Microscopy is one of the oldest lab techniques, originating in 17th century Europe. The earliest microscopes used only a single lens to focus light, but the technique evolved into more elaborate systems of lenses. Although improvements in design and optics allowed microscopes to achieve greater magnifications, there is a physical limit to the magnification that can be achieved by an ordinary light microscope. The so-called diffraction limit restricted the power of a microscope to resolving features that are about the same size as the wavelengths of light. In the case of visible light, this limit is roughly a few hundred nanometers.

Over the past few decades, a variety of techniques have been invented that allow optical microscopy to surpass the diffraction limit. Primarily developed by academic researchers, these technologies have been licensed to microscope manufacturers. It is difficult to summarize the zoo of acronyms and abbreviations that comprise the field of super-resolution microscopy. Some techniques are wide field, while others use confocal geometry or scanning probe technology. Most of the newer techniques make use of complicated interactions between light and fluorophores, chemical tags that cause molecules of interest to fluoresce so that they can be specifically imaged. Typically, these imaging agents are used to image cell components.

Three commercialized super-resolution microscopy techniques are SIM (structured illumination microscopy), STED (stimulated emission depletion) and STORM (stochastic optical reconstruction microscopy). SIM uses multiple patterns of laser light to illuminate the sample. These patterns interfere in a characteristic moiré pattern, which is the key to attaining greater resolution. Software analysis combines multiple images under different conditions into a single SIM image. STED is a method that involves using two competing illumination sources. One source provides a bright spot to excite the fluorescent compounds in the sample. The STED illumination returns the fluorophores to their ground states but only in an annular area around the focus of the microscope. Excited fluorophores that can then be imaged remain only at the center. This process improves resolution by a factor of about ten over conventional confocal microscopy. STORM makes use of the quantum resonance between two fluorescent compounds to localize individual fluorophore molecules. Repeated measurement allows the full image to be built up. STORM and many other super-resolution techniques based on confocal imaging have evolved into full three-dimensional techniques.

Leica Microsystems, Carl Zeiss, Nikon and Olympus dominate the realm of super-resolution microscopy, as many smaller vendors cannot produce or license the technologies. Some smaller vendors exist, such as Andor Technology and Applied Precision, which was acquired by GE Healthcare (see IBO 4/30/11). The rapidly growing market for super-resolution microscopy was estimated at about $350 million in 2010.

Super-Resolution Microscopy

at a Glance:

Leading Suppliers

• Leica (Danaher)

• Carl Zeiss

• Nikon

Largest Markets

• Academia

• Government

• Pharmaceutical

Instrument Cost

• $750,000–$2,000,000