How Light-Sheet Microscopy Has Evolved With Cutting-Edge Technology

Light microscopy is one of the most essential technologies in life science research. Advances in optoelectronic detectors and the widespread availability of laser-based light sources have led to the development of a diverse range of microscopic techniques.

This progress is particularly evident in the unique design of light-sheet microscopes, which represents a significant departure from the conventional microscope design that has remained largely unchanged for centuries.

Fig 1. Light-sheet microscope, OpenSPIM concept configurations. Image Credit: https://openspim.org/Gallery#OpenSPIM_data

The basic concept behind all light-sheet microscopes is that a thin sheet of light, usually perpendicular to the observation axis, illuminates the sample. Compared to conventional epi- or transillumination designs, this decoupling of the illumination and detection allows for greater design flexibility.

Fig 2. Light-sheet microscopy principle, based on Jan Krieger, CC BY-SA 3.0. Image Credit: https://commons.wikimedia.org/w/index.php?curid=22333698

When combined with fluorescence staining, light-sheet microscopy offers several advantages, including 3D resolution and enhanced contrast by eliminating out-of-focus light. Its low photobleaching and phototoxicity make it a gentle technique, preserving both signal quality and sample integrity.

Acquisition can be swift with camera-based detection, limited only by the maximum frame rate of the camera. The flexible design of light-sheet microscopes enables specialization for particular samples, from a few centimeters to the single-cell level.¹

Although such benefits promote the replacement of conventional microscopes, it is important to remember that they have some drawbacks, including sample mounting, which can hinder certain applications with established protocols.

Due to the flexibility in instrument design, some experiments may become unnecessarily complex, which is certainly advantageous in many situations. Furthermore, although there have been examples of combining light-sheet microscopy with super-resolution techniques, the highest resolution is still achieved with regular microscopes or with super-resolution approaches, even though the resolution may be diffraction-limited.

Fig 3. Tubulin assembly in cytoplasmic droplet, acquired on Viventis LS1 light-sheet microscope with Orca Fusion BT camera, (scale 100 μm). Image Credit: Dr. Michael Riedl (Jan Bruges Group) and Dr. Vanessa Carlos (PoL Microscopy), Cluster of Excellence “Physics of Life”, TU Dresden

Origins of Light-Sheet Microscopy

Although the fundamental concept of using a light sheet for illumination dates back over a century, the technique only gained prominence twenty years ago. Its success is ascribed to both the widespread availability of laser illumination and the development of quick digital cameras that allow for 3D image reconstruction.

The first light-sheet microscope was created in 1903 by Siedentopf & Zsigmondy.² Their goal to analyze sub-resolution gold particles earned them the first-ever Nobel prize awarded for a microscopy technique in 1925. Their dark field light-sheet setup, named “Ultramicroscope”, was designed to visualize particles as small as 4 nm.

In addition to demonstrating the light-sheet microscope for the first time, they spawned the field of colloid research, which some consider to be the cornerstone of nanotechnology.³

Using tissue-clearing techniques to reveal the inner structure of the tissues, Voie et al. revived the technique almost a century later and used it for the first time on a biological sample—the inner ear cochlea of a guinea pig.⁴

Interestingly, they selected an application (imaging of cleared tissue) that developed into a significant field twenty years later with the advent of modern tissue-clearing protocols.

Fig 4. Epithelial cells stained for E-Cadherin and Actin, imaged on Zeiss Lattice Light-Sheet 7 with ORCA^® Fusion cameras. Image Credit: Dr. Anastasiia Gabrielyan (Natalie Dye Group) and Dr. Bert Nitzsche (PoL Microscopy), Cluster of Excellence “Physics of Life”, TU Dresden

Modern Light-Sheet Microscopy

Light-sheet microscopy ultimately gained popularity in 2004 when Jan Huisken developed selective plane illumination microscopy (SPIM), enhancing earlier light-sheet microscopes by incorporating a microscope objective to the illumination arm and a Hamamatsu Photonics ORCA-ER B/W CCD digital camera.

This novel method improved the 3D resolution and reduced photobleaching, demonstrating low phototoxicity and high speed in observing the heartbeat of medaka (Japanese rice fish) embryos and Drosophila melanogaster (fruit fly) embryogenesis.⁵

Over the next decade, light-sheet microscopy became an established tool in developmental biology, leading to innovations such as digitally scanned light-sheet microscopy and creative adaptations like oblique plane microscopy. This approach merged the benefits of light-sheet microscopy with traditional microscope designs, making its advantages accessible for standard samples on glass slides .⁶

Technological acceleration in the early 2010s brought about the introduction of scientific CMOS cameras (sCMOS), surpassing previous EMCCDs with more pixels at higher speeds and increased sensitivity—the perfect combination for large, sensitive specimens.

What was initially seen as a drawback of CMOS cameras—the rolling shutter—became an advantage in light-sheet microscopy. Synchronizing the excitation beam in digitally scanned light sheets with the rolling shutter helped reduce stray light, enhance contrast, and improve axial resolution.⁷

Additionally, the rolling shutter could be synchronized with a tunable lens in axially swept light-sheet microscopy, ensuring a uniform light sheet across large fields of view.⁸

In order to simplify the synchronization of the rolling shutter with external equipment, Hamamatsu developed the patented ‘Light-sheet readout mode.’ With the release of Flash 4.0 V2 in 2013, sCMOS technology reached maturity, and a burst occurred in the development of light-sheet microscopy.

