Coelho Neto José, Agero Ubirajara, Gazzinelli Ricardo T, Mesquita Oscar N
Departamento de Física, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
Biophys J. 2006 Aug 1;91(3):1108-15. doi: 10.1529/biophysj.105.073783. Epub 2006 Apr 14.
Defocusing microscopy (DM) is a recently developed technique that allows quantitative analysis of membrane surface dynamics of living cells using a simple bright-field optical microscope. According to DM, the contrast of defocused images is proportional to cell surface curvature. Although, until now, this technique was used mainly to determine size and amount of membrane shape fluctuations, such as ruffles and small random membrane fluctuations, in macrophages, its applications on cell biology extend beyond that. We show how DM can be used to measure optical and mechanical properties of a living macrophage, such as cell refractive index n, membrane bending modulus K(c), and effective cell viscosity eta for membrane-actin meshwork relaxation. Experimental data collected from defocused images of bone marrow-derived macrophages were used to evaluate these parameters. The obtained values, averaged over several different macrophages, are n = (1.384 +/- 0.015), K(c) approximately 3.2 x 10(-19) J, and eta approximately 459 Pa.s. We also estimate the amplitude of the small fluctuations to be of the order of 3 nm, which is around the step size of a polymerizing actin filament.
散焦显微镜（DM）是一种最近开发的技术，它允许使用简单的明场光学显微镜对活细胞的膜表面动力学进行定量分析。根据DM技术，散焦图像的对比度与细胞表面曲率成正比。尽管到目前为止，该技术主要用于确定巨噬细胞中膜形状波动的大小和数量，如褶皱和小的随机膜波动，但其在细胞生物学上的应用不止于此。我们展示了如何使用DM来测量活巨噬细胞的光学和力学特性，如细胞折射率n、膜弯曲模量K(c)以及膜-肌动蛋白网络松弛的有效细胞粘度η。从骨髓来源的巨噬细胞的散焦图像中收集的实验数据用于评估这些参数。在几个不同的巨噬细胞上平均得到的值为n = (1.384 ± 0.015)，K(c)约为3.2×10^(-19) J，η约为459 Pa·s。我们还估计小波动的幅度约为3 nm，这与聚合肌动蛋白丝的步长相近。
