Abstract
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.
Highlights
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 [1,2,3,4]
In order to compare hippocampal neurons cultured in 2D and 3D environments, we examined axon and dendrite formation by staining Tau and microtubule-associated protein 2 (MAP2) proteins at an early stage (4 days in vitro (DIV)), and the formation of synapses using synaptotagmin to label presynaptic terminals and pbarer:s1y0nμampt.i(cBt)eTrhme idniafflesreanntidatipoonsotfsy3Dnacpulttiucreddenesuitryonpsrotein 95 (PSD-95) for postsynaptic terminal staining at a later stage (15 DIV) (Figure 1)
In order to improve the efficiency in drug screening applications, organs-on-chips, which integrate 3D cultures with microfluidic systems, may provide more reliable results, because they better represent the native microenvironment within tissues
Summary
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 [1,2,3,4]. 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 [5,6,7,8]. High-speed calcium imaging from 2D or 3D cultured neurons can be recorded at a fixed focal plane [20]. Recording neuronal activity in 3D cultures requires the development of high-speed volumetric imaging techniques. 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 [26,27]. In LLSM, a pair of microscope objectives with perpendicular orientation shares the same focal point, where the sample is placed The specimen and both tapered ends of the objective are immersed in the medium filled, temperature-controlled chamber. In this study, we utilized LLSM to monitor neuronal activity via subcellular imaging of voltage and calcium dynamics
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