Novel technologies for studies of structural and functional connections

Abstract: To understand the mechanisms underlying the correct functioning of an organ it is important to study its architecture and how the interactions between cells are leading to a specific function. Specifically, the connections that form in the brain are related to the pattern of activation that neurons have, and can help to understand what is the function of each region. Combining the structural knowledge with functional studies is crucial to understand how the cells communicate and propagate the depolarization. Another way to understand the mechanisms underlying tissue functionality is to try to replicate its features and inspect if the resulting behavior is similar to the original one. In this thesis I am describing different tools that we developed to inspect the cells connectivity from the architectural and functional point of view, focusing on imaging, analytic and engineering techniques. To inspect the connection within the brain, in paper I we developed a microscopy system capable of performing fast volumetric imaging of large cleared samples (called LSTM, Light Sheet Theta Microscopy). LSTM is built upon the LSM (Light Sheet Microscopy) system, but instead of illuminating the sample from the sides –which leads to a physical constrain to the samples lateral dimension or depth– the light sheet is scanned on the imaging plane from an angle smaller than 90°. Therefore this approach eliminates the constraints on the lateral size without compromising the image quality and speed. Furthermore, it overcomes the LSM limitation that leads to huge scattering on the center part of the sample. In fact, LSTM images each plane with the same intensity leading to homogeneous x-y acquisition throughout the whole dept. This system can help to create maps of long ranging connections of neurons of intact rodents organs (eg brain) and can theoretically be used to acquire un entire human brain, slab by slab, in a reasonable amount of time. In paper II we propose a tool to inspect the evolution of living cultures for an extended period of time. To do so, we developed a mini-microscope to be placed in the incubator that performs long lasting recordings and automatically detects the Regions Of Interest (ROI), calculates the intensity profiles, and compresses the data after every time-point. This system (called XDscope) is designed to limit the user interaction with the culture, minimize the light exposure and to ease the process of getting the desired information out of the experiment and store as little data as possible. Using the XDscope we performed long term monitoring of GCaMP6 expressing neurosphere (NSP) networks for over 2 weeks, showing that the cells behavior is not affected by the long acquisition. Furthermore we used the system to evaluate the uptake mechanism of p-HTMI, an LCO (Luminescent Conjugated Oligothiophene) over the NSP network, showing that the targeted cells are progenitor cells as expected, since the fluorescent cells are mainly located around the spheres. Finally we investigated further the specific target of p-HTMI within the cells performing double labeling with proteins that seemed to be in the targeted area. From the results it seems like GM130/Golga2, a protein that facilitates the transportation between ER and Golgi apparatus has a high percentage of overlap with the molecule. Finally in paper III we tried to mimic the features and the cell spatial arrangement of a leaving tissue to infer similar properties to an engineered construct. We propose an innovative strategy to integrate a patterned gold microelectrode into a flexible biomimetic hybrid actuator with double muscle-like patterned layers made using PEG (Polyethylene Glycol) and CNT-GelMA (Carbon Nano Tubes- Gelatin Methacryloyl). The CNT-GelMA patterned layer acted as a substrate for cell culture to induce maturation of cardiac muscle cells, while the PEG layer acts as the backbone of the whole membrane. The resulting muscle-like biohybrid actuator showed excellent mechanical integrity with an inserted Au microelectrode and advanced electrophysiological functions with strong muscle contractions. Therefore, we successfully fabricated a biomimetic hybrid actuator with muscle-like pattern, and controllable movement under an electrical field produced by integrated electrodes.

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