The observation of growth cones with advanced optical methods

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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.