Biology at the speed of life
Image: SCAPE 2.0 dual-colour 3D imaging sheds new light on freely moving C. elegans [Venkatakaushik Voleti/Hillman lab/Columbia’s Zuckerman Institute]
Researchers from the University of Columbia, US, have unveiled a new version of 3D SCAPE – SCAPE 2.0 - using the microscope to image previously unseen details of living creatures.
By combining a high-speed camera system with the latest 'Swept Confocally Aligned Planar Excitation' microscope design, the researchers have achieved high-resolution volumetric imaging at over 300 volumes per second and more than 1.2 GHz pixel rates.
From neurons firing inside a wriggling worm to the 3D dynamics of the beating heart of a fish embryo, the researchers reckon they can capture detail with far superior resolution and at speeds up to 30 times faster than their original demonstration.
SCAPE 2.0 dual-colour 3D imaging of zebrafish embryo and C. elegans. [Venkatakaushik Voleti/Hillman lab/Columbia’s Zuckerman Institute (C. elegans), Venkatakaushik Voleti/Kimara Targoff/Hillman lab/Columbia’s Zuckerman Institute (zebrafish)]
"The processes that drive living things are dynamic and ever-changing, from the way an animal's cells communicate with one another, to how a creature moves and changes shape," says Professor Elizabeth Hillman from Columbia's Mortimer B. Zuckerman Mind Brain Behavior Institute.
"The faster we can image, the more of these processes we can see, and imaging fast in 3D lets us see the whole biological system, rather than just a single plane, offering a clear advantage over traditional microscopes," she adds.
Hillman and colleagues first introduced SCAPE - Swept Confocally Aligned Planar Excitation - microscopy four years ago to image living tissues at high speeds.
"Most microscopes that image living samples scan a small spot of laser light around the sample, but the point-scanning approach is slow, giving only a short time to see each spot," points out Dr Venkatakaushik Voleti from the Hillman Lab. "Our system uses an oblique, or angled, sheet of light to illuminate an entire plane within the sample, and then sweeps this light sheet across the sample to form a 3D image."
As Voleti explains SCAPE can rapidly move the light sheet and focus the image of this sheet back to a stationary camera using a single moving mirror, making it simple and fast.
In addition, SCAPE is gentle on living samples because it uses only a fraction of the light that point-scanning microscopes would need to get images at comparable speeds.
The system achieves this through a single, stationary objective lens, opening up space for a wide array of samples compared to conventional light-sheet microscopes that require complex sample chambers surrounded by many lenses.
"People are often surprised at how compact, simple and easy to use SCAPE is," says Hillman.
"In our new paper, we show how SCAPE 2.0 can track individual neurons firing in a whole animal as it crawls around, giving us a new window into how neural activity guides behaviour," she adds.
Hillman recently partnered with paediatric cardiologist Professor Kimara Targoff from Columbia's Vagelos College of Physicians and Surgeons to use SCAPE 2.0 to study how the heart develops.
Targoff's lab uses zebrafish as an animal model to decipher the genetic mutations that can cause heart malformations in the embryo.
"The problem with imaging the beating heart is that it beats fast, changing its shape as blood flows through it in a wide range of directions," says Targoff. "With SCAPE 2.0, we can image the zebrafish embryo's beating heart in 3D and in real-time, allowing us to see how calcium signals sent between heart cells cause the heart wall to contract, or how red blood cells flow through the heart's valves beat after beat.”
“Using this knowledge, we can track how a particular genetic mutation affects normal heart development in an environment that most closely recapitulates the heart's natural state," he adds.
To image at high speeds, Hillman and colleagues used a HiCAM Fluo camera from Lambert Instruments.
This camera was used to capture images at more than 18,000 frames per second in the zebrafish embryo's beating heart.
This new configuration opened the door to recording individual neurons firing in a freely moving C. elegans worms, giving the first view of an animal's complete nervous system in action.
SCAPE 2.0's other upgrades include improved light efficiency, a larger field of view and much improved spatial resolution, enabling researchers to image samples created using tissue clearing and tissue expansion at record speeds.
SCAPE 2.0 imaging of a mCUBIC cleared mouse brain. The 8.4 x 9 x 0.4 mm xyz volume was acquired within 4 minutes with 1 x 1.37 x 1.14 micron per pixel sampling density. Rendered using Imaris (Bitplane). Sample provided by Pavel Osten Lab. [Kripa Patel/Hillman lab/Columbia’s Zuckerman Institute].
Using these methods, researchers could study structures and connections deep inside intact samples, from whole mouse brains to tumours and human biopsies.
"The limitations of tools and techniques often constrain what scientists think they can study," says Hillman. "SCAPE 2.0 opens up a new landscape of things that we can see. I hope our new results will inspire scientists to think of what new questions can be asked, and what new avenues of scientific discovery we can explore next."
Hillman is currently working with Leica Microsystems, which has licensed SCAPE and is currently developing a commercial version of the system.
Research is published in Nature Methods.