In its broadest sense, correlative microscopy refers to the combination of multiple microscopy techniques to observe the same structure or event. Recent announcements by two major microscopy providers suggest that correlative microscopy will be a focus of their businesses in coming years. Although correlative microscopy can take many forms, the use of light microscopy (LM) with scanning electron microscopy (SEM) is one of the most popular combinations. Whereas light microscopy can scan large areas and observe dynamic processes, SEM can provide higher resolution and structural information of targeted areas. One of the fastest-growing correlative microscopy markets is the use of fluorescence light microscopy and SEM in cell biology, where applications include the study of protein localization and cell surface morphology. As SEM providers’ products demonstrate, the solutions for correlative microscopy range from integrated instruments to software to transport systems.

One company that has made its intentions clear to grow the correlative microscopy market is electron microscopy provider FEI. Last month, the company acquired TILL Photonics, a developer of fluorescence microscopy systems for life science applications (see IBO 11/15/11), positioning FEI to combine the respective technologies. As Thomas Wohlfarth, director of Product Marketing for FEI’s Life Science Business Unit, told IBO, “TILL will play a significant role in developing the next-generation sophisticated correlated microscopy solution. I think this will certainly apply significantly to our EM [electron microscopy] business when it comes to, for instance, 2D and 3D correlation applications.”

Earlier this month, FEI announced its first dedicated products for correlative microscopy: a version of its new MAPS (Modular Automated Processing System) software designed for cell biology and a forthcoming system that integrates fluorescence microscopy and transmission electron microscopy (TEM). LM and TEM correlative microscopy applications include cryo-electron tomography for 3D imaging of cells and tissues. Asked how this system compares to other LM and EM integrated systems, Mr. Wohlfarth said, “First of all, and very important, we do not compromise the performances of either the light microscope or the electron microscope. Furthermore, the integrated light and electron microscope is automated and easy to use. By having those features, it will also be accessible for non-EM customers.” Also, the system specifically is designed for cell biology. “We are aiming at cell biologists, who are primarily users of light microscopes.”

With the MAPS software for cell biology, information from any light microscope is imported and imaging of a specific area correlated with SEM information. “This is a high-throughput correlative workflow software for all FEI SEM and DualBeam [focused ion beam/SEM] tools,” explained Mr. Wohlfarth. MAPS software can also be used with the company’s scanning TEMs. The software “stitches” together “tiles” of acquired images. “It offers features like ‘Tiling & Stitching’ from a large field of view down to the highest level of content (in the gigapixel range for EM resolution of 2–3 nm) and the correlation of either imported images from other modalities or images from different detectors in the EM in multilayer mode,” he explained.

According to Mr. Wohlfarth, SEM is gaining new attention from cell biologists. “If you look at cell biologists, [and you ] look 10 years back in time, at that point in time . . . confocal microscopy took some of the business from electron microscopy away because [biologists] thought ‘the resolution is good enough, it’s easier to use, it’s not as expensive,’” he explained. “But nowadays you see it’s actually the other way around. They have reached their limit of information, and they realize that, basically, if they would like to add contextual, higher-resolution information to when they see a green spot, in order to simplify that, you need to apply the electron microscope.”

Although the new products are focused on cell biology, Mr. Wohlfarth believed correlative microscope’s impact will be broader. “I think, within science, we’ve reached a point in time where different technologies have been used more in island mode,” he stated. “The future lies really in correlating data coming from different technologies in order to have more useful information—the next level of information that basically brings those together; a bridging approach between different scientific technologies.”

The commercialization of an integrated LM and EM system is not new. JEOL introduced such a system, the ClairScope JASM-6200, in 2010. The system consists of an atmospheric SEM with an optical microscope on top. A thin film separates the sample from the vacuum. “What is unique about this—rather than going from a fluorescence microscope and running into your SEM lab and preparing your sample to be vacuum compatible for SEM imaging—is that the SEM imaging is done at atmosphere. This is very different from ESEM [environmental SEM] technology in which the sample is exposed to some level of vacuum,” explained Donna Guarrera, assistant director of JEOL’s SM Division. “In the ClairScope, the sample is completely open to room temperature and atmospheric pressure. So you can really do dynamic studies.”

