Materials Science and Technology Division, Naval Research Laboratory, Washington DC, USA.
Biophys J. 2013 Aug 6;105(3):602-8. doi: 10.1016/j.bpj.2013.06.022.
Protein secretions from individual cells create spatially and temporally varying concentration profiles in the extracellular environment, which guide a wide range of biological processes such as wound healing and angiogenesis. Fluorescent and colorimetric probes for the detection of single cell secretions have time resolutions that range from hours to days, and as a result, little is known about how individual cells may alter their protein secretion rates on the timescale of minutes or seconds. Here, we present a label-free technique based upon nanoplasmonic imaging, which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from single hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell and from the bursts we estimated the diffusion constant of the secreted antibodies through the extracellular media. The design also incorporates transmitted light and fluorescence microscopy capabilities for monitoring cellular morphological changes and intracellular fluorescent labels. We anticipate that this technique can be adapted as a general tool for the quantitative study of paracrine signaling in both adherent and nonadherent cell lines.
个体细胞分泌的蛋白质在细胞外环境中形成空间和时间上变化的浓度分布，从而指导广泛的生物学过程，如伤口愈合和血管生成。用于检测单细胞分泌的荧光和比色探针的时间分辨率范围从数小时到数天，因此，对于单个细胞如何在数分钟或数秒的时间尺度上改变其蛋白质分泌速率，我们知之甚少。在这里，我们提出了一种基于纳米等离子体成像的无标记技术，该技术能够实时测量单个细胞的分泌。当应用于检测单个杂交瘤细胞分泌的抗体时，增强的时间分辨率揭示了两种分泌模式：一种是细胞连续分泌，另一种是抗体集中释放，与细胞长达一分钟的形态收缩同时发生。从连续分泌测量中，我们确定了与细胞最接近的传感阵列处的抗体局部浓度，并且从爆发中我们估计了分泌抗体通过细胞外介质的扩散常数。该设计还包含透射光和荧光显微镜功能，用于监测细胞形态变化和细胞内荧光标记。我们预计，该技术可以作为一种通用工具，用于研究粘附和非粘附细胞系中旁分泌信号的定量。
