Mesoscopic imaging method offers unprecedented cell views

A new microscopy technique developed by a team at Johns Hopkins University (Baltimore, MD) offers a remarkable and more complete view than ever before of cell interaction in its most natural state. This could ultimately lead to notable advancements including new, more effective treatments for diseases.

“Optical microscopy is fundamentally important in biomedical research,” says lead researcher Ji Yi, an assistant professor with the Johns Hopkins Wilmer Eye Institute and the Biomedical Engineering Department at the Whiting School of Engineering. “Understanding cellular structures and how they interact is crucial to understanding life as we know it.”

Their technique—mesoscopic oblique plane microscopy with a diffractive light sheet, a type of light-sheet microscopy that involves a sheet of laser light to illuminate a thin sample labeled with fluorescent markers—captures up to 3X more resolvable image points in 3D than is possible with conventional microscopy systems (see video).

Realizing large-scale dynamic connectivity within a living organism requires volumetric imaging over a large field of view at biologically relevant speed and resolution, the researchers say in their study, published in Optica. But with conventional microscopy systems, there is a tradeoff between field of view and axial resolution.

“This makes observation of highly dynamic processes at cellular resolution in 3D across mesoscopic scales quite challenging,” Yi says. “Our technique overcomes this limitation and allows much higher resolution over a larger field of view.”

As part of their study, the mesoscopic oblique plane microscopy method was used to image 3- to 4-mm-long zebrafish larvae, which captured whole-body volumetric recordings of its neural activity for a more comprehensive view of neural circuits throughout its entire central nervous system. The researchers captured recordings of the larvae’s blood flow dynamics with 3D cellular resolution, enabling, for the first time, tracking of single cells within an entire circulation system.

“Augmenting the illumination angle of a diffractive light sheet with a transmission grating optical component improved the axial resolution approximately six-fold over existing methods and approximately two-fold beyond the diffraction limitation of the primary objective lens,” Yi says. “This, in turn, enhanced depth sectioning and resolution. Demonstrating a larger field of view and higher resolution allows volumetric imaging of 3D cellular structures with a single scan.”

From there, the researchers can perform imaging at the mesoscopic scale, viewing biological systems in a larger context, while maintaining high cellular resolution throughout all dimensions. “This is just the beginning,” Yi says.

Yi’s team also plans to explore 4D information from brain or whole biological model systems to better understand some fundamental questions.

“How does the brain process the myriad of sensory input (light, sound, touch) to decide actions or focus attention? What are the critical cellular components for specific biological functions?” Yi says. “Designing rigorous experiments by combining advanced imaging and biological tools, for example, gene editing, sequencing, and transcriptomics, to answer these questions is exciting and what we are striving for.”

Their microscopy method could target neurodegeneration diseases such as Parkinson’s, multiple sclerosis, glaucoma, and others that involve dysfunction of neural systems in the brain or eye.

“4D dynamics imaging at the cellular level could provide useful insight into pathologies, quantify phenotypes, and assist therapeutic developments to combat these conditions,” Yi says. “The advance in 4D dynamics imaging reveals a large-scale time sequence that can be impactful for other diseases, too, such as using patient-derived organoids (i.e., mini organs in a dish) to study cancers.”