Innovations in Super-Resolution Microscopy

Imaging has officially shifted from microscopes to nanoscopes. Frost & Sullivan has identified important companies in this space and predicts the road ahead for the field.

An imaging system is judged on two parameters: magnification and resolution. The allure of zoom often eclipses the importance of image resolution. There is little value in enlarging an image of a live cell if it appears blurry rather than as a distinctive subject with clearly delineated features. Resolution is an imaging system’s ability to capture as much visual detail about a subject as possible.

While the resolving power of cameras and microscopes can be improved through product design, it cannot be increased indefinitely. The resolving power of optical imaging systems is limited by a phenomenon known as Abbe diffraction limit. German physicist Ernst Abbe (1840–1905) theorized that the resolution of any optical imaging device is directly proportional to the wavelength of incident light (λ) and inversely proportional to the numerical aperture (NA) of the lens used. Based on this law, while the resolution can be increased through innovations in the lens design that can lower the NA, ultimately the obstacle to improving resolution is the light itself. Even the best-in-class lens systems are limited in their ability to provide highly resolved images by the nature of light used. These high-end microscopy systems can produce a resolution of 250 nanometers (nm); unfortunately, life science applications (and increasingly, semiconductors, electronics and metallurgy) frequently come across subjects that are much smaller in size. Some of the most common laboratory processes for which super-resolution imaging is required are single cell imaging, live-cell imaging, quantitative cellular imaging and counting, sub-cellular interaction imaging, protein formation studies, epigenetic mapping, single molecule localization and neuronal imaging.

From Microscopes to Nanoscopes

Since it is not physically possible to breach the diffraction limit, instrumentation engineers have designed systems that work around it. Super-resolution imaging is not a marketing buzzword; it refers to a family of mathematical approaches to improve image quality. These approaches are broadly categorized as true super-resolution and functional super-resolution. The first approach captures hitherto-undetected information contained in evanescent waves. The energy contained in these waves decreases exponentially, making it very difficult for conventional microscopes to capture them as they leave the subject. Naturally, this requires complex instrumentation that is expensive and difficult to operate. As a result, functional super-resolution is instead employed, using experimental instrumentation tweaks to digitally reconstruct images into a high-resolution output. This approach uses special fluorescent tags known as fluorophores that display distinct emission properties, providing spatial and temporal differentiation.

Of the many methods that constitute super-resolution imaging, two in particular— photoactivated localization microscopy (PALM) and stimulated emission depletion (STED)—were rewarded with the 2014 Nobel Prize in Chemistry. The Royal Swedish Academy for Science, in its press release announcing the award, noted that these approaches have “ingeniously circumvented [Abbe’s diffraction] limitation.” By breaching the diffraction limit and making it possible to view structures in the nanometer dimension, the technology has officially shifted from microscopes to nanoscopes. Frost & Sullivan has identified important companies in this space; brief profiles of their imaging products are provided below.

Nikon Instruments (Melville, N.Y.)

Nikon Instruments is the microscopy division of the Japan-based Nikon Corporation. The company has two super-resolution microscopy products, N-SIM and N-STORM, in its portfolio. N-SIM is based on the principle of structured illumination microscopy (SIM), a technology that has been licensed from the University of California, San Francisco. The N-SIM device reportedly produces images with a resolution of 115 nm. N-STORM is built on a technology known as STochastic Optical Reconstruction Microscopy (STORM), licensed from Harvard University. N-STORM detects signals from thousands of fluorophores to produce images with a resolution of up to 20 nm—nearly 10 times greater than the image quality of conventional microscopes.

Leica Microsystems (Weltzar, Germany)

Leica’s HyVolution aims for a balance between high-resolution images and high-speed imaging—features that are inversely related. Due to the volume of information that has to be processed, the waiting time and the computing requirements for image processing are quite high. HyVolution produces images with resolution of 140 nm—a fraction lower than competing products—but at an image acquisition rate of minutes, reportedly 10 times faster. HyVolution also allows for multicolor fluorescence tags, a feature that enhances differentiation of subcellular structures.

Bruker Corporation (Billerica, Mass.)

Bruker’s Vutara 350 is a super-resolution microscope that has been developed for live cell imaging, deep-tissue imaging, and for single-molecule localization. The system enables high resolution up to a depth of 350 microns through the z-axis stage and an axial resolution of as high as 40 nm. Vutara Inc. was an independently operating company with a line of digital and super-resolution microscopes. Bruker, known more for its line of industrial measurement systems and electron microscopes, acquired Vutura in 2014 to strengthen its portfolio of advanced microscopy systems in order to compete with companies such as Nikon and Leica.

Oxford Nanoimaging (Oxford, England)

Oxford Nanoimaging, a spin-out from Oxford University, is in the business of designing and manufacturing low-cost, compact super-resolution imaging without the high maintenance and infrastructure requirements of conventional super-resolution microscopes. While other advanced microscopy products are bulky in their architecture and cost millions of dollars to build, the company’s flagship Nanoimager has a footprint of a desktop computer. Measuring 21 cm x 21 cm (nearly 30 times smaller than other imaging systems), the system still produces images with an axial resolution of 50 nm and lateral resolution of 20 nm.

The Road Ahead

Improvements in image quality automatically translate into increases in image size. This immediately raises concerns about storage and management of large data files. Making sense of high-resolution images is an entirely different challenge.

Frost & Sullivan strongly believes that the future of microscopy—indeed, the future of medical imaging—is incomplete without the presence of data companies. Large data sets and the need for advanced analytics open opportunities for data companies and necessitate the integration of information technology tools such as big data analytics and artificial intelligence into the imaging suite.

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