadboard subsystems identified to be high in technical risk. Generate a preliminary data processing and reconstruction design.
PHASE II: Design, build, and test a prototype tomographic nanoscale cell imaging system. Optimize the processing and reconstruction algorithms for near real time. Fully characterize the system. Demonstrate a series of relevant cellular measurements, including a time-resolved series of a cells response to external stimuli. Deliver the prototype system to the government.
PHASE III: During Phase III, refine a final design tailored for a specific application or platform, incorporating design modifications based on results from tests conducted during Phase II, and improving engineering/form-factors, processing/reconstruction, and manufacturability designs to meet requirements of the Joint Chemical and Biological Defense Program and end-user requirements.
This type of technology will also find use in medical research laboratories for cell and tissue diagnostics, biomarker discovery and identification and in pharmaceutical laboratories for drug discovery.
REFERENCES: 1: M.G.L. Gustafsson, Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy, J. Microsc. 198, 82-87 (2000).2: B.J. Davis,1, M. Dogan, B.B. Goldberg, W.C. Karl, M. S. Ãnlü, and A.K. Swan, 4Pi spectral self-interference microscopy, JOSA A 24(12), 3762-3771 (2007).3: Y. Cotte, F. Toy, P, Jourdain, N. Pavillon, D. Boss, P. Magistretti, P. Marquet, and C. Depeursing, Marker-free phase nanoscopy, Nat. Photonics 7, 113-117 (2013).4: E. Cuche, P. Marquet, and C. Depeursinge, Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms, Appl. Opt. 38(34), 6994-7000 (1999).5: P. Marquest, et al., Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with sub-wavelength accuracy, Opt. Lett. 30, 468 (2005).6: D. Naumann, et al., The characterization of microorganisms by Fourier Transform Infrared Spectroscopy (FTIR), in Modern Techniques for Rapid Microbiological Analysis, W.H. Nelson, ED., VCH Publishers, New York, 1991.7: D. Whelan, et al., Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscopy, Nucleic Acids Research 39, 5439 (2011).
KEYWORDS: Nanoscopy, Spectroscopy, Tomography, Cell Imaging, Pathogen Identification, Sub-diffraction Scale
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