Open Access Open Badges Methodology article

Multispectral fingerprinting for improved in vivo cell dynamics analysis

Paul M Kulesa12*, Jessica M Teddy1, Miranda Smith1, Richard Alexander1, Cameron HJ Cooper1, Rusty Lansford3 and Rebecca McLennan1

Author Affiliations

1 Imaging/Kulesa Lab, The Stowers Institute for Medical Research, (50th St) Kansas City, (64110), USA

2 Department of Anatomy and Cell Biology, The University of Kansas School of Medicine, (Rainbow Blvd), Kansas City, (66160), USA

3 Division of Biology, California Institute of Technology, (E. California Blvd), Pasadena, (91125), USA

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BMC Developmental Biology 2010, 10:101  doi:10.1186/1471-213X-10-101

Published: 24 September 2010



Tracing cell dynamics in the embryo becomes tremendously difficult when cell trajectories cross in space and time and tissue density obscure individual cell borders. Here, we used the chick neural crest (NC) as a model to test multicolor cell labeling and multispectral confocal imaging strategies to overcome these roadblocks.


We found that multicolor nuclear cell labeling and multispectral imaging led to improved resolution of in vivo NC cell identification by providing a unique spectral identity for each cell. NC cell spectral identity allowed for more accurate cell tracking and was consistent during short term time-lapse imaging sessions. Computer model simulations predicted significantly better object counting for increasing cell densities in 3-color compared to 1-color nuclear cell labeling. To better resolve cell contacts, we show that a combination of 2-color membrane and 1-color nuclear cell labeling dramatically improved the semi-automated analysis of NC cell interactions, yet preserved the ability to track cell movements. We also found channel versus lambda scanning of multicolor labeled embryos significantly reduced the time and effort of image acquisition and analysis of large 3D volume data sets.


Our results reveal that multicolor cell labeling and multispectral imaging provide a cellular fingerprint that may uniquely determine a cell's position within the embryo. Together, these methods offer a spectral toolbox to resolve in vivo cell dynamics in unprecedented detail.