Shining A Light On Light Sheet Microscopy

By John Oncea, Editor

Tracing its origins back to 1903, light sheet microscopy has developed into an invaluable tool in many fields. Learn more about Richard Zsigmondy and his role in creating this tool.

Richard Adolf Zsigmondy’s father, Adolf, passed away when he was 15 but his mother, Irma, made sure he received a good education. While in high school he developed an interest in chemistry and physics, conducting experiments in his home laboratory.

Upon graduation, Zsigmondy attended the University of Vienna Medical Faculty before moving on to the Technical University of Vienna and, later, the University of Munich where he conducted research on indene before receiving his Ph.D. from the University of Erlangen in 1889.

He became an assistant professor at Graz University of Technology before taking a job at the Schott Glass factory in 1897. Zsigmondy left Schott Glass three years later to give private lectures and conduct research before, in 1903, developing the ultramicroscope with the help of Henry Siedentopf and instrument manufacturer Zeiss.

Zsigmondy’s ultramicroscope is a microscope with a system that lights the object in a way that allows viewing of tiny particles via light scattering, not light reflection or absorption. When the diameter of a particle is below or near the wavelength of visible light the particle cannot be seen in a light microscope with the usual illumination methods. The “ultra” in ultramicroscope refers to the ability to see objects whose diameter is shorter than the wavelength of visible light, on the model of the “ultra” in ultraviolet.

The ultramicroscope proves to be suitable for the determination of small particles and establishes the foundation for light sheet microscopy while at the same time laying the groundwork for the field of nanotechnology. Twelve years after developing it, Zsigmondy is awarded the Nobel Prize for Chemistry for his work.

From Ultramicroscope To Light Sheet Microscopy

The ultramicroscope saw minor use in the 1960s in photomacrography, popularized by the Dynaphot microscope. In 1993, Voie and Burns pioneered Orthogonal-Plane Fluorescence Optical Sectioning microscopy, applying it to image guinea pig cochlea. A decade later, Ernst Stelzer’s group introduced Selective Plane Illumination Microscopy, marking a significant breakthrough in developmental biology.

Today, thanks to the ultramicroscope’s evolution to light sheet microscopy, phototoxicity is reduced due to the need to illuminate only a thin plane of the sample, minimizing photobleaching and phototoxicity, while enabling long-term imaging of live samples. There are other benefits as well, including:

• Enhanced 3D resolution: The technique provides improved three-dimensional resolution, particularly in the axial direction.
• Faster imaging: Camera-based detection allows for swift image acquisition, limited only by the camera's maximum framerate.
• Improved signal-to-noise ratio: The orthogonal illumination approach significantly increases the signal-to-noise ratio compared to traditional techniques.
• Versatility: Light sheet microscopy can be adapted for various sample sizes, from single cells to entire organisms.
• Deep tissue imaging: When combined with tissue clearing techniques, light sheet microscopy enables imaging of thick samples with minimal light scattering.

Thanks to Zsigmondy’s pioneering work, light sheet microscopy has become an invaluable tool in various fields, including developmental biology, neuroscience, and cellular dynamics research, allowing researchers to observe biological processes with unprecedented detail and minimal sample disruption.

How Light Sheet Microscopy Works

Light sheet microscopy is a fluorescence imaging technique that utilizes a sheet of laser light to illuminate only a thin slice of the sample. According to Nature, the basic technical principle is a wide-field fluorescence microscope, placed perpendicular to the light sheet, that collects the fluorescence signal and images of the observed region using a full-frame camera.

The orthogonal arrangement that decouples the illumination from the detection enables intrinsic 3D optical sectioning, as compared to other fluorescent imaging techniques like confocal and spinning disc microscopy. As a result, the method features drastically reduced overall acquisition duration, photobleaching effects, and phototoxicity, as well as yields excellent signal-to-noise ratio and enables high temporal and 3D-spatial resolution.

Light sheet microscopy can be utilized to image a huge variety of fixed, live, or cleared biological samples. Applications of light sheet microscopy can range from imaging of subcellular structures and rapid inter- and intracellular processes to the acquisition of the long-term development of a model system to the complete visualization of a macroscale cleared sample.

According to Microscopy U, light sheet microscopy offers several advantages over traditional microscopy techniques including:

• Reduced phototoxicity: By illuminating only a thin plane of the sample, light sheet microscopy minimizes photobleaching and phototoxicity, enabling long-term imaging of live samples.
• Enhanced 3D resolution: The technique provides improved three-dimensional resolution, particularly in the axial direction.
• Faster imaging: Camera-based detection allows for swift image acquisition, limited only by the camera's maximum framerate.
• Improved signal-to-noise ratio: The orthogonal illumination approach significantly increases the signal-to-noise ratio compared to traditional techniques.
• Versatility: Light sheet microscopy can be adapted for various sample sizes, from single cells to entire organisms.
• Deep tissue imaging: When combined with tissue clearing techniques, light sheet microscopy enables imaging of thick samples with minimal light scattering.

These benefits have made light sheet microscopy an invaluable tool in various fields, including developmental biology, neuroscience, and cellular dynamics research, allowing researchers to observe biological processes with unprecedented detail and minimal sample disruption.

Recent Advancements In Light Sheet Microscopy

Recent advancements have been made in light-sheet microscopy including the development of the Benchtop mesoSPIM. According to Nature, researchers introduced a next-generation open-source light-sheet microscope with improved capabilities. This new version offers:

• Increased field of view
• Higher resolution (1.5 μm laterally, 3.3 μm axially)
• Greater throughput
• More affordable cost
• Simpler assembly

A second advancement, writes optics.org, is the introduction of the soTILT3D platform. A team at Rice University developed an innovative imaging platform that enhances 3D visualization of cellular structures. Key features include:

• Angled light sheet for improved signal-to-background ratio
• Nanoprinted microfluidic system for precise sample environment control
• Advanced computational tools for improved imaging speed and precision

Finally, the Friedrich Miescher Institute for Biomedical Research writes a new light-sheet microscope for multicellular systems has been unveiled by researchers at the FMI and Viventis Microscopy. The innovative microscope is capable of imaging different multicellular systems, including organoids and entire animals3.

Future developments in light-sheet microscopy may include further integration of artificial intelligence and deep learning algorithms to enhance image processing and analysis, continued improvements in spatial and temporal resolution, allowing for even more detailed imaging of cellular structures and processes, and the development of more user-friendly and accessible light-sheet microscopy systems, potentially leading to wider adoption in research laboratories.