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A new three-photon microscope developed at the Picower Institute at the Massachusetts Institute of Technology (MIT) can deliver rapid, short, low-power light pulses capable of reaching deep targets within the brain without causing functional disturbance or physical damage. It can then detect the resulting fluorescence emitted by cells with high efficiency and produce images with sharp resolution and a fast frame rate.
Three-photon microscopy allows scientists to see deeper into the brain because lower-energy, higher- wavelength photons are less susceptible than higher-energy, shorter-wavelength photons to being scattered by cellular molecules such as lipids.
Three-dimensional rendering of a sequence of 450 lateral three-photon images acquired with 2-μm increment from the visual cortex (layer 1 on the left to the subplate on the right). Green color represents GCaMP6s signal, and magenta color represents label-free THG signal generated in the blood vessels and myelin fibers in the white matter. Scale bar, 100 μm. Courtesy of Murat Yildirim et al.
The researchers optimized a custom three-photon microscope to image a vertical column of the cerebral cortex greater than 1 mm in depth in awake mice, using less than 20-mW average laser power. They measured physiological responses and tissue-damage thresholds to define pulse parameters and safety limits for damage-free three-photon imaging.
With the delivery of lower energy levels as their goal, the scientists developed a pre-chirp system to compensate for pulse broadening in the microscope and were able to reduce the pulse width to 40 fs on the sample. They implemented a delay line to increase the pulse repetition rate and improve the frame rate and designed the optics in the excitation and emission paths to maximize the efficiency of the microscope.
The team developed two label-free methods to characterize optical properties of the live mouse brain and used these properties to design the collection optics.
After validating that the optimized three-photon scope’s measurements agreed with those of two-photon microscopes and electrophysiology, the researchers used their microscope to observe neural activity in all cortical layers of awake, behaving mice, going more than 1 mm deep to see how the cells reacted to standard visual input.
The researchers were able to image labeled cortical layer neurons with less than 10-mW average laser power, and subplate neurons with less than 20-mW average laser power — nearly an order of magnitude less average power than that required to image layer 5 neurons via two-photon microscopy with wavefront shaping.
They observed that layer 5 neurons respond to a wide variety of orientations and had more spontaneous activity than cells in other layers and more connections to deeper parts of the brain. Layer 6 neurons had somewhat sharper orientation tuning than neurons in other layers, meaning they are more specific in their response to distinct orientations. Subplate neurons, located in the white matter below cortical layer 6 and characterized by this team for the first time, showed low visual responsivity and broad orientation selectivity. This finding was of interest, the researchers said, in that many scientists had believed that the subplate neurons were mostly active during development.
“We were motivated to show what we could do with three-photon microscope technology for an animal in an awake condition so we could ask important questions of neuroscience,” researcher Murat Yildirim said. “You could think you have the best microscope in the world, but until you ask those questions you don’t know what results you are going to get.”
“By optimizing the optical design and other features for parameters for making measurements in the live brain, we were able to actually make novel discoveries that were not possible before,” said professor Mriganka Sur.
The research was published in Nature Communications (https://doi.org/10.1038/s41467-018-08179-6).
Plunge all the way through the visual cortex from the surface to the subplate beneath in this movie made using a new three-photon microscope developed at the Picower Institute for Learning and Memory. Green indicates neural activity while magenta labels blood vessels and white matter fibers. Courtesy of Murat Yildirim et al.READ MORE