Two-Photon Holographic Mesoscope Probes Activity Across Large Brain Regions

Recent advancements in two-photon (2P) mesoscopes have extended the field of view (FOV) for deep brain imaging, making it possible to image brain regions as large as 25 mm². Until now, though, mesoscopes could only measure neural signals; they could not stimulate separated regions of the brain.

To enable investigators to probe the inter-areal communication signals central to brain function, a team at the University of California, Berkeley (UC Berkeley) combined its aeroPULSE FS50 ultrafast fiber laser for photostimulation with a 2P mesoscope from Thorlabs Inc. that has a 5- × 5-mm FOV. The all-optical platform from UC Berkeley can stimulate a 1-mm² area while recording activity in a 25-mm² region of the brain.

Researchers at UC Berkeley developed a platform that combines the aeroPULSE FS50 ultrafast fiber laser for photostimulation with a 2P mesoscope to provide the platform with a larger FOV. The new system will allow neural activity to be observed across various scales and species and will enable scientists to better understand feed-forward and feed-back processing in distributed neural circuits. Courtesy of the University of California, Berkeley.

By providing precise control and monitoring of neural activity, the 2P holographic mesoscope could provide fresh insight into brain function, synchronization, and communication between brain areas. It has the potential to enhance brain-machine interfaces by optimizing signal transmission with single-cell resolution and millisecond precision. This could be used to improve, for example, prosthetic limb control.

The wide FOV of the mesoscope makes it suitable for studies across various scales and species, including larger-brained species. By providing the means for whole new classes of experiments, the mesoscope could enable explorations of theories of brain function that were previously beyond reach.

According to the researchers, the 2P holographic mesoscope is the first technology to offer targeted photostimulation along with simultaneous large-scale recording of neural activity across several cortical areas in a nominal 25-mm² FOV. It simultaneously reads and writes neural activity patterns across large regions of the brain with single-cell resolution, providing insight into feed-forward and feed-back processing in distributed neural circuits, which could have a substantial impact on neuroscience research and potential therapeutic applications.

To achieve high holographic resolution optogenetics in a 2P mesoscope, the researchers first had to address the low numerical aperture of the optical system and the need for mechanical rotation of the entire imaging system for accessing lateral brain areas. Also, since existing commercial 2P mesoscopes are not designed for the integration of holographic systems, the new tool required a significant redesign of the mesoscope build.

The compact 3D holographic module uses temporal focusing to confine excitation axially. To preserve all translational axes of the system, the team built the holographic module on an extension breadboard attached to the movable main frame of the microscope.

Sketch of 2P holographic mesoscope optical setup. EOM: electro-optic modulator; BE: beam expander; P: prism; QWP: quarter waveplate; Dn: dichroic mirror; BS: polarization beamsplitter; VC: voice coil; SLM: spatial light modulator. M6 mirror in the original mesoscope design has been replaced with the D2 dichroic. Courtesy of the University of California, Berkeley.

The femtosecond pulses from the aeroPULSE FS50 ultrafast fiber laser enter collinearly with the imaging laser at 920 nm. Both laser beams are separated using a long-pass, dichroic beamsplitter. The optics required for 3D Sparse Holographic Optogenetics with Temporal Focusing (3D-SHOT) are positioned on the breadboard, which moves along with the entire scope’s rotation. The two beams are recombined using another dichroic before entering the objective path.

The new platform does not compromise the wide FOV of the mesoscope or the 4-axis movement capabilities of the 2P random access mesoscope system that are required to execute various neurobiological experiments.

The researchers tested and validated the optical capabilities of the new platform to measure and re-create highly specific patterns of activity in the brain within a large FOV. They demonstrated the system’s ability to co-activate user-defined groups of neurons, selected based on their spatial or functional properties, while simultaneously recording neural activity from several downstream areas. The researchers could identify individual downstream neurons that displayed time-locked excitatory or inhibitory responses hundreds or even thousands of microns away. The results showed that the holographic 2P mesoscope could be used to probe how both local and long-range functional interactions relate to specific neuronal computations.

The mesoscope’s nominal 5- × 5-mm FOV; its fast, remote focusing system for FOV curvature correction and multiplane imaging; and its four automated axes of motion for positional flexibility make it a powerful tool for studying inter-areal communication.

The research is awaiting publication and is available in preprint on bioRxiv (www.doi.org/10.1101/2023.03.02.530875).