3.1. Neuron growth and transfection
To obtain neurons with mature axon and dendritic structure, primary hippocampal neurons were isolated and cultured for 5 days (Figure S1). Immunofluorescence assay revealed that the neurons were all Tuj1-positive and possessed a mature axon of approximate 0.74 μm width (Figure S1A). After that, the Tuj1-positive neurons were transfected to express mCherry proteins by a plasmid encoding CMV promoter-driven mCherry gene (Figure 1A). A continuous fluorescence imaging of a single neuron found that mCherry protein could be detected and evenly distributed in the cell soma and axon of neurons at 48 h after transfection (Figure 1B and Figure S1B). And then the clear granular fluorescence signal was visible between 60 h and 72 h, suggesting the formation of mCherry aggregations during this period. Finally, the fluorescence signal of the whole neuronal axon became discrete at 78 h. These results suggested an optimal axonal transport observing window between 60 h and 72 h after transfection, considering that the over-expression of mCherry protein for more than 72 h would affect the physiological state of neurons. It also demonstrated that the sparse transfection strategy can serve as a facile strategy to produce and image fluorescent protein aggregates in a single living neuron.