Elizabeth Hillman, associate professor of biomedical engineering at Columbia University Medical Center (CUMC), has developed a new 3D microscope prototype dubbed "SCAPE" (Swept Confocally Aligned Planar Excitation Microscopy), which requires no mounting of samples or other special preparation, and is capable of imaging freely moving living samples at speeds 10 to 100 times faster than current laser-scanning microscopes.

SCAPE is a variation on light-sheet microscopy, however, unlike conventional light-sheet microscopes that utilize a pair of cumbersomely-positioned objective lenses, SCAPE uses a single-objective lens, with a light sheet sweeping across the field of view to capture 3D images without moving the sample or the objective.

"This combination makes SCAPE both fast and very simple to use, as well as surprisingly inexpensive," said Professor Hillman. "We think it will be transformative in bringing the ability to capture high-speed 3D cellular activity to a wide range of living samples."

In developing SCAPE, Professor Hillman worked with graduate student Matthew Bouchard to repurpose an old polygonal mirror she had in her lab as the integral component. After several years of experimentation, Hillman and Bouchard were finally able to captured 3D images in great detail.

"It wasn’t until we built it that we realized it was a light-sheet microscope," said Professor Hillman. "It took us a while to realize how versatile the imaging geometry was, how simple and inexpensive the layout was – and just how many problems we had overcome."

The Columbia team has already imaged an amazing array of subjects, including zebrafish larvae, where the SCAPE system was able to peer through the entire creature. Tracking these miniscule creatures in 3D at high speeds as they swim around has meant that SCAPE has been able to image not only cellular structure and function, but real-time behavior as well.

SCAPE can also be used to capture neurons flashing in a living brain using fluorescent protein techniques, as well as combined with optogenetics and other tissue manipulations performed as imaging takes place. This is because, unlike other 3D imaging systems, SCAPE doesn't need to constantly readjust either the imaging objective lens or the sample to create a 3D image, and the subject is able to move freely throughout the imaging process.

"The ability to perform real-time 3D imaging at cellular resolution in behaving organisms is a new frontier for biomedical and neuroscience research," said Professor Hillman. "With SCAPE, we can now image complex, living things, such as neurons firing in the rodent brain, crawling fruit fly larvae, and single cells in the zebrafish heart while the heart is actually beating spontaneously – this has not been possible until now."

Even though confocal and two-photon microscopy can capture images in single planes inside a living sample, capturing events like neurons firing has been largely impossible due to the fact that these imaging systems are unable to acquire enough of these layers at a fast enough rate to create a 3D image. And though SCAPE doesn't yet possess the penetration depth capability of standard two-photon microscopy, the CUMC team has still been able to use the system to witness neurons firing in 3D neuronal dendritic trees (branched projections of a neuron) in the shallow top layers of a mouse brain.

Many further applications for SCAPE are envisaged by the CUMC researchers, including capturing images of cellular replication, function, and motion in living tissue, and imaging 3D cell cultures, as well as capturing 3D images of dynamics in microfluidic and flow-cell cytometry structures. These areas may be particularly enhanced by SCAPE, according to the team, as these are applications where molecular biology imaging methods have fallen behind the latest tools and techniques.

Professor Hillman also believes that future generations of SCAPE currently in development will provide even faster speeds, better resolution, increased sensitivity, and further penetration depth. All of which stands SCAPE in good stead to be harnessed in clinical applications like video-rate 3D microendoscopy and intrasurgical imaging.

Working with a wide range of collaborators as a member of the Zuckerman Institute and the Kavli Institute for Brain Science at Columbia, Professor Hillman is also seeing other researchers starting to use the SCAPE system in their research on brain function.

"Deciphering the functions of brain and mind demands improved methods for visualizing, monitoring, and manipulating the activity of neural circuits in natural settings," said Thomas M. Jessell, co-director of the Zuckerman Institute. "Hillman’s sophistication in optical physics has led her to develop a new imaging technique that permits large-scale detection of neuronal firing in three-dimensional brain tissues. This methodological advance offers the potential to unlock the secrets of brain activity in ways barely imaginable a few years ago."

Professor Hillman’s new technology has been made available for licensing from Columbia Technology Ventures and CUMC says it has attracted interest from a range of companies. A patent relating to this technology has also been issued to its inventors.

The research has been published in the journal Nature Photonics

The short video below, complete with pumpin' soundtrack, shows actual images captured with SCAPE.

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