3-D Superresolution Imaging in Living Cells with Single-Molecule Active Control Recent advances in microscopic imaging techniques with single molecules have led to superresolution information, that is, the ability to observe objects with resolution beyond the standard diffraction limit. These methods involve wide-field imaging, and require active control of the molecules in order to either turn emitters on or turn emitters off in order to maintain the concentration of emitters low enough to digitize the point-spread functions of individual molecules. By many imaging, photobleaching, and reactivation cycles, a superresolution image is obtained, but only for a two-dimensional projection of the actual three-dimensional sample. These methods may be collectively termed Single-Molecule Active Control Microscopy (SMACM), and have previously been applied primarily to fixed cells. However, many samples of biomedical interest, such as cells, are thick enough that two-dimensional imaging is a severe limitation. The primary goal of this research program is to achieve three-dimensional superresolution imaging in living cells using SMACM. This research will attack the problem of 3-D superresolution imaging with three thrusts. First, the optical illumination used to achieve active control will be tailored in its intensity as a function of time, in order to increase the efficiency of the reactivation and imaging process and eventually enable observation of time- dependent changes. Second, the microscope will be redesigned to utilize rotating point-spread functions. This relies on forcing the image of a single emitter to have a shape at the detector which rotates for different z- positions of the single molecule in the sample. The effect of this will be to enable much more precise determinations of the z positions of various single-molecule labels in the sample, which, when combined with precise localization in the x-y plane, will yield three-dimensional image information beyond the diffraction limit. Third, the research will implement multi-plane imaging in addition to rotating point-spread-functions, which will enable acquisition of 3D information over a greater depth into the sample. The results of this research will be to enable a new type of optical microscopy of cells, where three- dimensional superresolution information can be obtained in a noninvasive fashion about cellular substructures, including single molecules. The power of a single fluorophore as a nanoscale light source will then be used to its maximum benefit. By providing a new method for three-dimensional high resolution optical imaging in living cells, this research will bear directly upon biotechnological and biomedical applications as these fields currently utilize optical fluorescence microscopy of cells in many diagnostic situations. Current trends are pushing toward smaller and smaller spatial scales for analysis of the behavior and morphology of individual cellular structures. The ability to specifically and noninvasively analyze mutant or toxic behaviors of organelles and other tiny cellular structures will allow precise assessment of the utility of targeted drug treatments, which will help drive the future of medical interventions exactly at the point of disease.

Public Health Relevance

By providing a new method for three-dimensional high resolution optical imaging in living cells, this research will bear directly upon biotechnological and biomedical applications as these fields currently utilize optical fluorescence microscopy of cells in many diagnostic situations. Current trends are pushing toward smaller and smaller spatial scales for analysis of the behavior and morphology of individual cellular structures. The ability to specifically and noninvasively analyze mutant or toxic behaviors of organelles and other tiny cellular structures will allow precise assessment of the utility of targeted drug treatments, which will help drive the future of medical interventions exactly at the point of disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085437-04
Application #
8119132
Study Section
Special Emphasis Panel (ZGM1-GDB-7 (EU))
Program Officer
Lewis, Catherine D
Project Start
2008-08-01
Project End
2013-09-29
Budget Start
2011-08-01
Budget End
2013-09-29
Support Year
4
Fiscal Year
2011
Total Cost
$298,166
Indirect Cost
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Backer, Adam S; Lee, Maurice Y; Moerner, W E (2016) Enhanced DNA imaging using super-resolution microscopy and simultaneous single-molecule orientation measurements. Optica 3:3-6
Shechtman, Yoav; Weiss, Lucien E; Backer, Adam S et al. (2016) Multicolour localization microscopy by point-spread-function engineering. Nat Photonics 10:590-594
Backlund, Mikael P; Arbabi, Amir; Petrov, Petar N et al. (2016) Removing Orientation-Induced Localization Biases in Single-Molecule Microscopy Using a Broadband Metasurface Mask. Nat Photonics 10:459-462
Milenkovic, Ljiljana; Weiss, Lucien E; Yoon, Joshua et al. (2015) Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1. Proc Natl Acad Sci U S A 112:8320-5
Backlund, Mikael P; Joyner, Ryan; Moerner, W E (2015) Chromosomal locus tracking with proper accounting of static and dynamic errors. Phys Rev E Stat Nonlin Soft Matter Phys 91:062716
Moerner, W E; Shechtman, Yoav; Wang, Quan (2015) Single-molecule spectroscopy and imaging over the decades. Faraday Discuss 184:9-36
Backer, Adam S; Moerner, W E (2015) Determining the rotational mobility of a single molecule from a single image: a numerical study. Opt Express 23:4255-76
von Diezmann, Alex; Lee, Maurice Y; Lew, Matthew D et al. (2015) Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy. Optica 2:985-993
Shechtman, Yoav; Weiss, Lucien E; Backer, Adam S et al. (2015) Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions. Nano Lett 15:4194-9
Backlund, Mikael P; Lew, Matthew D; Backer, Adam S et al. (2014) The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging. Chemphyschem 15:587-99

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