Microscope Takes 3-D Images From Inside Moving Subjects

A new kind of microscope enables rapid 3-D imaging of living and moving samples, potentially offering advantages over laser-scanning confocal, two-photon and light-sheet microscopy.

Developed by Columbia University professor Dr. Elizabeth Hillman and graduate student Matthew Bouchard, swept confocally aligned planar excitation (SCAPE) microscopy involves simplified equipment and does not require sample mounting or translation. The microscope scans a sheet of light through the sample, making it unnecessary to position the sample or the microscope’s single objective.

“The ability to perform real-time, 3-D imaging at cellular resolution in behaving organisms is a new frontier for biomedical and neuroscience research,” Hillman said. “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.”

SCAPE yields data equivalent to conventional light-sheet microscopy, but using a single, stationary objective lens; no sample translation; and high-speed 3-D imaging. This unique configuration permitted volumetric imaging of cortical dendrites in the awake, behaving mouse brain. Courtesy of Elizabeth Hillman/Columbia Engineering.

Conventional light-sheet microscopes use two orthogonal objectives and require that samples be in a fixed position. Confocal and two-photon microscopes can image a single plane within a living sample, but cannot generate 3-D images quickly enough to capture events like neurons firing.

SCAPE does have one drawback: Using a 488-nm laser, it cannot penetrate tissue as deeply as two-photon microscopy.

The new technique could be combined with optogenetics and other tissue manipulations, the researchers said. It could also be used for imaging cellular replication, function and motion in intact tissues, 3-D cell cultures and engineered tissue constructs; as well as imaging 3-D dynamics in microfluidics and flow-cell cytometry systems.

Hillman next plans to explore clinical applications of SCAPE, such as video-rate 3-D microendoscopy and intrasurgical imaging.

Funding for the project came from the National Institutes of Health, Human Frontier Science Program, Wallace H. Coulter Foundation, Dana Foundation and the U.S. Department of Defense.

The research was published in Nature Photonics (doi:10.1038/nphoton.2014.323).

For more information, visit www.engineering.columbia.edu.