Zamir Eli, Frey Christoph, Weiss Marian, Antona Silvia, Frohnmayer Johannes P, Janiesch Jan-Willi, Platzman Ilia, Spatz Joachim P
Department of Cellular Biophysics, Max Planck Institute for Medical Research , Jahnstraße 29, D-69120 Heidelberg, Germany.
Department of Biophysical Chemistry, University of Heidelberg , Im Neuenheimer Feld 253, D-69120 Heidelberg, Germany.
Anal Chem. 2017 Nov 7;89(21):11672-11678. doi: 10.1021/acs.analchem.7b03108. Epub 2017 Oct 20.
Fluorescence correlation spectroscopy (FCS) is a sensitive technique commonly applied for studying the dynamics of nanoscale-labeled objects in solution. Current analysis of FCS data is largely based on the assumption that the labeled objects are stochastically displaced due to Brownian motion. However, this assumption is often invalid for microscale objects, since the motion of these objects is dominated by Stokes drag and settling or rising effects, rather than stochastic Brownian motion. To utilize the power of FCS for systems with nonstochastic displacements of objects, the collection and analysis of FCS data have to be reconceptualized. Here, we extended the applicability of FCS for the detection and analysis of periodically passing objects. Toward this end, we implemented droplet-based microfluidics, in which monodispersed droplets containing fluorescent marker are flowing equally spaced within microchannels. We show by simulations and experiments that FCS can sensitively quantify the flow-rates, variability, and content of rapidly passing droplets. This information can be derived at high temporal resolution, based on the intensity fluctuations generated by only 5-10 passing droplets. Moreover, by utilizing the periodicity of the flowing droplets for noise reduction by averaging, FCS can monitor accurately the droplets flow even if their fluorescence intensity is negligible. Hence, extending FCS for periodically passing objects converts it into a powerful analytical tool for high-throughput droplet-based microfluidics. Moreover, based on the principles described here, FCS can be straightforwardly applied for a variety of systems in which the passing of objects is periodic rather than stochastic.
荧光相关光谱法（FCS）是一种灵敏的技术，常用于研究溶液中纳米级标记物体的动力学。目前对FCS数据的分析很大程度上基于这样一种假设，即标记物体由于布朗运动而随机位移。然而，对于微尺度物体，这种假设往往是无效的，因为这些物体的运动主要受斯托克斯阻力以及沉降或上升效应的支配，而非随机的布朗运动。为了将FCS的功能应用于物体具有非随机位移的系统，必须重新构思FCS数据的采集和分析方法。在此，我们扩展了FCS在周期性通过物体的检测和分析方面的适用性。为此，我们采用了基于微滴的微流控技术，其中含有荧光标记物的单分散微滴在微通道内等间距流动。我们通过模拟和实验表明，FCS能够灵敏地量化快速通过微滴的流速、变异性和含量。基于仅5 - 10个通过微滴产生的强度波动，就可以在高时间分辨率下获得这些信息。此外，通过利用流动微滴的周期性进行平均降噪，即使微滴的荧光强度可忽略不计，FCS也能准确监测微滴流动。因此，将FCS扩展应用于周期性通过的物体，使其成为基于微滴的高通量微流控技术的强大分析工具。此外，基于此处所述原理，FCS可直接应用于各种物体通过是周期性而非随机性的系统。
