Optical microscopy remains a very active area of technological innovation, and one of the most exciting techniques to achieve commercialization in the past decade is light-sheet microscopy. Other common names for the technique include Light-Sheet Fluorescence Microscopy (LSFM) and Selective Plane Illumination Microscopy (SPIM). The technique offers a number of significant advantages for imaging live cells and even entire intact organisms, and this has stimulated great interest in developmental biology and other life science fields of research.

As the names suggest, the key to the technology is the illumination, which is confined to a relatively thin plane or sheet that intersects the sample. This is achieved with the use of a cylindrical lens and other optical components to confine the illumination from the source into a thin plane. Alternatively, a “plane” can be built up over time by scanning a linear beam across the sample. Typically, there will be a locus where the sheet is thinnest, and placing the sample there provides the best imaging. Light-sheet microscopy is a fluorescence technique, so the illumination excites fluorescent molecules and the resulting fluorescence is detected with a scientific camera.

Another distinguishing feature of the technique is that this planar illumination is perpendicular to the detection optics. Thus, the detection optics receive the fluorescent signal from the entire thin slice of the sample. As the sample is moved vertically through the light sheet, an entire 3D reconstruction of the sample can be achieved. Because the illumination is limited to a specific plane, phototoxic effects on living samples is reduced.

At the same time, there are some challenges. Because the light has to pass perpendicularly through the sample, large samples may not be suitable, as the light source is absorbed or scattered. Even small features that block a portion of the light can lead to “stripes” in the images. Combining multiple views of the sample can eliminate these artifacts, and the use of multiphoton techniques allow the use of longer wavelengths that can penetrate farther into larger samples. This barely scratches the surface of the many different wrinkles on the technique that exist.

Applications are primarily life science oriented and come largely from academia. Samples range from single cells to cleared tissue samples to complete small live organisms. The ability to image the same embryo at different stages of its development has enabled advances in developmental biology.

Since the technology was originally developed in various academic research groups, many systems continue to be home-built. OpenSPIM, an open-source group, provides researchers with a parts lists and instructions, so that with an outlay of effort and perhaps $40,000 in parts, a skilled microscopist can build a functional system. However, commercial systems have also begun to proliferate. LaVision Biotec is generally credited as having the first system in 2009, and the company continues to be a leading vendor, now with the second generation of its UltraMicroscope. ZEISS also entered the market quite early with the Lightsheet Z.1. Leica Microsystems has adapted one of its confocal microscopes with a clever system of mirrors to add light-sheet imaging capability. Other market participants are generally smaller firms focusing directly on the development and elaboration of the technique. These include Intelligent Imaging Innovations (3i), PhaseView and Applied Scientific Instrumentation, which partners with Nikon. Another vendor, Luxendo, was recently acquired by Bruker (see Executive Briefing). Vendors of scientific cameras and microscopy components, such as Andor Technology (Oxford Instruments) and Hamamatsu, also contribute to the market.

Light Sheet Microscopy At a Glance:

Leading Suppliers:

• ZEISS
• LaVision Biotec
• Leica Microsystems (Danaher)

Largest Markets:

• Academia
• Government
• Biotechnology

Instrument Cost:

$40,000–$300,000