The observation of growth cones with advanced optical methods

将消逝光照明引入多成像显微镜，构建了一种新型的全内反射荧光显微镜(TIRFM)。该显微镜结合了TIRFM、高分辨率视频增强差示干涉对比(DIC)、荧光、干涉反射(IR)和最先进的延时显微镜图像采集系统。用该TIRFM系统研究了大鼠海马神经元生长锥细胞-底物接触的动态变化。生长锥体用膜标记荧光染料(DiI或十八烷基罗丹明B)染色，并利用该系统的多重成像能力进行观察。当高钾溶液的压力喷射刺激生长锥区时，生长锥基膜上的TIRF强度增加，这表明当激发时，生长锥基膜接近玻璃衬底。在IR模式下，我们还观察到兴奋刺激时腹膜向底物的靠近。讨论了细胞/底物接触在生长锥体中的功能重要性。

Evanescent light illumination was introduced to a multi-imaging microscope to construct a new type of total internal reflection fluorescence microscope (TIRFM). The microscope combined TIRFM, high resolution video-enhanced differential interference contrast (DIC), epifluorescence, interference reflection (IR) with a state-of-the-art image acquisition system for time-lapse microscopy. Dynamic changes of the cell-substrate contact of the neuronal growth cone of a rat hippocampal neuron were studied with this TIRFM system. Growth cones were stained with membrane labeling fluorescence dyes (DiI or octadecyl rohdamine B) and observed using the multi-imaging capacities of this system. When growth cone regions were stimulated by pressure ejection of a high potassium solution, TIRF intensity at the basal membrane of the growth cone increased, suggesting that basal membrane of growth cone approaches the glass substrate when excited. The approach of the ventral membrane to the substrate during excitatory stimulation was also observed in IR mode. The functional importance of cell/substrate contact in growth cones is discussed.