Advancing our understanding of the human brain will require new insights into how neural circuitry works in mammals, including laboratory mice. These investigations require monitoring brain activity with a microscope that provides resolution high enough to see individual neurons and their neighbors.
Two-photon fluorescence microscopy has significantly enhanced researchers’ ability to do just that, and the lab of Spencer LaVere Smith, an associate professor in the Department of Electrical and Computer Engineering at UC Santa Barbara, is a hotbed of research for advancing the technology. As principal investigator on the five-year, $9 million NSF-funded Next Generation Multiphoton Neuroimaging Consortium (Nemonic) hub, which was born of President Obama’s BRAIN Initiative and is headquartered at UCSB, Smith is working to «push the frontiers of multi-photon microscopy for neuroscience research.»
In the Nov. 17 issue of Nature Communications, Smith and his co-authors report the development of a new microscope they describe as «Dual Independent Enhanced Scan Engines for Large Field-of-view Two-Photon imaging (Diesel2p).» Their two-photon microscope provides unprecedented brain-imaging ability. The device has the largest field of view (up to 25 square millimeters) of any such instrument, allowing it to provide subcellular resolution of multiple areas of the brain.
«We’re optimizing for three things: resolution to see individual neurons, a field of view to capture multiple brain regions simultaneously, and imaging speed to capture changes in neuron activity during behavior,» Smith explained. «The events that we’re interested in imaging last less than a second, so we don’t have time to move the microscope; we have to get everything in one shot, while still making sure that the optics can focus ultrafast pulses of laser light.»
The powerful lasers that drive two-photon imaging systems, each costing about $250,000, deliver ultrafast, ultra-intense pulses of light, each of which is more than a billion times brighter than sunlight, and lasts 0.0001 nanosecond. A single beam, with 80 million pulses per second, is split into two wholly independent scan engine arms, enabling the microscope to scan two regions simultaneously, with each configured to different imaging parameters.
In previous iterations of the instrument, the two lasers were yoked and configured to the same parameters, an arrangement that strongly constrains sampling. Optimal scan parameters, such as frame rate and scan region size, vary across distributed neural circuitry and experimental requirements, and the new instrument allows for different scan parameters to be used for both beams. The new device, which incorporates several custom-designed and custom-manufactured elements, including the optical relays, the scan lens, the tube lens and the objective lens, is already being broadly adopted for its ability to provide high-speed imaging of neural activity in widely scattered brain regions.
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Materials provided by University of California — Santa Barbara. Original written by James Badham. Note: Content may be edited for style and length.