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Researchers at MIT have developed an inexpensive way of using ordinary light microscopes to look at virus particles.1 Normally, as viruses are objects on the nanometer length scale, they are generally too small to image directly with optical microscopy techniques. However, with a combination of expansion microscopy and sample preparation techniques, a standard light microscope is now sufficient to see individual viruses.
Expansion Microscopy
The diffraction limit determines the maximum possible resolving power and this limit scales inversely with wavelength. To achieve the highest possible resolution, using shorter wavelengths is preferable and is why some of the key imaging techniques in structural biology have been based on X-ray radiation.
What this inverse scaling means for the use of optical microscopes is that the diffraction limit is on the order of a few hundred nanometers and the resulting resolution is too poor to image many nanoscale structures. Some of the largest viruses have diameters and lengths on the order of a few hundred nanometers, but many other viral particles are less than a hundred nanometers in diameter for which this level of resolving power is insufficient to identify individual particles or determine various structural features.
To address this, super-resolution microscopy has become an incredibly active area of research with new microscopy schemes and techniques being regularly developed. However, such methods are often very technically demanding; many rely on the use of suitable fluorophores and the data can be complex to process and interpret.
Expansion microscopy takes another approach to recover sub-diffraction limit information on a sample. Rather than trying to improve the resolving power of the microscope, or use sampling and statistics, it instead relies on expanding the sample itself to enlarge it and make it easier to image. Since its development in 2015, several sample preparation protocols have been created by many different research groups that are compatible with different cell or sample types.2
Sample preparation works by using a polymer matrix that swells in water to pull apart the cells equally in all directions. With the use of a suitable matrix and careful injection, it is possible to expand the cell to a size that is greater than the diffraction limit, without distorting the cell structure.
However, finding matrices that manage a truly isotropic expansion is challenging. The potential resolution of conventional expansion microscopy had been hampered by the swelling process of the polymers on the introduction of water not being truly uniform and resulting in small distortions of the original structure. One of the key developments in this recent work is the discovery that tetra-gel polymers can be used to create more predictable and lattice-like structures for the expanded systems.
Resolution Boosts
With the new gels (with their improved uniformity), the team achieved an effective resolution of 10 nm, making it possible to image the herpes simplex virus type 1 (HSV-1). Normally, viral imaging is performed using electron microscopy, which achieves spatial resolutions as high as 50 pm and magnification factors exceeding one million.
However, electron microscopes are incredibly costly and bulky pieces of equipment and there is a huge demand for cheaper imaging techniques with better resolution than standard optical microscopes.
The ability of expansion microscopy to now visualize samples as small as viruses using more standard optical microscopes has very important potential in making nanoimaging more widely accessible. The sample preparations for expansion microscopy are inexpensive, particularly when compared to the costs associated with microscopy equipment with similar resolutions.
Scientific Possibilities
As well as making imaging of nanostructures more affordable, this improvement in the uniformity of the polymer matrix for the expansion process will enable deep examination of cells. The team also hopes this method can be enhanced so that single molecules can be imaged, opening up a wealth of possibilities for examining biomarkers and signaling processes.
The current tetra-gels have also proved suitable for use with human kidney and mouse brain cells. Moving the methodology towards single-molecule imaging will indicate the need for smaller (10 to 20 nm) labels or finding techniques to attach the labels after the expansion process has occurred.
The team is currently investigating other polymer species suitable for use as matrices and whether modifications of the tetra-gels could further enhance the effective resolving power of expansion microscopy.
References and Further Reading
- Gao, R., Yu, C. C., Gao, L., Piatkevich, K. D., Neve, R. L., Munro, J. B., … Boyden, E. S. (2021). A highly homogeneous polymer composed of tetrahedron-like monomers for high-isotropy expansion microscopy. Nature Nanotechnology. https://doi.org/10.1038/s41565-021-00875-7
- Bellow, S. and Florindo, C, (2020) What is expansion microscopy? https://andor.oxinst.com/learning/view/article/what-is-expansion-microscopy-getting-the-most-from-expanded-samples, accessed 1 May 2021
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