Recent advances in super-resolution microscopy have provided many exciting opportunities for researchers; enabling them to better understand the detailed inner workings of the cell. But not all super-resolution microscopes are created equal, and choosing the correct technology for your needs is key to the success of your research. Choose correctly, and super-resolution technologies can be a powerful tool; allowing you to understand cellular structure and dynamics at a level conventional microscopy could only dream.
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Super-resolution is the umbrella term for technologies that break the diffraction limit, producing images of greater resolution than conventional light microscopy can offer. They work by circumventing the limit imposed by point-spread function—either by modulating the excitation light temporally or spatially, or reducing the effective size of the point spread function. The technologies are split into two basic categories, depending on whether they exploit these effects at an ensemble (SIM and STED) or single-module (STORM, PALM) level.
SIM
Ensemble techniques increase resolution by shaping excitation light. The main types are SIM and STED. Structured illumination microscopy (SIM) enhances spatial resolution and works by illuminating the sample with patterned light. As Renée M. Dalrymple, Product Marketing Manager—Life Sciences Lattice Line, ZEISS Research Microscopy Solutions, explains, “SIM has become a go-to super-resolution fluorescence microscopy technique for live imaging, due to its comparatively gentle illumination and compatibility with standard fluorophores allowing easy multi-color experiments.”
Lattice SIM, which works by illuminating the sample with a lattice pattern instead of grid lines, extends live imaging capabilities even further—increasing imaging speed and extending lateral resolution. This enhanced resolution allows observations at a sub-organelle level. Lattice SIM “opens the door to formerly unattainable applications by enabling the observation of rapid sub-organelle structural changes and inter-organelle interactions without the need to modify your standard sample preparation protocols,” Dalrymple explains.
STED
Stimulated emission depletion (STED) is a popular super-resolution imaging technique. It enhances resolution by employing two lasers; a STED laser, used alongside an excitation laser, suppresses any fluorophores surrounding the excitation focus, creating a small focal spot. A major advantage of this technique is the depth of tissue it can penetrate, meaning that applications such as neuroscience often favor STED.
STORM and PALM
Single-molecule techniques by contrast, enhance resolution by selectively exciting only a small subset of the fluorescent molecules present—repeating this process over time and then assembling the localized points. The main types are STORM and PALM. Stochastic optical reconstruction microscopy, or STORM, uses photoswitchable fluorophores, while photoactivated localization microscopy (PALM) utilizes photoactivatable fluorophores—that until activated, exist in a “dark” state. Both techniques are popular in the study of gene expression—revealing insights into both where and when genes are expressed or transcribed.
How to choose
So, with the growing number of super-resolution technologies available, how do you choose a system to best aid your research? According to Dalrymple, “It may sound obvious but, choose the easiest to use technique that allows you to answer your scientific question.” While it may be tempting to reach for the technique with the highest potential resolution, in practice this can be very difficult to achieve and often requires time to perfect special sample preparation protocols. “Many questions can be answered much more easily by techniques such as Lattice SIM”— which can be performed without the need to change your sample-preparation method.
“Choosing the correct objective is also critical to take full advantage of the improved resolution of these imaging methods,” says Jake Jones, Associate Product Manager for Life Science Microscopy at Evident Scientific. An objective with low numerical aperture, or one that does not use an imaging medium to reduce the index of refraction mismatch between a biological sample and the optics, can reduce resolution.
Success also comes from your choice of fluorophore. Ideally probes for super-resolution technologies should be as bright as possible, as well as being highly photostable, which reduces photobleaching. They should also be capable of alternating between light or dark states, or from one wavelength to another. Specific buffers can help facilitate this switching, with two of the most commonly used buffers containing either mercaptoethylamine or β-mercaptoethanol. Although, as Jones explains, while solutions “like STORM and PALM previously required a chemical understanding of fluorophores and buffers to properly prepare imaging samples, the creation of readily available sample-preparation kits has greatly streamlined this process and increased accessibility to researchers of all backgrounds.”
Recent developments continue to expand the promise and usability of super-resolution techniques among the scientific world. However, embracing these technologies is not without its challenges, and improvements are still to be made. A future goal of super-resolution technology, says Dalrymple, "is to combine the highest resolution with speed, gentleness, and ease of use in order to minimize any trade-offs that currently exist.” Developments in 3D imaging are also required in order to penetrate deeper into tissue while maintaining resolution.
Increasing the accessibility of the technologies is also key, Jones says. “Combining traditional imaging modalities with super-resolution microscopy in single systems is expected to become more prevalent.” Being able to “switch between these modalities in a unified software package without changing samples, buffers, or fluorophores” is ideal for multi-user microscopy units, while simultaneously providing new users with seamless access to super-resolution imaging.