What is being achieved through super-resolution microscopy?

Andrew Williams explores the rise of super-resolution microscopy and finds out what it’s being used to accomplish

Over the past few months, a string of universities and research institutes around the world have announced breakthroughs in the use of microscope technology for animal and human brain imaging. So what have been some of the key recent developments? And what are the benefits of such innovative approaches to microscopy?

Optical biopsy

One of the most interesting recent developments has been at the Barrow Neurological Institute in Arizona, USA, where a team of researchers has employed innovative confocal laser endomicroscopy (CLE) to collect real-time images from experimental brain tumours in mice, as well as from human brain tumours during their surgical removal and from human brain tumour biopsies. As part of the research, mice were injected with fluorescein sodium (FNa) before imaging and human patients received FNa intraoperatively, enabling the surgical team to view images immediately in the operating room.

In the ongoing quest for better imaging quality, super resolution optical microscopy – particularly a version known as stochastic optical reconstruction microscopy (STORM) – is emerging as a key enabling tool. In recognition of this fact, a growing number of research centres around the world now use the technology, including the University of Pittsburgh and ETH Zurich in Switzerland. So, what exactly is STORM? What microscopy technology does it use? What are the main advantages of using STORM technology for imaging? And what are the current and potential applications?

Breaking the limits

According to some observers, the emergence of super resolution optical microscopy is helping to enable a step change in the way that scientists approach research across a number of disciplines – and is rapidly becoming a vital tool in the laboratory. As Dr Dorothea Pinotsi, staff scientist - Single-Molecule, Super Resolution and Spectroscopy Applications in the ScopeM Scientific Centre for Optical and Electron Microscopy at ETH Zürich, explains, optical super-resolution microscopy techniques have ‘revolutionised’ research in life sciences. In collaboration with other members of the team at ETHZ, Pinotsi has successfully deployed a variety of innovative technologies to further the study of Parkinson’s and Alzheimer’s diseases – and she continues working in the fields of super-resolution microscopy and spectroscopy for physical, chemical and life science applications.

“These techniques have broken the limits of conventional microscopy imposed by the diffraction of light and allow us to ‘see’ molecular mechanisms and biological processes with nanometre resolution, even in live specimens,” she says.

“One such technique is STORM – with which we can achieve unprecedented spatial resolution, for a light microscopy technique, down to 20-30nm,” she adds.

Nuclear membrane

Another of the most interesting recent developments in this field is at the University of Pittsburgh, where a team of researchers has employed STORM microscopy to investigate the structure of the nuclear envelope – which is made up of the inner and outer nuclear membrane and a protein meshwork, known as the nuclear lamina, that lies just beneath the inner nuclear membrane.

As Dr Quasar Padiath, associate professor in the Department of Human Genetics at Pitt Graduate School of Public Health, explains, the nuclear lamina is composed of distinct proteins called lamins and is found in all metazoan cells. In addition to playing a critical role in regulating the shape and structural integrity of the nuclear envelope, it also has important functions in regulating gene expression through chromatin interactions and integrating cytoskeletal dynamics within the cell. In vertebrates, there are two major classes of lamins, the A and the B type. While previous research has suggested that the A and B type lamins form independent networks, whether they exhibit any distinct spatial organisation or were randomly distributed was unknown.

“We attempted to elucidate the organisation of the distinct lamin proteins using STORM microscopy, which provides a significantly higher resolution than conventional light microscopy, while allowing for the simultaneous identification of multiple molecular species that are possible with other high-resolution imaging techniques such as electron microscopy (EM),” says Padiath.

“STORM is based on single molecule localisation approach. In this work, we used both a custom-built STORM system and a commercially available STORM system from Nikon (N-STORM). This approach relies on turning ‘on’ a small subset of densely labelled fluorescent emitters, followed by precise localisation of their central positions at the precision of 10-20nm – a process that is repeated for thousands of frames to reconstruct the final super-resolved image,” he adds.

Molecular discrimination

Amongst other key findings, Padiath explains that, using STORM, his teams’ studies revealed that the A and B type lamins form concentric but overlapping networks, with lamin B1 forming the outer concentric ring located adjacent to the INM. These results were consistent across multiple cells lines, such as mouse and human fibroblasts and human He La cell lines. For Padiath, the main advantage of using STORM is its ‘superior resolution,’ with the ‘most surprising’ discovery enabled by the technology being the ‘spatial separation between lamin B1 and lamin A/C at a scale of ~15-20nm.’ Such precise localisation of the two lamin species was achieved primarily by employing aberration-free two-colour super-resolution imaging.

Padiath also explains that the use of STORM microscopy was also important in enabling he and his research team to discover that the more peripheral localisation of lamin B1 is mediated by its carboxyl-terminal farnesyl group– and that ‘Lamin B1 localisation is
also curvature- and strain-dependent, while the localisation of lamin A/C is not.’ STORM was also used to demonstrate that lamin B1’s outer-facing localisation stabilises nuclear shape by restraining outward protrusions of the lamin A/C network.

“In summary, we have identified a model for the spatial organisation of the nuclear lamina based on two critical principles: firstly, that lamin B1 forms a looser, outer meshwork facing the nuclear membrane, while lamin A/C forms a tighter, inner meshwork facing the nucleoplasm and, secondly, that lamin B1’s meshwork is more curvature and strain-responsive than lamin A/C’s meshwork, which affects its localisation in tightly curved structures,” he says.

“Imaging at two different wavelengths often suffers from chromatic aberration and a complete correction of chromatic aberration is often difficult to achieve, especially when the imaging target is farther away from the coverslip surface. We used the same reporter dye in the activator-reporter dye pairs in two-colour STORM imaging to eliminate any chromatic aberration,” he adds.

In the future, Padiath reveals that he and his team plan to make use of STORM to further elucidate the organisation and structure of the nuclear lamina in numerous diseases that are associated with this structure and to ‘understand how the nuclear lamina is altered with age.’

“STORM is best suited for applications where high-resolution imaging needs to be coupled with molecular discrimination. The ability to distinguish multiple molecular species by labelling them with fluorophores of different ‘colours’ makes this technique critical in visualiing complex cellular structures at the nanometre scale,” he adds.

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