This proposal is directed toward development of a fluorescent spectral imaging system for simultaneous high resolution sub-cellular microscopy of multiple fluorescence probes in living cells. Recent developments in fluorescent probes, imaging instrumentation and micro- fabrication now permit building for an Image Slicing Spectrometer (ISS) for real time quantitative spectral imaging. We propose to combine our expertise in microscopy, optical design, fabrication, and imaging with newly available large format CCD cameras and fabrication techniques to develop an ISS system. ISS is a widefield method that is capable of acquiring full spectral information simultaneously from every pixel. This approach works by spatially redirecting image zones to obtain space between image lines. Next, by using a diffractive element, ISS obtains wavelength spread on the CCD camera (for more details on the system principle see Sections C and D). In this way, we unambiguously map x,y, ?;data onto the 2-D image sensor.
The specific aims of the project are: (1) to construct the ISS with an initial wavelength range of 450 to 700 nm and (2) to test the Image Slicing Spectrometer against currently available spectral imaging systems in several live cell imaging applications. Work on development of imaging spectrometers for cellular imaging has thus far been hampered either by small fields of view, limited temporal-spatial-spectral resolution, requirement of extensive computations, or limited light efficiency. The Image Slicing Spectrometer proposed here is based on a concept borrowed from the astronomy field, and addresses the major difficulties previously connected with construction of a snapshot spectrometer. The Image Slicer transforms a rectangular Field of View (FoV) into a series of mini """"""""slits"""""""", and rearranges them to create sufficient area for spectral spread and acquisition in the snapshot mode. No complicated processing is necessary and only simple remapping is sufficient to obtain a complete x,y, ?;data cube. The core of the system will be a custom-made redirecting mirror fabricated with diamond turning technology. The instrument will employ a Hamamatsu CCD camera with 4000 x 2624 pixel elements, Peltier cooling, and low-noise readout (C4742-98-24HR). Using this large format CCD, the final image data cube will be 400 x 260 x 50 (X, Y, ?) with a spectral resolution of 5 nm and ~0.5

Public Health Relevance

The project targets the development of a modern spectrometer called Image Slicing Spectrometer enabling high resolution spectral imaging in real time. In consequence researchers will be able to rapidly advance the investigation of live cells with multiple fluorescent contrasts. The instrument's principle allows obtaining spectral information for entire image without scanning and thus improve signal to noise ratio. It also allows also more efficient investigation of transient biological events. Technologies applied in the project and their low cost may potentially allow access of larger group of scientists to spectral imaging instrumentation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21EB009186-02
Application #
7695567
Study Section
Microscopic Imaging Study Section (MI)
Program Officer
Zhang, Yantian
Project Start
2008-09-30
Project End
2011-08-31
Budget Start
2009-09-01
Budget End
2011-08-31
Support Year
2
Fiscal Year
2009
Total Cost
$185,488
Indirect Cost
Name
Rice University
Department
Biomedical Engineering
Type
Schools of Engineering
DUNS #
050299031
City
Houston
State
TX
Country
United States
Zip Code
77005
Konecky, Soren D; Wilson, Robert H; Hagen, Nathan et al. (2015) Hyperspectral optical tomography of intrinsic signals in the rat cortex. Neurophotonics 2:045003
Bedard, Noah; Hagen, Nathan; Gao, Liang et al. (2012) Image mapping spectrometry: calibration and characterization. Opt Eng 51:
Elliott, Amicia D; Gao, Liang; Ustione, Alessandro et al. (2012) Real-time hyperspectral fluorescence imaging of pancreatic ?-cell dynamics with the image mapping spectrometer. J Cell Sci 125:4833-40
Gao, Liang; Tkaczyk, Tomasz S (2012) Correction of vignetting and distortion errors induced by two-axis light beam steering. Opt Eng 51:043203
Gao, Liang; Smith, R Theodore; Tkaczyk, Tomasz S (2012) Snapshot hyperspectral retinal camera with the Image Mapping Spectrometer (IMS). Biomed Opt Express 3:48-54
Hagen, Nathan; Kester, Robert T; Gao, Liang et al. (2012) Snapshot advantage: a review of the light collection improvement for parallel high-dimensional measurement systems. Opt Eng 51:
Gao, L; Hagen, N; Tkaczyk, T S (2012) Quantitative comparison between full-spectrum and filter-based imaging in hyperspectral fluorescence microscopy. J Microsc 246:113-23
Hagen, Nathan; Gao, Liang; Tkaczyk, Tomasz S (2012) Quantitative sectioning and noise analysis for structured illumination microscopy. Opt Express 20:403-13
Hagen, Nathan; Tkaczyk, Tomasz S (2011) Compound prism design principles, III: linear-in-wavenumber and optical coherence tomography prisms. Appl Opt 50:5023-5030
Kester, Robert T; Bedard, Noah; Gao, Liang et al. (2011) Real-time snapshot hyperspectral imaging endoscope. J Biomed Opt 16:056005

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