A new paradigm in three-dimensional microscopic imaging will be formulated, tested, and evaluated to address two specific issues in structural biology; Namely, the issue of multifunctional imaging (the parallel acquisition of images with complementary information, e.g. fluorescence, absorption, quantitative phase), and the issue of measurement of specifically labeled intracellular targets with sub-nanometer accuracy. Multifunctional imaging is essential in quantitative structural biology for identifying correlations between specific morphological features and functional characteristics. Sub-nanometer accuracy in distance measurement is needed in e.g. structural genomic research, as well as in other areas of nanoscience. The new paradigm is based on implementing the steps of the imaging process in a reverse order compared to that of the conventional model. Specifically, a spatially structured and temporally modulated three-dimensional interference pattern is created by the objective, projected on the specimen, and scanned in a two-dimensional raster. A series of non-imaging detectors tuned to the modulation frequency collect, in parallel, the transmitted, scattered, and fluorescent light. The scanning pattern is designed to synthesize, after decoding the data, a point-spread-function with specific characteristics leading to specific imaging modalities. Detector masks allow simultaneous imaging in coherent mode (for quantitative phase measurements), and incoherent mode (for intensity and fluorescence measurements). The primary aim is to build a laboratory prototype to (1) demonstrate parallel multifunctional imaging, (2) demonstrate simultaneous coherent and incoherent (phase and fluorescence) imaging, (3) demonstrate the synthesis of point-spread-functions leading to improved spatial resolution, extended depth-of-focus, and axial sectioning, (4) demonstrate the possibility of distance measurement between point-like targets with sub-nanometer accuracy by combining spatially structured illumination with temporal modulation and phase-sensitive detection, (5) evaluate quantitatively and critically the capabilities and limitations of the proposed paradigm.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21RR018440-01
Application #
6596434
Study Section
Special Emphasis Panel (ZRR1-BT-1 (01))
Program Officer
Farber, Gregory K
Project Start
2003-08-01
Project End
2005-07-31
Budget Start
2003-08-01
Budget End
2004-07-31
Support Year
1
Fiscal Year
2003
Total Cost
$102,240
Indirect Cost
Name
Virginia Polytechnic Institute and State University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
003137015
City
Blacksburg
State
VA
Country
United States
Zip Code
24061
Indebetouw, Guy; Tada, Yoshitaka; Rosen, Joseph et al. (2007) Scanning holographic microscopy with resolution exceeding the Rayleigh limit of the objective by superposition of off-axis holograms. Appl Opt 46:993-1000
Indebetouw, Guy; Tada, Yoshitaka; Leacock, John (2006) Quantitative phase imaging with scanning holographic microscopy: an experimental assessment. Biomed Eng Online 5:63
Rosen, Joseph; Indebetouw, Guy; Brooker, Gary (2006) Homodyne scanning holography. Opt Express 14:4280-4285
Indebetouw, Guy; Zhong, Wenwei (2006) Scanning holographic microscopy of three-dimensional fluorescent specimens. J Opt Soc Am A Opt Image Sci Vis 23:1699-707
Indebetouw, Guy (2006) A posteriori quasi-sectioning of the three-dimensional reconstructions of scanning holographic microscopy. J Opt Soc Am A Opt Image Sci Vis 23:2657-61
Indebetouw, Guy; Zhong, Wenwei; Chamberlin-Long, David (2006) Point-spread function synthesis in scanning holographic microscopy. J Opt Soc Am A Opt Image Sci Vis 23:1708-17
Indebetouw, Guy; El Maghnouji, Alouahab; Foster, Richard (2005) Scanning holographic microscopy with transverse resolution exceeding the Rayleigh limit and extended depth of focus. J Opt Soc Am A Opt Image Sci Vis 22:892-8