Neural transplant staining with DiI and vital imaging by 2-photon laser-scanning microscopy

We are developing a multielectrode silicon "neuro-probe" for maintaining a long-term, specific, two-way electrical interface with nervous tissue. Our approach involves trapping a neuron (from an embryonic rat hippocampus) in a small well with a stimulation/recording electrode at its base. The well i s covered with a grillwork through which the neuron's processes are allowed to grow, making synaptic contact with the host tissue, in our case a cultu red slice from a rat hippocampus. Each neuroprobe can accommodate 15 neuron s, one per well. As a first step in studying neurite outgrowth from the neu roprobe, it was necessary to develop new staining techniques so that neurit es from the probe neurons can be distinguished from those belonging to the host, without interference from non-specific background staining. We virtua lly eliminated background staining through a number of innovations involvin g dye solubility, cell washing, and debris removal. We also reduced photobl eaching and phototoxicity, and enhanced imaging depth by using a 2-photon l aser-scanning microscope. We focused on using the popular membrane dye, DiI , however a number of other membrane dyes were shown to provide clear image s of neural processes using pulsed illumination at 900 mm. These techniques will be useful to others wishing to follow over time the growth of neurons in culture or after transplantation in vivo, in a non-destructive way.