Wang Wenqian, Ma Yuanqing, Bonaccorsi Simone, Cong Vu Thanh, Pandžić Elvis, Yang Zhengmin, Goyette Jesse, Lisi Fabio, Tilley Richard D, Gaus Katharina, Gooding J Justin
School of Chemistry, University of New South Wales, Sydney 2052, Australia.
Australian Centre for NanoMedicine, University of New South Wales, Sydney 2052, Australia.
Anal Chem. 2021 Mar 2;93(8):3803-3812. doi: 10.1021/acs.analchem.0c04285. Epub 2021 Feb 16.
How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.
纳米颗粒如何在活细胞中分布以及如何克服细胞屏障是药物载体设计中的重要标准。对相关显微镜技术是一种对共焦线扫描获得的荧光强度波动进行的相关性分析，它可以量化纳米颗粒扩散的动态特性，包括可移动纳米颗粒的数量、扩散系数以及跨越空间距离的通过时间。由于纳米颗粒性质的潜在异质性以及细胞环境的复杂性，对平均自相关和对相关图谱的量化可能会掩盖对纳米颗粒给药能力的重要见解。为了克服这个问题，我们使用相量分析开发了一种数据标准化方法，该方法可以根据扩散将扫描线分割成几个子区域，并解决细胞内移动的纳米颗粒的空间异质性问题。相量分析是一种免拟合方法，它在所谓的相量图上表示每个像素相对于自由扩散的自相关图谱。然后相量图可用于选择可以分别进行自相关和对相关分析的亚群。我们展示了用于对相关显微镜技术的相量分析，以研究16纳米、Cy5标记的二氧化硅纳米颗粒在MCF-7细胞中跨质膜的扩散以及绿色荧光蛋白（GFP）跨核膜的扩散。
