Scientists at Purdue University and collaborators say they have developed a new technology that allows the visualization of nanoscale structures inside whole cells. The team’s technology is described in a paper “Three-dimensional nanoscopy of whole cells and tissues with in situ point spread function retrieval” in Nature Methods, and a video showing an animated 3D super-resolution.

“Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets, and therefore allows their locations to be pinpointed with precision that achieves the Cramér-Rao lower bound and uncompromised fidelity,” write the investigators.

“We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures, from mitochondrial networks and nuclear pores in mammalian cells to amyloid-β plaques and dendrites in brain tissues and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extracellular targets deep inside tissues.”

“Our technology allows us to measure wavefront distortions induced by the specimen, either a cell or a tissue, directly from the signals generated by single molecules – tiny light sources attached to the cellular structures of interest,” said Fang Huang, PhD, an assistant professor of biomedical engineering in Purdue’s College of Engineering. “By knowing the distortion induced, we can pinpoint the positions of individual molecules at high precision and accuracy. We obtain thousands to millions of coordinates of individual molecules within a cell or tissue volume and use these coordinates to reveal the nanoscale architectures of specimen constituents.”

“During three-dimensional super-resolution imaging, we record thousands to millions of emission patterns of single fluorescent molecules,” added Fan Xu, PhD, a postdoctoral associate in Huang’s lab and a co-first author of the publication. “These emission patterns can be regarded as random observations at various axial positions sampled from the underlying 3D point-spread function describing the shapes of these emission patterns at different depths, which we aim to retrieve. Our technology uses two steps: assignment and update, to iteratively retrieve the wavefront distortion and the 3D responses from the recorded single molecule dataset containing emission patterns of molecules at arbitrary locations,” according to the researchers.

“This advancement expands The Purdue technology allows finding the positions of biomolecules with a precision down to a few nanometers inside whole cells and tissues and therefore, resolving cellular and tissue architectures with high resolution and fidelity the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extra-cellular targets deep inside tissues,” said Donghan Ma, PhD, a postdoctoral researcher in Huang’s lab and a co-first author of the publication. “This newfound capacity of visualization could allow for better understanding for neurodegenerative diseases such as Alzheimer’s, and many other diseases affecting the brain and various parts inside the body.”

Other members of the research team include Gary Landreth, PhD, a professor from Indiana University’s School of Medicine; Sarah Calve, PhD, an associate professor of biomedical engineering in Purdue’s College of Engineering (currently an associate professor of mechanical engineering at the University of Colorado Boulder); Peng Yin, PhD, a professor from Harvard Medical School; and Alexander Chubykin, PhD, an assistant professor of biological sciences at Purdue.

“This technical advancement is startling and will fundamentally change the precision with which we evaluate the pathological features of Alzheimer’s disease,” Landreth said. “We are able to see smaller and smaller objects and their interactions with each other, which helps reveal structure complexities we have not appreciated before.”

Calve pointed out that the technology is a step forward in regenerative therapies to help promote repair within the body. “This development is critical for understanding tissue biology and being able to visualize structural changes.,”

Chubykin, whose lab focuses on autism and diseases affecting the brain, said the high-resolution imaging technology provides a new method for understanding impairments in the brain.

“This is a tremendous breakthrough in terms of functional and structural analyses,” Chubykin said. “We can see a much more detailed view of the brain and even mark specific neurons with genetic tools for further study.”

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