Photothermal raster image correlation spectroscopy of gold nanoparticles in solution and on live cells.
Photothermal raster image correlation spectroscopy of gold nanoparticles in solution and on live cells.
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DOI：
10.1098/rsos.140454
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发表时间：
2015-06
影响因子：
3.5
通讯作者：
Lévy R
中科院分区：
文献类型：
--
作者：
Nieves DJ;Li Y;Fernig DG;Lévy R
光栅图像相关光谱 (RICS) 通过分析图像内的相关性来测量共焦显微镜图像堆栈中荧光标记分子的扩散情况。与其他单分子方法相比，RICS 能够观察更大的生物系统区域，从而更具代表性。金纳米粒子的光热显微镜可以对相同标记的分子进行长期成像，而无需光漂白。在这里，我们在光热显微镜上实施 RICS 分析。演示了使用压电驱动光热外差显微镜在足够短的 RICS（60μs）像素停留时间下对单个金纳米颗粒进行成像（光热光栅图像相关光谱，PhRICS）。作为原理证明，PhRICS 用于测量金纳米粒子在甘油 : 水溶液中的扩散系数。 PhRICS 测量的纳米颗粒的扩散系数与其通过透射电子显微镜测定的尺寸一致。然后使用 PhRICS 探测金纳米颗粒标记的成纤维细胞生长因子 2 (FGF2) 与硫酸乙酰肝素结合在活成纤维细胞的细胞周基质中的扩散速度。这些数据与之前 FGF2 在这些细胞上扩散的单纳米粒子追踪研究一致。重要的是，这些数据揭示了以前通过光热追踪无法实现的更快的 FGF2 运动，并表明结合的 FGF2 分布的不均匀性是动态的。
Raster image correlation spectroscopy (RICS) measures the diffusion of fluorescently labelled molecules from stacks of confocal microscopy images by analysing correlations within the image. RICS enables the observation of a greater and, thus, more representative area of a biological system as compared to other single molecule approaches. Photothermal microscopy of gold nanoparticles allows long-term imaging of the same labelled molecules without photobleaching. Here, we implement RICS analysis on a photothermal microscope. The imaging of single gold nanoparticles at pixel dwell times short enough for RICS (60 μs) with a piezo-driven photothermal heterodyne microscope is demonstrated (photothermal raster image correlation spectroscopy, PhRICS). As a proof of principle, PhRICS is used to measure the diffusion coefficient of gold nanoparticles in glycerol : water solutions. The diffusion coefficients of the nanoparticles measured by PhRICS are consistent with their size, determined by transmission electron microscopy. PhRICS was then used to probe the diffusion speed of gold nanoparticle-labelled fibroblast growth factor 2 (FGF2) bound to heparan sulfate in the pericellular matrix of live fibroblast cells. The data are consistent with previous single nanoparticle tracking studies of the diffusion of FGF2 on these cells. Importantly, the data reveal faster FGF2 movement, previously inaccessible by photothermal tracking, and suggest that inhomogeneity in the distribution of bound FGF2 is dynamic.