Despite competition, the light microscope remains the most important instrument in the laboratory. And it's quickly evolving.

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This detail of a mouse embryo’s heart was obtained with Leica’s TCS SP5 II super-resolution confocal microscope. Photo: Dr. Elisabeth Elher, Department of Medicine, Kings College, London, UK

Galileo Galilei is celebrated for his celestial observations and his struggles with the Catholic church; but few realize he is often credited with one of the most important and ubiquitous instruments used in today's laboratories.

In 1609, he reportedly made an early type of compound microscope with a convex and a concave lens. Though unsophisticated, the invention was the first recorded device to be given the name "microscope".

Today's versions of Galileo's crude instrument outnumber the telescope—which he also invented—by a large margin. Optical (or light) microscopy is still the dominant imaging tool used by laboratory researchers worldwide. Of the $2.7 billion global market in 2010, optical microscopes accounted for $1.1 billion, according to a June 2011 market report from MarketsandMarkets.

Barbara Foster, president and senior strategic consultant, The Microscopy & Imaging Place Inc., McKinney, Texas, says the strength of fluorescence techniques and the breadth of applications—from biological to materials science—has ensured that about 80% of laboratory researchers use upright light microscopes, easily outpacing all other types. About 70% use stereomicroscopes, she says, and about 40% use inverted microscopes.

Despite the popularity of light microscopy, and a recent victory over the diffraction limit through advanced confocal techniques, competition abounds. Scanning probe and electron microscopes are becoming cheaper and smaller, and offer characterization into the nanoscale. Unanticipated challenges have come from the digital camera industry as well. But after decades of few advances, light microscopes have in recent years seen major evolutionary changes.

Quantitation arrives for light microscopy
As researchers have pushed the limits of optical resolution in live cell research, the information gathered has grown exponentially. Fields such as genomics, proteomics, and metabolomics have arisen to handle this growth in information. The ability to quantitatively compare and combine this information has become more and more critical.

Previously, such information was the domain of scanning probe microscopy (SPM), which relies on mechanical forces to obtain feature information. But light microscopy has benefited greatly from photonics technologies such as charge-coupled devices (CCDs), avalanche photodiodes (APDs), advanced laser sources, and improved photomultipliers (PMTs). These advances have allowed many new light microscopes to adopt a standard unit, the photoelectron, a move that eliminates sources of variation and allows quantitative comparisons of different data sets.

A good example of this approach is found in Quant-View imaging, first developed for Photometrics' Evolve 512 EMCCD camera. According to James Joubert, an applications specialist, at the Tucson, Ariz.-based company, the technology has improved quantitation, reproducibility, and certainty in data comparisons by moving away from arbitrary image intensities that vary from camera to camera and with different gain states, electron multiplication gains, and bit depths.

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Nikon Metrology Inc. combined some features of a handheld digital camera with the laboratory performance capabilities of an upright light microscope to create the mobile Shuttlepix. The microscope is intended for a variety of industrial applications, but is also suitable for some research purposes.

Additionally, other modifications and modes of imaging have been added to Photometrics' cameras to allow for high-speed, multiplex imaging, which is especially important for dynamic multi-color, super-resolution microscopy. One such addition is the ability to rapidly switch between different exposure times when capturing image streams.

"This can be extremely useful when capturing images of cells labeled with several dyes that have differing concentrations or quantum yields, and thus require different exposure times to obtain the same signal-to-noise image quality," says Joubert.

Trends in quantitation have reached other forms of light microscopy. According to market analysis conducted by Foster, approximately 45% of researchers now use confocal imaging for its ability to maximize both optical resolution and contrast. Confocal microscopy has been popular in part because of its ability to maximize both optical resolution and contrast. New designs featuring advances in illumination and electronics are putting high-performance systems in the hands of researchers with more conservative budgets.

Leica Microsystems, Buffalo Grove, Ill., a long-time manufacturer of high-end optics and microscopy systems for the R&D market, recently introduced two microscopes that reflect the changing demands of light microscopy users. The TCS SP5 II Confocal imaging system is a development of the company's three-detector spectral acquisition system, which captures multiple fluorescent channels simultaneously.

Central to the system's capability is a multiple-laser setup that extends the lifetime of both the dye and living cells. For particularly sensitive studies, photon counting is possible with an optional hybrid detector (Leica's HyD), which combines the capabilities of a PMT with that of an APD. Photon counting is a technique that relies on the accumulation of photons—which are read and counted by a detector—in the pixels of a photodetector. A color-coded image is scaled by wavelength on the basis of these counts and delivered as a quantified, statistical analysis image.

The key to the HyD technology is four sensitive detectors that record photon streams at a rate of hundreds of counts per second. A high quantum efficiency of 45% at 500 nm helps supply the detectors with low noise and large dynamic range, allowing fixed single molecule analysis, imaging of delicate yeast samples, and real-time tasks such as monitoring the embryogenesis of Caenorhabditis elegans.

The mobile microscope
For most of its history, the microscope has been confined to the bench top. Most traditional systems were designed for use in a static laboratory environment where samples remain undisturbed and easy to handle.

The form factor changed dramatically, however, with the advent of the digital microscope. Companies such as Keyence Corporation, Woodcliff Lake, N.J., were able to free the optics of its traditional stage, moving the viewing area to a flat screen and allowing the optics to rotate against a fixed sample. The move, as exemplified in the R&D 100 Award-winning VHX-1000 instrument, has proven itself successful in the marketplace.

The notion of moving the microscope and not the sample isn't new, but only recent innovations in machine vision and camera technology, as well as the introduction of light-emitting diodes, have allowed microscope designers to truly liberate the optical microscope from the bench top. Nikon Metrology Inc., Brighton, Mich., made waves earlier this year with the release of the ShuttlePix P-400R Digital Microscope.

