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Abstract Mapping 3D plasma membrane topology in live cells can bring unprecedented insights into cell biology. Widefield-based super-resolution methods such as 3D-structured illumination microscopy (3D-SIM) can achieve twice the axial ( ~ 300 nm) and lateral ( ~ 100 nm) resolution of widefield microscopy in real time in live cells. However, twice-resolution enhancement cannot sufficiently visualize nanoscale fine structures of the plasma membrane. Axial interferometry methods including fluorescence light interference contrast microscopy and its derivatives (e.g., scanning angle interference microscopy) can determine nanoscale axial locations of proteins on and near the plasma membrane. Thus, by combining super-resolution lateral imaging of 2D-SIM with axial interferometry, we developed multi-angle-crossing structured illumination microscopy (MAxSIM) to generate multiple incident angles by fast, optoelectronic creation of diffraction patterns. Axial localization accuracy can be enhanced by placing cells on a bottom glass substrate, locating a custom height-controlled mirror (HCM) at a fixed axial position above the glass substrate, and optimizing the height reconstruction algorithm for noisy experimental data. The HCM also enables imaging of both the apical and basal surfaces of a cell. MAxSIM with HCM offers high-fidelity nanoscale 3D topological mapping of cell plasma membranes with near-real-time ( ~ 0.5 Hz) imaging of live cells and 3D single-molecule tracking.
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4Pi stimulated Raman scattering for label-free super-resolution chemical imaging
Super-resolution fluorescence microscopy has transformed biological imaging beyond the diffraction limit. However, many biomolecules, nanostructures, drug molecules, and metabolites cannot be easily tagged, requiring a label-free imaging approach. Stimulated Raman scattering (SRS) microscopy is a powerful platform for super-resolution label-free imaging, yet current super-resolution SRS approaches rely on photoswitching, saturation, or sample expansion, which are limited by labeling, photodamage, or signal dilution, respectively. Here, we combine SRS with 4Pi interferometry to enhance axial resolution nearly sevenfold. We report on improvements in imaging sensitivity and axial resolution using 80-nanometer polystyrene beads. Harnessing the improved axial resolution, we demonstrate super-resolution 4Pi-SRS imaging in resolving small lipid droplet structures in mammalian cells and lipid membranes inEscherichia colicells. Because 4Pi-SRS uses interferometry to improve axial resolution, it is orthogonal to all previous super-resolution SRS techniques; thus, it is straightforward to integrate it with existing methods to achieve much higher resolution chemical imaging than previously possible.
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- Award ID(s):
- 1846503
- PAR ID:
- 10660339
- Publisher / Repository:
- American Association for the Advancement of Science
- Date Published:
- Journal Name:
- Science Advances
- Volume:
- 12
- Issue:
- 1
- ISSN:
- 2375-2548
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1126/sciadv.aec0523
