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Neural Transplant Staining with DiI and Vital Imaging by 2-Photon Laser-Scanning Microscopy

Potter, Steve M. and Pine, Jerome and Fraser, Scott E. (1996) Neural Transplant Staining with DiI and Vital Imaging by 2-Photon Laser-Scanning Microscopy. Scanning Microscopy, 10 . pp. 189-199. ISSN 0891-7035. PMCID PMC2585498.

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We are developing a multielectrode silicon “neuroprobe” 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 is 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 cultured slice from a rat hippocampus. Each neuroprobe can accommodate 15 neurons, one per well. As a first step in studying neurite outgrowth from the neuroprobe, it was necessary to develop new staining techniques so that neurites from the probe neurons can be distinguished from those belonging to the host, without interference from non-specific background staining. We virtually eliminated background staining through a number of innovations involving dye solubility, cell washing, and debris removal. We also reduced photobleaching and phototoxicity, and enhanced imaging depth by using a 2-photon laser-scanning microscope. We focused on using the popular membrane dye, DiI, however a number of other membrane dyes were shown to provide clear images of neural processes using pulsed illumination at 900 nm. These techniques will be useful to others wishing to follow the growth of transplanted neurons over time, in a non-destructive way.

Item Type:Article
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URLURL TypeDescription CentralArticle
Fraser, Scott E.0000-0002-5377-0223
Additional Information:Author manuscript; available in PMC 2008 November 20. Presented at the XIV Pfefferkorn Conference: The Science of Biological Specimen Preparation for Microscopy, Aug, 1995, Belleville, IL. Proceedings to appear in Scanning Microscopy International. We thank Sheri McKinney for her expert technical assistance; Pete Vanderklish for teaching us hippocampal slice preparation; Yu-Chong Tai, Svetlana Tatic-Lucic and John Wright for neuroprobe design and fabrication; Gyuri Buzsaki and Anatol Bragin for their continued collaboration on the Neural Prosthesis project; The NINDS at the NIH for funding the Neural Prosthesis project; and Molecular Dynamics, a Silvio Conte Center award from the NIMH, and the Beckman Institute for support of the 2-photon imaging facility.
Funding AgencyGrant Number
National Institute of Neurological Disorders and Stroke (NINDS)UNSPECIFIED
National Institute of Mental Health (NIMH)UNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Subject Keywords:Neural prosthesis; cell suspension staining; cellular debris; phototoxicity; non-destructive imaging; rat hippocampus; carbocyanine dye; grafting; two-photon fluorescence; neuroprobe
PubMed Central ID:PMC2585498
Record Number:CaltechAUTHORS:20160413-092402353
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:66107
Deposited By: Tony Diaz
Deposited On:13 Apr 2016 17:49
Last Modified:03 Oct 2019 09:53

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