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To meet the higher-sensitivity requirements of modern super-resolution microscopes, PI (Physik Instrumente) LP has adopted capacitive sensors for its PInano line of scan stages to achieve higher quality scans with less noise than piezoresistive sensors.

Geared toward industrial applications that require fast, repetitive readings, the ShuttlePix functions much like a digital camera. Designed for on-site analysis of samples, it serves as a handheld microscope that shoots high-resolution images quickly and stores them digitally. Stationary use is also possible. The microscope pairs with a 17-in touchscreen monitor to let users interact more easily with the images captured by the 20X zoom.

"The versatility of the ShuttlePix system means the user can bring the microscope to large samples, such as an aircraft frame, turbine casting, or pipe work that often cannot be reached with a standard microscope," says Bob Wasilesky, senior vice president, Nikon Metrology.

ShuttlePix offers a magnification range of 20X to 400X. The CCD is rated at 2.11 megapixels and data storage is held by data cards.

According to Atsushi Takeuchi, mechanical engineer at Nikon Metrology who was project manager of the ShuttlePix development team, a research group was formed in 2007 to begin development of the instrument and conducted four months of market research. The priority for users was mobility, as well as ease of operation and all-in-one shooting-to-measurement capability.

Because conventional microscopes generally consist of a wired camera and monitor and weigh about 10 kg, he says, designing a handheld device proved challenging and required years of development. The zoom head on the Shuttlepix, for example, would not have been possible without the use of light-emitting diodes that form the built-in, four-section ring illumination.

"Normally, we design a product’s outlook after fixing the lens and mechanical functions. However, we felt that if we developed the lens and mechanical functions first, we would risk not achieving the development of a handy microscope," Takeuchi says.

Super-resolution and the hybridization of light microscopy
Even as light microscopes break free of the traditional stand, bench-top microscopes are benefiting from a revolution in capabilities. In 1999, Foster predicted the eventual union of two formerly well-segregated technologies.

"I saw an eventual collision between microscopy and spectrometry. It took some time but it has happened. The inclusion of Raman information in microscopes has greatly advanced the field," she says.

A prime example of this shift is CytoViva, Auburn, Ala., which launched a Dual Mode Fluorescence system in 2008 that allows researchers to observe interactions between fluorescently labeled nanoparticles or bacteria and live unlabeled cells. The company later added hyperspectral imaging.

Hybridization has also allowed the resolution barrier to be broken.

"In terms of recent breakthroughs, the most significant advance for optical microscopy could easily be the breaking of the diffraction resolution limit through the advent of super-resolution microscopy. Until recently, the closest that features could be to each other and still be resolved by a traditional optical microscope was set by the diffraction limit of the microscope optics at around 250 nm," says Joubert.

Super-resolution uses sub-pixel shifts between multiple low-resolution images of the same scene to create an improved resolution image fusing information from all low-resolution images. Carl Zeiss MicroImaging Inc., Thornwood, N.Y., has integrated two methods of super-resolution into one, turn-key platform. Their ELYRA system can be configured for super-resolution structured illumination (SR-SIM) and/or photoactivated localization microscopy (PALM). It can also be combined with its laser scanning microscopes.

PI (Physik Instrumente) LP, Auburn, Mass., has added two higher-performance models equipped with direct-measuring capacitive its PInano super-resolution (SR) microscope stage series.

This type of sensor can provide higher linearity and long-term stability than other lower-cost piezoresistive sensor-equipped stages. Capacitive sensors are also less sensitive to noise due to a high-frequency measurement principle as opposed to the DC-based sensing technique used in piezoresistive sensors. The net effect is similar to the higher quality and lower noise on FM radio channels compared to AM radio.

Light microscopy staves off challenges
Despite numerous innovations—super-resolution, mobility, quantitation, and hybridization—some still believe light microscopy's market is under threat. One conclusion in the MarketsandMarkets report was that light microscopy is losing ground to higher resolution scanning probe and electron microscopy. As Foster reports, it's an opinion that has persisted over the last decade as SPM, AFM, and SEM instruments improve and become smaller and more affordable. Nanotechnology funding has supported this perception as companies race to meet new research demands through the use of non-optical instruments.

"People have been saying this for decades now, but what’s really interesting is that light microscopy continues to reinvent itself," says Foster, pointing to PALM systems that are technically light microscopes but are able to follow biological entities to 10 to 20 nm, well within the nanorealm.

According to Joubert, the one advantage electron microscopes have over optical microscopes is the ability to resolve finer details, but the development of super-resolution optical microscopy has chipped away at that advantage.

"It would be difficult to state that optical microscopes are losing ground to electron microscopes when, in fact, the reverse is likely to be true," he says, adding that the cost of electron microscopy is dramatically higher, they require more expertise, and sample preparation is time-consuming and difficult despite efforts of instrument manufacturers to circumvent that process.

Additionally, he says, electron microscopes require vacuum to avoid electron scatter and are thus unable to image live cells in vivo or in vitro to track dynamic events.

"As much of current cutting-edge bioresearch is moving away from before-and-after imaging of fixed cells and toward imaging dynamic cellular events as they happen, this is a significant advantage for optical microscopy," says Joubert.

The threat, if there is any, may be coming from an entirely unexpected direction. Foster has observed that a wave of cheaper CCD cameras have taken over some industrial and semiconductor arenas.

"Many people don’t realize that machine vision has been eroding some of light microscopy's market," says Foster.

Ultimately, these cheaper solutions may be what forces light microscopy's continued evolution.