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Up until the last 15 years, researchers and scientists have been largely constrained by a long-established optical resolution limit related to biological microscopy systems. This limit was first stipulated by Ernst Abbe more than 140 years ago and established the maximum resolution of an optical system to be around 200 nm in the XY direction. What this limit has always meant for microscope users was that many subcellular structures were not large enough for detailed observation. The imaging of intracellular structures and activities has typically been restricted by the optical resolution limit of available imaging systems.
Today, scientists are using powerful super-resolution microscopy techniques to image living samples well beyond the established theoretical limit, allowing the study of far-ranging biological phenomena. The primary benefit of super-resolution is enhanced resolving power, with several super-resolution options available that allow the imaging of samples below 200 nm. Some super-resolution techniques have reported improvements in resolution ranging up to an order of magnitude, bringing details into focus on a cellular or molecular level that were previously entirely invisible.
Selecting the right super-resolution technique
Super-resolution is an ideal solution for scientists who need to resolve structures beyond the diffraction limit of optical microscopy, including specimens that are incompatible with electron or atomic force microscopy. There are a number of super-resolution techniques, each with its own advantages and disadvantages. It is helpful to consider and compare the super-resolution options currently available as part of commercial imaging systems:
Total internal reflectance fluorescence microscopy (TIRFM)
If sample features are located within 100 nm of a coverslip or tissue culture container, TIRFM and related evanescent wave methods can be used to dramatically enhance image resolution. TIRFM provides excellent high-contrast imaging of single-molecule fluorescence and membrane dynamics.
- Typical resolution: ~230 nm (XY); ~100 nm (Z)
- Speed: very high
- Advantages: works well with living cells; can be combined with SIM
- Disadvantages: resolution only improved in Z axis; imaging can only be done close to coverslip.
Photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)
These super-resolution techniques rely on the calculated localization of fluorescent molecules in the sample. PALM and STORM offer high spatial resolution, but have low temporal resolutions ranging from 30 seconds to 30 minutes, depending on the application, sample, and fluorochromes used. Recent advances in camera technology offer the potential for even higher signal-to-noise (SNR) ratios with these techniques.
- Typical resolution: >50 nm (XY); ~30 nm (Z)
- Speed: very low
- Advantage: maximum lateral resolution
- Disadvantages: special fluorophores required; phototoxicity associated with multiple imaging/quenching cycles; imaging close to coverslip (usually combined with TIRFM).
Stimulated emission depletion (STED) microscopy
STED microscopy is a powerful super-resolution technique that uses two lasers in sequence, stimulating and then canceling out light. The first laser excites the sample and stimulates fluorescent emissions. The second laser depletes all fluorescence except for that occurring in a sub-resolution volume of the sample. STED microscopy can present complex optical alignment challenges, but does provide excellent results.
- Typical resolution: 80 nm (XY); ~50 nm (Z)
- Speed: medium
- Advantage: reasonable speed
- Disadvantages: system complexity; phototoxicity associated with intense quenching beam.
Structured illumination microscopy (SIM) and saturated SIM
SIM involves exciting a sample with repeating bands of light that interact with the sample structure to create moiré patterns (Figures 1 and 2). Subsequent algorithmic processing of these patterns results in higher-resolution images than would be possible with TIRFM or traditional unstructured widefield microscopy. Saturated SIM, known as (S)SIM, is a related super-resolution technique that introduces nonlinear fluorochrome responses to further enhance SIM’s capabilities.
Figure 1 – Confocal image. Stress fibers of Hela cell: Antibody staining with Phalloidin-Alexa 488 (green) for actin; Alexa 568 (red) for myosin heavy chain. (Image courtesy of Keiju Kamijo, Ph.D., Division of Anatomy and Cell Biology, Faculty of Medicine, TOHOKU Medical and Pharmaceutical University.)
Figure 2 – Same image as Figure 1, made using Olympus SD-OSR super-resolution SIM mode.- Typical resolution: SIM 90–140 nm (XY)
- Speed: low
- Advantage: no need for special fluorophores
- Disadvantages: limited super-resolution; bleaching.
When selecting between different super-resolution tools and techniques, it is important to ask a number of questions to determine the best solution for the application at hand. Speed, resolution, and system complexity all come into play when deciding which super-resolution technique is best for your situation. In some cases, however, super-resolution may not be required to achieve the desired results.
In other instances, there may be issues regarding specimen integrity that rule out super-resolution as a viable option. The following questions can serve as good guidelines when considering a super-resolution imaging system:
- Is what I need to see so small that my research demands a super-resolution solution?