Although cell biology is the most common application for the ClairScope JASM-6200, the system has applications in many fields, including materials research. Asked about materials research applications, Ms. Guarrera told IBO that the system is used for “developing new materials as well as monitoring processes in manufacturing and anything that might be in a liquid or wet, where you don’t want to expose it to any type of vacuum.” The integrated system also has advantages over environmental capsules and films used with SEM. “[They] tend to be very small volume, so the diffusion [and] kinetics might not be the same as something that would happen in a bulk environment or on a larger scale,” she said.

Advancing the use of correlative microscopy in cell biology has been the development of labels, including combinatorial labels, which allow the same label to be imaged by both the light microscope and the SEM. Combinatorial labels include quantum dots and fluorophores combined with electron dense particles. Asked about these developments, Natasha Erdman, SM product manager at JEOL, said, “What is new is the access to newer materials: so the ability to combine, for example, two dyes of completely different properties and completely different sizes into one, and then tag your material with those two dyes.” Other advances include microscopy itself. “And then, of course, the other part of it is microscopy technology has advanced in terms of resolution but also in terms of automation.“

For correlative microscopy using different systems, JEOL offers the Desktop SEM Navigator software, which creates a color optical image using a digital camera that can be correlated with a JEOL SEM. “You don’t spend a long time trying to relocate a particular feature, and that’s really all in the automation of microscope stages and automation of imaging conditions,” said Ms. Guarrera. “And that’s all relatively easily done just by having a piece of software that imports positions that recorded with either your handheld camera or the digital camera on your high-end optical microscope.” She also cited the use of CCD cameras integrated into SEMs as another imaging mode.

Tools, such as these, that increase the accessibility of SEM to nonspecialists, as well as lower-priced systems, have shifted how biologists view SEM. “Especially, for the biological materials, resolution is not necessarily the limiting factor. On the biological side, it’s really the automation, and how fast you can do something that is the key,” explained Dr. Erdman. The case is different for materials science. “In the materials world, it becomes a little more complicated, depending on the material—it could be beam sensitive, it could have a very small structure but be pushing the resolution of the instrument, it may require very low accelerating voltages for imaging—so there are some challenges there.”

Electron and optical microscope provider Carl Zeiss offers another type of solution for correlative microscopy. The Shuttle & Find interface was introduced in 2009 for materials analysis. Last year, the company released a version for life science applications. It consists of a specimen holder for specimen transfer and software. “Shuttle & Find is the only commercially available offering for correlative light and electron microscopy in which one and the same software controls the required functions of the light and the electron microscope,” said Markus Wiederspahn, a public relations representative for Carl Zeiss Microscopy. “The light and electron microscopy systems themselves remain separate, keeping full flexibility and performance for all imaging modes without compromises.” The system is designed for use with Carl Zeiss microscopes. “Shuttle & Find is compatible with all Zeiss SEMs and CrossBeam FIB/SEM workstations in combination with all Zeiss widefield light microscopes that can be equipped with a motorized stage like Axio Imager, Axio Observer and SteREODiscovery.”

Highlighting the importance of the development of correlative microscopy, in October, Carl Zeiss formed a new business group that combines the company’s Nano Technology Systems division, which offers EM and was previously part of its Semiconductor Manufacturing technology group, and its Microscopy division, which offers optical microscopy. The new division incorporates a single sales organization and name. “Now, being one organizational unit, the future challenges of correlative microscopy can be addressed even better,” stated Mr. Wiederspahn. “Carl Zeiss Microscopy is the only company with a full portfolio in light and charged-particle microscopy. So we feel optimally prepared, with know-how and manpower, to take the next steps and make correlative microscopy a mainstream technology for all microscopy disciplines.”

Asked about the future of correlative microscopy in general, he told IBO, “We are pretty sure correlative microscopy will become a major trend and will open doors to new research areas and applications never thought of.” He added, “Future developments will comprise increasing automation, new interfaces (e.g., SEM with confocal light microscopes, TEM with light microscopes) and, of course, new sample preparation and labeling techniques.”