Braïni Céline, Mottolese Angelo, Ferrante Ivan, Monnier Sylvain, Villard Catherine
Laboratoire Physico-Chimie Curie, Institut Curie, Institut Pierre-Gilles de Gennes pour la microfluidique, Université PSL, CNRS.
UMR 144 Institut Curie, Université PSL, CNRS.
J Vis Exp. 2018 Mar 26(133):56923. doi: 10.3791/56923.
Volume is an important parameter regarding physiological and pathological characteristics of neurons at different time scales. Neurons are quite unique cells regarding their extended ramified morphologies and consequently raise several methodological challenges for volume measurement. In the particular case of in vitro neuronal growth, the chosen methodology should include sub-micrometric axial resolution combined with full-field observation on time scales from minutes to hours or days. Unlike other methods like cell shape reconstruction using confocal imaging, electrically-based measurements or Atomic Force Microscopy, the recently developed Fluorescence eXclusion method (FXm) has the potential to fulfill these challenges. However, although being simple in its principle, implementation of a high-resolution FXm for neurons requires multiple adjustments and a dedicated methodology. We present here a method based on the combination of fluorescence exclusion, low-roughness multi-compartments microfluidic devices, and finally micropatterning to achieve in vitro measurements of local neuronal volume. The high resolution provided by the device allowed us to measure the local volume of neuronal processes (neurites) and the volume of some specific structures involved in neuronal growth, such as growth cones (GCs).
在不同时间尺度上，体积是关于神经元生理和病理特征的一个重要参数。神经元因其延伸的分支形态而成为非常独特的细胞，因此在体积测量方面带来了若干方法学挑战。在体外神经元生长的特定情况下，所选用的方法应包括亚微米级轴向分辨率以及在从几分钟到几小时或几天的时间尺度上进行全场观察。与其他方法，如使用共聚焦成像进行细胞形状重建、基于电的测量或原子力显微镜不同，最近开发的荧光排除法（FXm）有潜力应对这些挑战。然而，尽管其原理简单，但为神经元实施高分辨率的FXm需要多次调整和专门的方法。我们在此介绍一种基于荧光排除、低粗糙度多隔室微流控装置以及最终微图案化相结合的方法，以实现对局部神经元体积的体外测量。该装置提供的高分辨率使我们能够测量神经元突起（神经突）的局部体积以及参与神经元生长的一些特定结构的体积，如生长锥（GCs）。
