M.Sc Thesis

M.Sc StudentPaluch Shir
SubjectOptical Probing of Three-Dimensional Neuronal Networks
DepartmentDepartment of Biomedical Engineering
Supervisor PROF. Shy Shoham
Full Thesis textFull thesis text - English Version


Spatio-temporal neuronal activity patterns are the fundamental representation of information within the nervous system. Recent developments in genetically encoded light sensitive proteins for imaging and stimulation make opto-physiology methods optimally suited for non-contact probing of three dimensional networks activity patterns. Multiphoton excitation methods and temporal focusing are commonly used for axially confined excitation deep inside 3D scattering tissues, using ultra-short amplified lasers operating at higher infrared wavelengths that scatter less. To advance its performance envelope, we recently developed a powerful new Hybrid Multiphoton microscope, allowing fast switching between scanning-line temporal-focusing (SLITE) and a two-photon laser scanning microscopy (TPLSM) subsystems.

In this work I present an advanced nonlinear microscopy system that enables monitoring and stimulating multiple cultured neurons.  I demonstrate its imaging and stimulation capabilities in a set of in vitro and characterization experiments. The novel developments presented in this work include integrating a new holographic stimulation subsystem for 3D targeting of multiple individual neurons simultaneously into a rapid functional imaging microscope, screening for new optogenetic probes performance under physiological imaging and stimulation conditions, and optimizing the illumination wavelengths for manipulating these probes. In addition, I present an implementation of a computational solution to the problem of registering volumetric images from the two imaging modalities, and for analytically evaluating and inverting tissue scattering effects (which will be implemented in future applications of in vivo neuronal imaging).

In a series of experiments, volumetric functional imaging was performed with mm-scale dimensions and μm resolution of 'Optonets' containing over 1000 developing cells and showing complex spontaneous activity patterns, as well as targeted optogenetic stimulation of multiple neurons.  These developments further advance this new tool that could prove useful for network dynamics studies and control of large neuronal populations in three dimensional neuronal circuits both in vitro and in vivo.