Tracing the technological advancements in light-sheet microscopy

The history of light-sheet microscopy is replete with constant innovation. For the last 30 years, the world has witnessed an incredible diversification in microscopic techniques spearheaded by the swift development of optoelectronic detectors and the extensive availability of laser-based light sources. The introduction of scientific CMOS cameras has accelerated technological advancement, a significant moment in the history of microscopy.

Light-sheet microscopy overview

Light microscopy is a cornerstone in life sciences technology, having experienced considerable diversification since its inception. This development becomes visible when considering the distinctive design of light-sheet microscopes, which deviate significantly from standard microscope designs that have remained mostly unchanged for hundreds of years.

Light-sheet microscopes illuminate samples at their core with a thin light sheet, usually perpendicular to the observation axis. This method decouples illumination from detection, producing more design freedom than traditional setups.

When combined with fluorescence staining, light-sheet microscopy offers enhanced 3D resolution and contrast while protecting signal integrity and sample integrity.

This delicate approach protects the signal due to low photobleaching and the sample integrity, thanks to low phototoxicity. Moreover, with camera-based detection, image acquisition can be quick, confined only by the camera's maximum framerate. The versatile design of light-sheet microscopes makes specialization tailored to specific samples possible from a few centimeters to the single-cell level.¹

Although the advantages of light-sheet microscopy motivate its adoption and potential substitution of standard microscopes, it is crucial to recognize some downsides. Sample mounting, for example, might bring about difficulties and impede some applications with established protocols.

Although superior in multiple instances, the flexibility in instrument design may present unwanted complications to specific experiments. Moreover, although the resolution reachable with light-sheet microscopy can be diffraction-limited, the maximum resolution is still often reached with standard microscopes or super-resolution approaches.

The origins of light-sheet microscopy

The origins of light-sheet microscopy can be traced back over a century. In 1903, Siedentopf and Zsigmondy2 built the world’s first light-sheet microscope, called the "Ultramicroscope." They aimed to examine sub-resolution gold particles, eventually leading to a Nobel Prize in 1925, the first awarded for a microscopy approach. Their work established the basis for light-sheet microscopy but also formed the foundations for the domain of nanotechnology.³

Almost 100 years later, Voie et al. brought back the approach and applied it for the first time to a biological sample: the inner ear cochlea of a guinea pig.

Using tissue-clearing approaches, they uncovered the inner structure of the tissues, opening novel routes for biological imaging. In particular, they selected an application that would later blossom into a significant application domain of lightsheet microscopy by introducing modern tissue-clearing protocols.⁴

The decisive moment for light-sheet microscopy arrived in 2004 when Jan Huisken et al. built selective plane illumination microscopy (SPIM). Integrating a microscope objective into the illumination arm and utilizing a fast digital camera improved previous light-sheet microscopes, attaining better 3D resolution and minimizing photobleaching. This development allowed high-speed imaging of dynamic biological approaches, including Drosophila melanogaster (fruit fly) embryogenesis and medaka (Japanese rice fish) embryos' heartbeat⁵.

The synergy of scientific research and technological advancements

Over the next 10 years, light-sheet microscopy became an accepted tool in developmental biology, ushering in developments such as digitally scanned light-sheet microscopy and creative advancements like "oblique plane microscopy”.⁶

Technological acceleration in the early 2010s saw the beginning of scientific CMOS cameras (sCMOS), exceeding prior EMCCDs in terms of pixel count, speed, and sensitivity.

Hamamatsu's sCMOS cameras, particularly the Flash 2.8 and 4.0 models, were critical in this shift. Even the originally perceived downside of CMOS cameras, the rolling shutter, became an upper hand in light-sheet microscopy.

To simplify synchronization with external equipment, Hamamatsu built the patented "Light-sheet readout mode."

With the advent of the Flash 4.0 V2 in 2013, sCMOS technology matured. Since then, sCMOS cameras have found extensive use in high-end light-sheet microscopy setups, such as those built by Nobel laureate Eric Betzig^7,8, Philip Keller⁹, Reto Fiolka¹⁰, Illaria Testa¹¹, and multiple others.

Later developments have brought about a complete range of sCMOS cameras, with many research projects utilizing Hamamatsu's ORCA series. Over 1,000 scientific papers have been published over the last four years using an ORCA sCMOS camera. Every camera delivers certain features, such as various dynamic ranges, shutter functionalities, and sensor dimensions, tending to the unique demands of various research pursuits.

Every advancement in the field takes humanity one step closer to understanding the mysteries of the life sciences. Technology and scientific discovery will likely continue to collaborate, presenting new pathways for investigation and understanding.

References and further reading

1. Girkin, J. M. (2018). The light-sheet microscopy revolution. Journal of Optics.
2. Siedentopf. H. and Zsigmondy, R. (1903). Über Sichtbarmachung und Größenbestimmung ultramikroskopischer Teilchen, mit besonderer Anwendung auf Rubingläser. Analen der Physik, 692 - 702.
3. Mappes, T. J. (2012). The invention of immersion ultramicroscopy in 1912—the birth of nanotechnology? Angewandte Chemie International Edition, 11208-11212.
4. Voie, A. H. (1993). Orthogonal‐plane fluorescence optical sectioning: Three‐dimensional imaging of macroscopic biological specimens. Journal of microscopy, 229 - 236.
5. Huisken, J. e. (2004). Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science, 1007-1009.
6. Dunsby, C. (2008). Optically sectioned imaging by oblique plane microscopy. Optics express, 20306 - 20316.
7. Chen, B.-C. e. (2014). "Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science, 1257998.
8. Liu, T.-L. e. (2018). Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms. Science, 6386.
9. Krzic, U. e. (2012). Multiview light-sheet microscope for rapid in toto imaging. Nature methods, 730-733.
10. Dean, K. M. (2015). Decon
11. Bodén, A. e. (2024). Super-sectioning with multi-sheet reversible saturable optical fluorescence transitions (RESOLFT) microscopy. Nature Methods, 1-7.
12. The research was made on Meltwater Media Monitoring from the year 2000 to 2023.

About Hamamatsu Photonics Europe

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