In any field of science, new discoveries often depend on finding answers to fundamental questions. In drug discovery, that often means uncovering the differences between healthy and diseased cells and tissues and understanding the biological basis of cellular function and dysfunction at the molecular level.
Microscopes and imaging technology have evolved in synchrony with advances in computers and software applications, detectors, light sources, and labeling and detection systems. Microscopes have an increasingly important role in research aimed at illuminating what exactly happens, for example, when a virus infects a host cell, a ligand binds to a membrane receptor, or a tumor cell metastasizes.
The abundance of published articles featuring advanced imaging techniques, and grant awards to support the establishment and expansion of imaging centers are evidence of the critical role of microscope technology in biomedical research.
For example, The HIV Center at the University of Utah recently received a $21.8 million grant from the NIH to develop methods to image and study the structural biology of the AIDS-causing virus and to understand how it infects host cells, replicates, and spreads.
The Molecular Imaging Center at Washington University School of Medicine in St. Louis received a $7.1 million grant from the NCI. And the Dana-Farber Cancer Institute received a $10 million grant from the Massachusetts Life Sciences Center to expand its cancer imaging research program and to help establish the Molecular Cancer Imaging Facility, a research initiative aimed at developing new molecular imaging probes.
These sizable grants also emphasize the high cost of imaging technology, and one overarching goal expressed by developers of microscope systems is to bring down the cost of ownership and expand accessibility of the technology.
While the most advanced technology is available to the top biologists in the world, “we need to make it more financially and technically accessible to all biologists,” says Paul Goodwin, science director at Applied Precision, a GE Healthcare company.
In surveying the microscopy industry, several key trends emerge, most prominently an emphasis on increased resolution and the emergence of super-resolution technology and dynamic imaging in live cells and organisms.
The super-resolution methods that have emerged represent an alphabet soup of sophisticated techniques including structured illumination microscopy (SIM), stochastical optical reconstruction microscopy (STORM), spatially modulated illumination (SMI), ground state depletion (GSD), and stimulated emission depletion (STED).
Live-cell/organism imaging requires faster image acquisition to capture biological and physiological processes as they are happening, such as protein-protein interactions, cell migration, morphologic changes related to cell division or apoptosis, and ion-channel activity. Growing interest in live-cell imaging has spurred developments in related fields such as label-free imaging to enable less intrusive and disruptive methods that do not require exposure to large amounts of light likely to affect the biology and compromise the viability of cells.
Additional trends focus on enhanced quantitation, co-localization, and correlative microscopy, and the integration of different microscopy techniques in unified multifunctional systems. Advances in deep-tissue imaging and 3D microscopy reflect a trend in cell biology away from studying cells grown in monolayers to examining multicellular and tissue constructs to explore biology in a more natural, dynamic environment.
The challenges lie mainly in the ability of tissues to absorb and scatter light, and other factors that contribute to poor signal-to-noise ratios when imaging through tissues and aberrations that can compromise resolution.
Goodwin describes a “renaissance in microscopy,” with multiple new technologies emerging across a relatively short time span to meet a variety of technical and application-oriented needs in the biopharma R&D arena.
When you get down to basics, “the glass lens of a microscope has not changed substantially in 100 years,” he explains. What has largely driven advances in microscopy systems has been the combination of sophisticated computational and sensor technology. This is evident across several of the key trends highlighted in this article, including super-resolution imaging technology, label-free imaging, and advances in correlative, quantitative, and 3D microscopy.
Advances in super-resolution imaging are enabling spatial and temporal resolution in the same sample and live-cell imaging, notes Goodwin, highlighting the company’s DeltaVision OMX® super-resolution system with the BLAZE™ 3D SIM module capable of imaging a 1 micron slice in <1 sec and widefield imaging at >400 fps.
He draws attention to a recently published paper by Strauss et al. (PLoS Biol), which described the use of 3D-SIM super-resolution microscopy to perform time-lapse imaging studies of cell division in bacteria and visualize protein dynamics and changes in protein distribution that contribute to cytokinesis.
Selecting a super-resolution technique can mean a trade-off between higher resolution versus dynamic imaging, explains Chris Vega, Ph.D., marketing manager, life science research at Leica Microsystems. With STED, for example, “you can image in a purely optical way” and typically achieve resolution in the 40−50 nm range, whereas with the GSD method, which involves the localization of individual fluorophores, higher resolution of about 20 nm is feasible using existing technology, but GSD performs better with fixed specimens than live samples.
