Multispectral fingerprinting for improved in vivo cell dynamics analysis.
Multispectral fingerprinting for improved in vivo cell dynamics analysis.
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DOI：
10.1186/1471-213x-10-101
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发表时间：
2010-09-24
影响因子：
--
通讯作者：
McLennan R
中科院分区：
文献类型：
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作者：
Kulesa PM;Teddy JM;Smith M;Alexander R;Cooper CH;Lansford R;McLennan R
当细胞轨迹在空间和时间上交叉，且组织密度使单个细胞边界模糊不清时，追踪胚胎中的细胞动态就变得极其困难。在此，我们以鸡神经嵴（NC）为模型，测试多色细胞标记和多光谱共聚焦成像策略以克服这些障碍。
我们发现，多色细胞核标记和多光谱成像通过为每个细胞提供独特的光谱特征，提高了体内神经嵴细胞识别的分辨率。神经嵴细胞的光谱特征使得细胞追踪更加准确，并且在短期的延时成像过程中保持一致。计算机模型模拟预测，与单色细胞核标记相比，三色标记在细胞密度增加时对物体计数有显著更好的效果。为了更好地分辨细胞间的接触，我们表明双色细胞膜标记和单色细胞核标记相结合，极大地改善了对神经嵴细胞相互作用的半自动分析，同时保留了追踪细胞运动的能力。我们还发现，对多色标记胚胎进行通道扫描而非波长扫描，显著减少了获取和分析大型三维体数据集的时间和工作量。
我们的研究结果表明，多色细胞标记和多光谱成像提供了一种细胞指纹，它可以唯一确定一个细胞在胚胎内的位置。总之，这些方法提供了一个光谱工具箱，以前所未有的细节解析体内细胞动态。
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.