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The Applications of Lattice Light-Sheet Microscopy for Functional Volumetric Imaging of Hippocampal Neurons in a Three-Dimensional Culture System

Research Center for Applied Sciences, Academia Sinica, Taipei 11529, Taiwan
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Micromachines 2019, 10(9), 599; https://doi.org/10.3390/mi10090599
Received: 12 August 2019 / Revised: 7 September 2019 / Accepted: 9 September 2019 / Published: 11 September 2019
(This article belongs to the Special Issue Organs-on-chips)
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. In this study, we report the applications of lattice light-sheet microscopy (LLSM) for monitoring neuronal activity in three-dimensional cell culture. We first established a 3D environment for culturing primary hippocampal neurons by applying a scaffold-based 3D tissue engineering technique. Fully differentiated and mature hippocampal neurons were observed in our system. 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. 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. 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. 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 μm2, with an image pixel size of 0.102 μm. When we analyzed the time-lapse volumetric images, we could quantify the voltage responses in different neurites in 3D extensions. View Full-Text
Keywords: lattice light-sheet microscopy; 3D cell culture system; functional neuron imaging lattice light-sheet microscopy; 3D cell culture system; functional neuron imaging
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Chen, C.-Y.; Liu, Y.-T.; Lu, C.-H.; Lee, P.-Y.; Tsai, Y.-C.; Wu, J.-S.; Chen, P.; Chen, B.-C. The Applications of Lattice Light-Sheet Microscopy for Functional Volumetric Imaging of Hippocampal Neurons in a Three-Dimensional Culture System. Micromachines 2019, 10, 599.

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