Imaging Cellular Spheroids with a Single (Selective) Plane Illumination Microscope
Abstract
In modern biology, most optical imaging technologies are applied to two-dimensional cell culture systems. However, investigation of physiological context requires specimens that display the complex three-dimensional (3D) relationship of cells that occurs in tissue sections and in naturally developing organisms. The imaging of highly scattering multicellular specimens presents a number of challenges, including limited optical penetration depth, phototoxicity, and fluorophore bleaching. Light-sheet-based fluorescence microscopy (LSFM) overcomes many drawbacks of conventional fluorescence microscopy by using an orthogonal/azimuthal fluorescence arrangement with independent sets of lenses for illumination and detection. The specimen is illuminated from the side with a thin light sheet that overlaps with the focal plane of a wide-field fluorescence microscope. Optical sectioning and minimal phototoxic damage or photobleaching outside a small volume close to the focal plane are intrinsic properties of LSFM. The principles of LSFM are implemented in the single (or selective) plane illumination microscope (SPIM). Cellular spheroids are spherical aggregations of hundreds to thousands of cells and they provide a useful model system for studies of 3D cell biology. Here we describe a protocol for imaging cellular spheroids by SPIM.
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Conventional wide-field fluorescence microscopy is limited in resolving fluorescence signals within thick or optically dense spheroids, reducing contrast and spatial resolution, particularly for large spheroids.
“conventional wide-field fluorescence microscopy is limited in its ability to resolve fluorescence signals within thick or optically dense spheroids as it collects out-of-focus light from the entire specimen and projects it onto a single focal plane. This results in reduced contrast and loss of spatial resolution, particularly for large spheroids [244].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [244]
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- Authors
- Swoger, J., Pampaloni, F., & Stelzer, E. H. K.
- Venue
- Cold Spring Harbor Protocols
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2013-12-26
- Volume
- 2014
- Issue
- 1
- Pages
- pdb.prot080176
- ISSN
- 1559-6095 (ISSN-L)
- 1940-3402
- OpenAlex
- W1978183395
- Cited by
- 13
- References
- 9
- Citations by year
- 2026: 1 · 2025: 1 · 2022: 1 · 2021: 2 · 2019: 1 · 2018: 1 · 2017: 1 · 2016: 2 · 2015: 1 · 2013: 2
- Type
- posted content
- Language
- en
- Open access
- closed
- Locations
- Cold Spring Harbor Protocols (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Digital Holography and Microscopy
- Keywords
- cellular spheroids, single plane illumination microscopy, light-sheet microscopy, 3D cell imaging, optical sectioning, phototoxicity, photobleaching, optical penetration depth
- MeSH terms
- Animals, Humans, Lighting, Microscopy, Fluorescence, Spheroids, Cellular, Imaging, Three-Dimensional, Optical Imaging
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