The Flash 4.0 found widespread use in high-end light sheets, including those by Nobel laureate Eric Betzig, Philip Keller’s sophisticated setups (MuVi SPIM, IsoView), Reto Fiolka’s innovative microscopy techniques, and Illaria Testa’s super resolution and light-sheet combinations.^9-14

Subsequent developments have spawned a wide range of sCMOS cameras, with numerous research projects utilizing Hamamatsu’s ORCA^® series.

Over the past four years, more than 1000 scientific papers have been published using an ORCA^® sCMOS camera. Each camera offers specific features, including different dynamic ranges, shutter functionalities, and sensor dimensions, catering to the unique demands of diverse research endeavors.

Fig 5. Spontaneous calcium wave in a live cardiomyocyte cell imaged at 25 volumes per second. (a) shows central volume planes in xy, xz and yz orientation, while (b) shows the expanded views of the datapoints indicated with triangles in (a). Image Credit: Dr. Hugh Sparks and Dr. Christopher Dunsby, Imperial College London, as published.¹⁵

Conclusions

The development of selective planar illumination microscopy (SPIM) in 2004 marked a turning point for the technology, which had been pioneered by Siedentopf & Zsigmondy in 1903 and revitalized by Voie et al. in 1993.

The ORCA^®-ER B/W CCD digital camera from Hamamatsu Photonics, which pioneered 3D resolution, low photobleaching and fast speed, was key to the SPIM’s success. Light-sheet microscopy reached new heights in the early 2010s as sCMOS cameras reached a technological apex, embodied by Hamamatsu's Flash 4.0 V2. This development made it possible to use Hamamatsu's ORCA^® series in a variety of research.

Today, a variety of light-sheet microscopy setups are available, ranging from simple DIY projects like OpenSPIM, which offers step-by-step tutorials, to more advanced open-hardware projects such as mesoSPIM, as well as commercial solutions. Notably, the flexibility of some of these approaches enables researchers to design microscopes around the sample rather than forcing the sample to fit a fixed microscope setup.

It is predicted that light-sheet microscopy will continue to advance technology and scientific discovery, with each new development bringing researchers closer to solving the mysteries of life sciences.

References and Further Reading

1. Girkin, J.M. and Carvalho, M.T. (2018). The light-sheet microscopy revolution. Journal of Optics, 20(5), p.053002. https://doi.org/10.1088/2040-8986/aab58a.
2. Siedentopf, H. and Zsigmondy, R. (1902). Uber Sichtbarmachung und Größenbestimmung ultramikoskopischer Teilchen, mit besonderer Anwendung auf Goldrubingläser. Annalen der Physik, 315(1), pp.1–39. https://doi.org/10.1002/andp.19023150102.
3. Mappes, T., et al. (2012). The Invention of Immersion Ultramicroscopy in 1912-The Birth of Nanotechnology? Angewandte Chemie International Edition, 51(45), pp.11208–11212. https://doi.org/10.1002/anie.201204688.
4. VOIE, A.H., BURNS, D.H. and SPELMAN, F.A. (1993). Orthogonal-plane fluorescence optical sectioning: Three-dimensional imaging of macroscopic biological specimens. Journal of Microscopy, 170(3), pp.229–236. https://doi.org/10.1111/j.1365-2818.1993.tb03346.x.
5. Huisken, J. (2004). Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy. Science, 305(5686), pp.1007–1009. https://doi.org/10.1126/science.1100035.
6. Dunsby, C. (2008). Optically sectioned imaging by oblique plane microscopy. Optics Express, 16(25), p.20306. https://doi.org/10.1364/oe.16.020306.
7. Baumgart, E. and Kubitscheck, U. (2012). Scanned light sheet microscopy with confocal slit detection. Optics Express, 20(19), p.21805. https://doi.org/10.1364/oe.20.021805.
8. Chen, B.-C., et al. (2014). Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution. Science, 346(6208), p.1257998. https://doi.org/10.1126/science.1257998.
9. Liu, T.-L., et al. (2018). Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms. Science, (online) 360(6386), p.eaaq1392. https://doi.org/10.1126/science.aaq1392.
10. Krzic, U., et al. (2012). Multiview light-sheet microscope for rapid in toto imaging. Nature Methods, 9(7), pp.730–733. https://doi.org/10.1038/nmeth.2064.
11. Chhetri, R.K., et al. (2015). Whole-animal functional and developmental imaging with isotropic spatial resolution. Nature Methods, 12(12), pp.1171–1178. https://doi.org/10.1038/nmeth.3632.
12. Dean, K.M., et al. (2015). Deconvolution-free Subcellular Imaging with Axially Swept Light Sheet Microscopy. Biophysical Journal, (online) 108(12), pp.2807–2815. https://doi.org/10.1016/j.bpj.2015.05.013.
13. Bodén, A., et al. (2024). Super-sectioning with multi-sheet reversible saturable optical fluorescence transitions (RESOLFT) microscopy. Nature methods, (online) 21(5), pp.882–888. https://doi.org/10.1038/s41592-024-02196-8.
14. Sparks, H., et al. (2020). Development a flexible light-sheet fluorescence microscope for high-speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure. Journal of biophotonics, (online) 13(6), p.e201960239. https://doi.org/10.1002/jbio.201960239.

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