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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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Roadmap on Label‐Free Super‐Resolution Imaging
Abstract Label‐free super‐resolution (LFSR) imaging relies on light‐scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super‐resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state‐of‐the‐art in this field, and to discuss the resolution boundaries and hurdles that need to be overcome to break the classical diffraction limit of the label‐free imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction‐limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super‐resolution capability that are based on understanding resolution as an information science problem, on using novel structured illumination, near‐field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere‐assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.
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- Award ID(s):
- 2052745
- PAR ID:
- 10515287
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Laser & Photonics Reviews
- Volume:
- 17
- Issue:
- 12
- ISSN:
- 1863-8880
- 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/10.1002/lpor.202200029
