The specimen and both tapered ends of the objective are immersed in the medium filled, temperature-controlled chamber. In LLSM, a pair of microscope objectives with perpendicular orientation shares the same focal point, where the sample is placed. Among them, lattice light-sheet microscopy (LLSM) has been shown to offer several advantages over other volumetric imaging tools, including less photobleaching and phototoxicity and better subcellular imaging resolution. Recently, selective plane illumination-based techniques have been demonstrated to be capable of high-speed volumetric imaging. However, recording neuronal activity in 3D cultures requires the development of high-speed volumetric imaging techniques. However, the penetration depth of light in 3D culture is always problematic, which may be partially resolved by the use of two-photon microscopy. Alternatively, multi-point scanning spinning disc microscopy also offers high speed imaging with low phototoxicity. At present, high-speed calcium imaging from 2D or 3D cultured neurons can be recorded at a fixed focal plane. However, it remains difficult to measure neuronal activity in 3D due to the limited light penetration and imaging speed. In general, the application of calcium indicators has yielded satisfactory results for primary 2D cultures, slices of brain tissue, and in vivo brain imaging. The function of neurons can be monitored through membrane potential or calcium dynamics using optical microscopic tools. Two-dimensional (2D) neuron cultures are the most studied systems for understanding neurodegenerative diseases, in which neuronal behavior can be manipulated and measured through various techniques. Among various diseases, neurodegenerative diseases are of great research importance because these diseases are currently considered incurable. One of the major advantages of the organs-on-chips is the capability to mimic diseases at the organ level on chips. Therefore, one of the key issues in the development of the organs-on-chips system is to monitor the spatiotemporal behavior of individual cells in the 3D cell culture. In the conventional approach, microfluidic devices are placed on an inverted microscope, where the scattering from multiple layers of cells hampers light penetration, thus leading to low imaging quality. In order to faithfully reconstruct the in vivo microenvironment, 3D culture systems are often used for the organs-on-chips, which raises a challenging issue in detecting the response of individual cells with high spatiotemporal resolution while minimizing the damage of cells during the observation process. With the integration of microfluidic systems and three-dimensional (3D) cell cultures, organs-on-chips have shown great potential in high throughput drug screening applications. In recent years, the rapid development of microfabrication techniques has enabled us to study cellular behavior in a well-controlled microenvironment, mimicking the native environment of the disease states. When we analyzed the time-lapse volumetric images, we could quantify the voltage responses in different neurites in 3D extensions. Neuronal volumetric images were sheet scanned along the axial direction and recorded at a laser exposure of 6 ms, which covered an area up to 4800 μm 2, with an image pixel size of 0.102 μm. From the volumetric images, it was found that the voltage indicators mainly resided in the cytosol instead of the membrane, which cannot be distinguished using conventional microscopy. We demonstrated that our system could study the membrane voltage and intracellular calcium dynamics at subcellular resolution in 3D under both chemical and electrical stimulation. With LLSM, we were able to monitor the behavior of individual cells in a 3D cell culture, which was very difficult under a conventional microscope due to strong light scattering from thick samples. Fully differentiated and mature hippocampal neurons were observed in our system. We first established a 3D environment for culturing primary hippocampal neurons by applying a scaffold-based 3D tissue engineering technique. In this study, we report the applications of lattice light-sheet microscopy (LLSM) for monitoring neuronal activity in three-dimensional cell culture. The characterization of individual cells in three-dimensions (3D) with very high spatiotemporal resolution is crucial for the development of organs-on-chips, in which 3D cell cultures are integrated with microfluidic systems.
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