M.Sc Thesis

M.Sc StudentMayblum Tom
SubjectAdvances in Spatiotemporally-Focused Bidirectional
Multiphoton Neuronal Interfaces
DepartmentDepartment of Biomedical Engineering
Supervisors DR. Inbar Brosh
PROF. Shy Shoham


Multiphoton optogenetics, the use of multi-photon excitation to precisely activate optogenetic probes, is currently the only way to achieve cellular-resolution optical stimulation and recording in three-dimensional, scattering brain tissue. Despite major advancements, this approach still has fundamental limitations: in imaging applications, standard single-point scanning has a trade-off between the volumetric imaging rate and the accessible field-of-view (FOV), while in stimulation applications, achieving an effective multiphoton excitation of large membrane patches is still nontrivial. Temporal focusing (TF) is a powerful solution which can be used to overcome these challenges. Here, we address fundamental aspects in the design of TF multiphoton optogenetics systems which are used for rapid, high spatial resolution imaging, and for generating temporally focused flexible three-dimensional patterns.

First, we implemented and examined the characteristics of two different solutions for simultaneously stimulating large patches of membrane. The first solution utilizes computer generated holography (CGH) to produce a cell-matched light patch consisting of numerous diffraction limited spots. While the axial sectioning of multiphoton holographic patches increases linearly with the patch’s lateral dimension, quickly exceeding cellular dimensions, the introduction of TF limits this effect, providing a regime with suitably cell matched patches. In the second solution, we employ simultaneous spatiotemporal focusing (SSTF) in a unique optical system design, which is used to shape a focal spot that approximately fits the size of a target nerve cell in both the lateral and axial dimensions.

Next, we examined the performance in scattering tissue of a hybrid imaging system combining a standard two-photon laser scanning microscope (TPLSM) with a TF setup. We demonstrate the registration of high spatial resolution volumetric images with high temporal resolution volumetric activity patterns acquired by the two modalities and the ability to observe spontaneous neuronal activity patterns in volumes over 150µm deep inside acute mouse brain slices.

Our work demonstrates the capability of spatiotemporal focusing systems to address fundamental issues in multiphoton optogenetic applications and to enable flexible control over spatial-temporal patterns, providing novel tools for bidirectional neuronal interfacing. These systems can be easily integrated into the optical path of multiphoton microscopes.