As we navigate the rich history of light-sheet microscopy, a story of constant innovation unfolds. Over the last three decades, we've witnessed a remarkable diversification in microscopic techniques, driven by the rapid development of opto-electronic detectors and the widespread availability of laser-based light sources. Our focus sharpens on the evolution catalysed by the advent of scientific CMOS cameras, a pivotal moment in the history of microscopy.
About light-sheet microscopy
Light microscopy stands as a cornerstone in life sciences technology, experiencing significant diversification since its inception. This development becomes evident in the distinctive design of light-sheet microscopes, deviating substantially from standard microscope designs that have remained largely unchanged for centuries.
Fundamentally, light-sheet microscopes illuminate samples with a thin sheet of light, typically perpendicular to the observation axis. This approach decouples illumination from detection, offering greater design freedom compared to traditional setups. Light-sheet microscopy, when combined with fluorescence staining, provides enhanced 3D resolution and contrast while preserving signal integrity and sample integrity. This gentle technique preserves both the signal, thanks to low photobleaching, and the sample integrity due to low phototoxicity. Additionally, with camera-based detection, image acquisition can be swift, limited only by the maximum framerate of the camera. The flexible design of light-sheet microscopes allows for specialisation tailored to specific samples, ranging from a few centimetres to the single-cell level1.
While the advantages of light-sheet microscopy encourage its adoption and potential replacement of standard microscopes, it's important to acknowledge certain drawbacks. Sample mounting, for instance, may present challenges and hinder certain applications with established protocols. The flexibility in instrument design, while advantageous in many scenarios, might introduce unwanted complexity to some experiments. Moreover, while the resolution achievable with light-sheet microscopy can be diffraction-limited, the highest resolution is still typically achieved with standard microscopes or super-resolution techniques.
The origins of light-sheet microscopy
The origins of light-sheet microscopy trace back more than a century. In 1903, Siedentopf & Zsigmondy2 pioneered the first light-sheet microscope, known as the "Ultramicroscope." Their objective was to analyse sub-resolution gold particles, which ultimately led to a Nobel Prize in 1925, the first awarded for a microscopy technique. Their work not only established the foundation for light-sheet microscopy but also laid the groundwork for the field of nanotechnology3.
Nearly a century later, Voie et al revitalised the technique and applied it for the first time to a biological sample: the inner ear cochlea of a guinea pig. By employing tissue-clearing methods, they revealed the inner structure of the tissues, opening new avenues for biological imaging. Interestingly, they chose an application that would later blossom into a major application field of light-sheet microscopy with the advent of modern tissue-clearing protocols4.
The turning point for light-sheet microscopy came in 2004 when Jan Huisken et al developed selective plane illumination microscopy (SPIM). By incorporating a microscope objective into the illumination arm and using a fast digital camera, they enhanced earlier light-sheet microscopes, achieving higher 3D resolution and reducing photobleaching. This innovation allowed for high-speed imaging of dynamic biological processes such as Drosophila melanogaster (fruit fly) embryogenesis and medaka (Japanese rice fish) embryos' heartbeat5.
The synergy of scientific research and technological advancements
In the following decade, light-sheet microscopy became a recognised tool in developmental biology, witnessing advancements like digitally scanned light-sheet microscopy and creative implementations like "oblique plane microscopy”6. Technological acceleration in the early 2010s saw the introduction of scientific CMOS cameras (sCMOS), surpassing previous EMCCDs in terms of pixel count, speed, and sensitivity. Hamamatsu's sCMOS cameras, particularly the ORCA Flash 2.8 and 4.0 models, played a significant role in this shift. Even the initially perceived drawback of CMOS cameras, the rolling shutter, was transformed into an advantage in light-sheet microscopy.
To simplify synchronisation with external equipment, Hamamatsu developed the patented "Light-sheet readout mode." With the release of the Flash 4.0 V2 in 2013, sCMOS technology reached maturity. Since then, sCMOS cameras have found widespread use in high-end light-sheet microscopy setups, including those developed by Nobel laureate Eric Betzig7,8, Philip Keller9, Reto Fiolka10, Illaria Testa11, and many others.
Subsequent developments have brought forth a whole range of sCMOS cameras, with numerous research projects utilising Hamamatsu's ORCA series. In fact, over the last four years, more than 1000 scientific papers have been published using an ORCA sCMOS camera12. Each camera offers specific features such as different dynamic ranges, shutter functionalities, and sensor dimensions, catering to the unique demands of diverse research endeavours.
Each milestone brings us closer to unravelling life sciences mysteries. Today, as we anticipate the future, we predict continued collaboration between technology and scientific discovery, opening new avenues for exploration and understanding.
References
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 software from the year 2000 to 2023.
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