Advances in Super-Resolution Microscopy Expand Applications

Recent advances in super-resolution microscopy continue to reveal exciting discoveries about the subcellular world. These were unattainable using conventional light microscopy, whose lateral resolution is limited to about 200 nm by the diffraction of light. With newer approaches that fall under the umbrella of super-resolution microscopy, this limit is circumvented and the resolution improved. The three main types of super-resolution microscopy include stimulated emission depletion (STED), which uses an excitation and a depletion laser to form a doughnut-shaped excitation pattern; structured illumination microscopy (SIM), which uses striped illumination patterns of changing orientation; and stochastic optical reconstruction microscopy (STORM), which activates photoswitchable fluorophores over time using a low-power laser. Numerous creative variations make for a wide variety of applications, with lateral resolution improved down to tens of nanometers. This article looks at how super-resolution microscopy advances are allowing researchers to peer into areas previously unseen.

STED imaging with live samples

At first, super-resolution techniques were mainly developed for fixed samples, which typically present fewer complications than using live tissue. However, steady technological improvements are easing the use of studying live tissue, and the lure of investigating new sample types has proved irresistible.

Super-resolution techniques like STED have become more compatible with live biological samples, such as in Leica Microsystems’ TauSTED approach. “TauSTED takes advantage of reduced laser power requirements for achieving super-resolution, thereby providing gentle illumination for extended imaging of delicate specimens using more frames, or of larger volumes using more planes,” says Haridas Pudavar, Product Performance Manager for Confocal Systems in the Life Science Division at Leica Microsystems Americas. The milder illumination protocols enable the use of more sensitive fluorophores and higher imaging speeds. “With the addition of TauSTED on our STELLARIS confocal platform, we are able to improve compatibility of STED technique with live samples, allowing super-resolved imaging of dynamic events for better understanding of biological processes.”

Indeed, the SARS-CoV-2 pandemic shone a brighter light on the importance of understanding viral infection mechanisms. A group from the Karolinska Institute in Sweden recently used STED super-resolution imaging, with the Leica SP8 STED 3X platform, to study how the influenza-A virus targets epithelial cells of the respiratory tract. “The use of optical super-resolution techniques over conventional electron microscopy techniques significantly simplified the sample-preparation methods for the scientists, allowing them to follow the infection mechanism for viral entry via endocytotic pathway,” notes Pudavar.

STORM imaging and spatial ‘omics

Super-resolution microscopy is instrumental in new spatial profiling techniques that allow detailed measurements of where and when particular genes are expressed and/or transcribed. “Spatial genomic and expression profiling techniques, such as MERFISH or FISSEQ, combined with localization microscopy, have been one of the fastest growing recent applications in super-resolution,” says Adam White, Product and Logistics Manager in Advanced Microscopy at Nikon Instruments. “With high frame rates and incredible spatial resolution, Nikon’s N-STORM localization super-resolution systems are a perfect platform for this type of application.”

Super-resolution microscopy is enabling subcellular localization studies of RNA expression that aren’t possible with traditional methods, as recently shown by Xiaowei Zhuang’s research group at Harvard University. “In a disease state, it may be possible that the amount of RNA expression of a particular gene may not change, but subcellular localization may be disrupted,” says White. “This sort of question has been studied on a more macro scale with older in situ techniques, but super-resolution allows scientists to see more subtle changes.”

White notes that advances in super-resolution microscopy are linked to developments in artificial intelligence. Software using AI-based tools can improve detection and analysis in super-resolution microscopy. “Nikon’s NIS-Elements software platform offers a variety of AI-based tools that can be applied to all of our super-resolution offerings,” he says. AI-based software can support live-cell super-resolution imaging by minimizing the required information for useful images. “This could mean collecting fewer frames to make a localization-based image, increasing overall frame rates, and decreasing phototoxicity,” White says, “Or it could mean shorter exposure times are needed to produce quality images.”

SIM for sub-organelle imaging in live samples

Structured illumination microscopy (SIM) is a super-resolution fluorescence microscopy technique for imaging live samples with resolution of about 120 nm laterally and 300 nm axially. The new ZEISS Elyra 7 with Lattice SIM² platform improves resolution in live samples (60 nm laterally, and 200 nm axially), with faster frame rates while maintaining a large field of view. “Lattice SIM² 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,” says Renée Dalrymple, Product Marketing Manager for Life Sciences Lattice Line at ZEISS Research Microscopy Solutions.

Image. Vibrant interactions between the endoplasmic reticulum (Calreticulin-tdTomato, magenta) and mitochondria (Tomm20-mEmerald, green) in a COS-7 cell can be observed with sub-100 nm resolution utilizing ZEISS Elyra 7 with Lattice SIM².

The Lattice SIM²’s improved spatial resolution in live samples, large field of view, fast frame rates, and multi-channel imaging, make experiments possible at the sub-organelle (< 100 nm) level. “There are many biological questions that reside below the 100 nm limit, and up until now researchers had to either limit themselves to imaging these structures in fixed samples, adapt their sample preparation procedures to fit the super-resolution technique, or both,” says Dalrymple. “The ability to observe structures in a live sample at these resolution levels will allow new questions to be answered,” such as whether the location of a protein of interest changes dynamically through the cell cycle or after a drug treatment, and how organelles interact in real time.

The creative tenacity of scientists seeking to improve resolution further, or to combine better resolution with techniques for localizing proteins or RNA in live samples, is still driving advances forward. “The most important advances in super-resolution techniques involve pushing capabilities to apply super-resolution into broader application spaces, to expand the biological questions that can be answered with the technology,” says Dalrymple. “Importantly, the true advances come when you can combine all of these abilities simultaneously.”

Hero image. Simultaneous super-resolution imaging of the endoplasmic reticulum (Calreticulin-tdTomato, magenta) and microtubules (EMTB-3xGFP, green) in a Cos-7 cell reveals highly dynamic interaction of these organelles down to 60 nm when imaged with ZEISS Elyra 7 with Lattice SIM².