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Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
Published on: June 30, 2018
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Localization microscopy: mapping cellular dynamics with single molecules
1Department of Physics and Astronomy and Institute for Molecular Biophysics, University of Maine, Orono, Maine, U.S.A.
Journal of Microscopy
|March 12, 2014
Summary
Superresolution fluorescence localization microscopy (SRFLM) achieves nanoscale resolution by localizing individual fluorescent molecules. This technique surpasses optical diffraction limits, enabling visualization of cellular structures and dynamics at the molecular level.
Area of Science:
- Biophysics
- Optical Microscopy
- Molecular Imaging
Background:
- Optical microscopes are limited by diffraction to ~200-250 nm resolution for visible light.
- Localization microscopy measures object positions with high precision, surpassing diffraction limits.
- Superresolution fluorescence localization microscopy (SRFLM) combines imaging and localization of fluorescent molecules.
Purpose of the Study:
- To review the concept, process, and recent advances in SRFLM.
- To highlight SRFLM's ability to overcome diffraction limits for molecular-level imaging.
- To discuss SRFLM's applications in understanding cell biology and dynamic processes.
Main Methods:
- Controlling fluorophore visibility to sparsely activate and spatially separate fluorescent tags.
- Acquiring time-lapse images (movies) of fluorescing molecules using a camera.
- Computer analysis to independently measure and record the positions of single molecules.
Main Results:
- SRFLM achieves spatial resolutions better than 20 nm, an order of magnitude improvement over diffraction-limited resolution.
- Specialized optics allow splitting signals into multiple channels for color, orientation, and 3D position information.
- SRFLM is compatible with living samples, enabling visualization of dynamic biological processes.
Conclusions:
- SRFLM has revolutionized molecular imaging since its 2006 inception.
- The technique's versatility allows investigation of biological questions below the diffraction limit.
- SRFLM significantly enhances the understanding of cell biology at the molecular scale.