- What limitations am I experiencing with my current imaging system? Are there any system enhancements that may help me get the results I need?
- Can I achieve my imaging goals by simply adjusting contrast?
- Do I have any concerns regarding specimen integrity versus increase in resolution?
- How important are system speed and flexibility, both now and in regard to future observations?
The ideal way to approach super-resolution imaging is to start with the specific needs of your study and consider the validity of all solutions, including all super-resolution techniques as well as widefield and confocal imaging. In the end, you should opt for whichever method produces the most usable fluorescence emission while providing all of the needed data. With confocal imaging, lowering the background intensity can improve fluorochrome visualization and enhance image analysis. In some cases, this enhanced contrast is enough to provide sufficient resolution. In other instances, super-resolution is required.
Some imaging systems allow the use of confocal and super-resolution modes within the same system. These systems allow the operator to select the mode that will provide the best results for a given application, and provide the advantage of a common stage. Using the same slide, the researcher can switch back and forth between confocal and super-resolution modes, producing two data sets for comparison. This added benefit allows multiple sources of information that can help confirm findings and lead to better certainty without artifacts.
An example of a dual confocal/super-resolution imaging solution, the Olympus SD-OSR microscope system (Olympus Corp., Waltham, MA) pairs a high-speed spinning disk with high-performance optics and objectives that can deliver imaging resolution up to twice that of a widefield microscope (Figures 3–5). In super-resolution SIM mode, the SD-OSR produces resolution-limited point scanning and optical oversampling to achieve 120-nm planar resolution at up to three frames per second. The system’s Airy disk over-sampling captures subtle high-frequency spatial components that are not visible in conventional spinning disk microscopy. Its super-resolution mode requires no special sample protocols—the same workflow utilized during traditional confocal imaging can be used to achieve super-resolution results.
Figure 3 – Widefield image made using the Olympus SD-OSR microscope system. Odf2 staining (Alexa Fluor 488) of cilia at the upper part of the basal body. (Image courtesy of Hatsuho Kanoh, Elisa Herawati, and Sachiko Tsukita, Ph.D., Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University.)
Figure 4 – Same image as Figure 3, made using Olympus SD-OSR confocal mode.
Figure 5 – Same image as Figure 3, made using Olympus SD-OSR super-resolution mode.Challenges associated with super-resolution microscopy
Despite its enormous power and potential, super-resolution microscopy presents some challenges, most of which increase as resolution becomes higher. As super-resolution systems image smaller and smaller details, the population of responding fluorochromes decreases, requiring the development of new fluorochromes with higher quantum yields. Vibration and spherical aberration are also a greater hindrance at higher resolutions. Additionally, some live samples are more adversely affected by super-resolution imaging than others because of high excitation intensity or extended exposure times. Super-resolution systems can eventually reach a point of diminishing returns, where specimen stress and its impact on data reliability can outweigh the benefits of higher resolution.
Lack of flexibility is another challenge encountered with many super-resolution systems—many hardware-based super-resolution systems cannot be easily adapted if an experimental protocol changes in the middle of an application. The quality of super-resolution images can also vary tremendously; images captured using the same resolution can appear dramatically different in detail and appearance. Lastly, super-resolution systems are much more expensive than other high-resolution imaging options. A super-resolution system can start at more than twice the price of a confocal imaging system and can cost as much as four times more. The difference in cost between a super-resolution system and a widefield fluorescence system is even more dramatic.
Looking forward to the next 15 years
The world of biological microscopy is based on the ongoing effort to see further and further into what cannot be seen by natural means—the most subtle details and processes that define life. Super-resolution, one of the newest tools available to researchers, provides a highly advanced means to see even deeper, and more clearly, into the dynamics of living cells. However, although great strides have been made in the world of super-resolution microscopy over the past 15 years, and there are now a variety of effective super-resolution techniques, super-resolution has not yet solidified its place as a primary tool for advanced imaging. Further advances in super-resolution will likely allow researchers to image even smaller structures for longer periods of time, and delve even deeper into living tissue. We look forward to seeing what the next 15 years bring to this increasingly popular microscopy technology.
Russell Ulbrich is product manager, Olympus Corporation of the Americas, Scientific Solutions Group, 48 Woerd Ave., Waltham, MA 02453, U.S.A.; tel.: 781-419-3900; e-mail: [email protected];www.olympus-lifescience.com.