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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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Quantitative Stimulated Raman Scattering Microscopy: Promises and Pitfalls
Since its first demonstration, stimulated Raman scattering (SRS) microscopy has become a powerful chemical imaging tool that shows promise in numerous biological and biomedical applications. The spectroscopic capability of SRS enables identification and tracking of specific molecules or classes of molecules, often without labeling. SRS microscopy also has the hallmark advantage of signal strength that is directly proportional to molecular concentration, allowing for in situ quantitative analysis of chemical composition of heterogeneous samples with submicron spatial resolution and subminute temporal resolution. However, it is important to recognize that quantification through SRS microscopy requires assumptions regarding both system and sample. Such assumptions are often taken axiomatically, which may lead to erroneous conclusions without proper validation. In this review, we focus on the tacitly accepted, yet complex, quantitative aspect of SRS microscopy. We discuss the various approaches to quantitative analysis, examples of such approaches, challenges in different systems, and potential solutions. Through our examination of published literature, we conclude that a scrupulous approach to experimental design can further expand the powerful and incisive quantitative capabilities of SRS microscopy.
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- Award ID(s):
- 1846503
- PAR ID:
- 10413776
- Date Published:
- Journal Name:
- Annual Review of Analytical Chemistry
- Volume:
- 15
- Issue:
- 1
- ISSN:
- 1936-1327
- Page Range / eLocation ID:
- 269 to 289
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1146/annurev-anchem-061020-015110