Leica’s latest addition to its super-resolution systems is the third-generation TCS SP8 STED instrument, which the company introduced at the Society for Neuroscience meeting in October. It includes a gated STED option that combines Leica’s HYD™ hybrid detection technology and white light laser to increase resolution and contrast.
Is the microscopy industry headed to ultra-resolution? Do cell and molecular biologists need to go beyond super-resolution to get the information of value to them? Inevitably, there will be groups in the research community that will strive to achieve ever higher resolution, and others will find applications for it.
At present, observes Goodwin, resolution limits depend mainly on the size of autofluorescent proteins such as green fluorescent protein and labeled antibodies. Systems can get down to about the 15−20 nm range, about the size of the labeled antibody that is associated with the target protein of interest. “I think we will get to the resolution of individual molecules,” predicts Goodwin.
“I guarantee you that someone is working on [single] nanometer or picometer resolution,” adds Brian Manning, Ph.D., application scientist at Chroma.
As a manufacturer of optical filters and mirrors for microscope developers and end-users, Chroma is attuned to changing demands related to application trends. For example, there continues to be a lot of interest in optogenetics and the introduction of rhodopsin proteins into neurons to enabling imaging of individual neurons during live animal studies.
These types of experiments are part of an effort to map neuronal networks and map signaling pathways. He also describes demand from customers for increasingly flat dichroic mirrors, primarily for total internal reflection fluorescence microscopy applications, in which the flatness of the mirror is the limiting factor in being able to minimize deformation of the beam.
What Goes Around
“What’s old is new again,” says Manning, to describe the concept that the basic foundation of technology development involves a “constant revisiting” of what came before. A good example is the resurgence of Raman microscopy and specifically the technique commonly referred to as CARS, or coherent anti-stokes raman scattering, in which imaging is based on the vibrational signatures of molecules. This label-free method eliminates the need for fluorophores.
Label-free imaging techniques are increasingly being used to study neural architecture and to understand processes such as myelination and remylelination of neurons that have a role in neurodegenerative disease.
According to Brendan Brinkman, senior product manager for the scientific equipment group at Olympus America, using a technique such as CARS, researchers can “intrinsically image myelin” and, essentially, any lipids, making it a useful tool for studying plaque formation in atherosclerosis, for example, or in the development of drugs targeting obesity.
Olympus has employed a multimodal optical imaging approach that combines colocalized label-free imaging using CARS and two-photon microscopy for detecting protein autofluorescence and has demonstrated the ability to differentiate, for example, healthy arterial wall, early atherosclerotic lesions, and advanced atherosclerotic plaques.
For looking at live samples, “within the last year, we have seen advances in marker technologies that make it easier to image cells without damaging them,” says Paul Jantzen, marketing manager, core microscopy and research imaging at Olympus, “such as the smaller and brighter luciferase enzymes from Promega.”
As the markers are brighter and no fluorescence is involved, the cells are not subjected to high intensity light and imaging can extend over longer periods of time. Olympus plans to introduce its Luminoview LV200 luminescence microscope on the U.S. market before the end of this year to support luciferase-based cellular imaging.
The ability to do label-free imaging in whole tissue sections using two-photon confocal as well as conventional microscopy has been facilitated by the development of clearing, or clarifying agents that allow the light to penetrate deeper into the tissue sample for visualizing 3D structures.
“We can now do two-photon microscopy at the resolution of light microscopy and go down to 8 mm to look at intact tissues,” says Brinkman. The company’s Scaleview-A2 clearing solution and Scaleview immersion objectives were designed to facilitate imaging of large tissue areas at high resolution with the FV1200MPE system.
Not only are researchers better able to image large areas of fixed tissue in the x and y dimensions, but now in the z dimension too. “Whole slide imaging has really come to the forefront,” says Jantzen. “They can do it faster and are better able to manage the large amounts of data generated.”
For imaging multicellular constructs and tissues, two-photon microscopy allows for the use of longer wavelengths, which are less likely to interact with cells and be scattered, resulting in deeper light penetration and better z-resolution. These types of applications have also led to greater use of fluorophores that are excited by or emit far red or near infrared photons, according to James Joubert, application scientist at QImaging.