Ph.D Thesis

Ph.D StudentDana Hod Michael
SubjectTemporal Focusing Multiphoton Microscopy for Rapid
Volumetric Neuronal Imaging
DepartmentDepartment of Biomedical Engineering
Supervisor PROF. Shy Shoham


The dynamic activity patterns of neuronal networks contain important information about network computations and connectivity. This type of information is essential in order to analyze the relation between stimulus and response at the network level. Optical imaging methods, mainly two-photon laser scanning microscopy (TPLSM) are routinely used for single-cell resolution neuronal imaging, usually in combination with functional fluorescence indicators. However, the spatially localized nature of excitation by nonlinear optical methods limits their ability to monitor more than a small number of neurons with a single action potential temporal resolution. Currently, the state-of-the-art solutions for rapid volumetric imaging are based on acusto-optic deflector systems, which rapidly jump between predefined cells' location.

      In this work we study an alternative imaging method, temporal focusing multiphoton microscopy, that enables the simultaneous excitation of an optically-sectioned plane, line, or flexible two-dimensional pattern, and therefore, to significantly enhance the imaging rate. First, we develop quantitative tools for analyzing and predicting temporal focusing performance as a function of the optical system’s parameters for two commonly-used applications based on widefield and line illumination.  We then show, using these tools, that the performance of temporal focusing methods is dramatically different than that of spatial focusing based methods, in both transparent and scattering media.

      Next, we present a new optical design solution for temporal focusing microscopes, based on a dual prism grating, which enables to increase the microscope's efficiency and to combine it with TPLSM. Moreover, the new design also enables to perform remote scanning of the focal plane, which was considered not feasible in previous studies. The multi-modal Scanning Line Temporal focusing (SLITE) multiphoton microscope is used for rapid functional imaging of three dimensional bio-engineered neural cultures. Simultaneous imaging of ~1000 cells with single cell specificity are demonstrated and analyzed and are seen to show synchronous network activity patterns.

      Finally, we discuss the necessary considerations for adapting SLITE microscopy to in vivo imaging experiments, where tissue scattering is significantly stronger. A simple algorithmic approach is presented for combining structural information, acquired by TPLSM, into a functional data extraction method. We test this approach on simulated data and show that functional activity may be extracted from heavily blurred images. 

      The presented theoretical, computational and design solutions and their implementation in a neuronal imaging system could play an important role in the engineering of systems for volumetric functional imaging of large neuronal networks under a wide range of experimental scenarios.