Lohr Christina, Kunding Andreas H, Bhatia Vikram K, Stamou Dimitrios
Bio-Nanotechnology Laboratory, Department of Neuroscience and Pharmacology and Nano-Science Center, University of Copenhagen, Copenhagen, Denmark.
Methods Enzymol. 2009;465:143-60. doi: 10.1016/S0076-6879(09)65008-4.
We describe in detail a simple technique to construct the size distribution of liposome formulations from single-object fluorescence measurements. Liposomes that are fluorescently labeled in their membrane are first immobilized on a surface at dilute densities and then imaged individually using epi-fluorescence microscopy. The integrated intensities of several thousand single liposomes are collected and evaluated within minutes by automated image processing, using the user-friendly freeware ImageJ. The mean intensity of the liposome population is then calculated and scaled in units of length (nm) by relating the intensity data to the mean diameter obtained from a reference measurement with dynamic light scattering. We explain the process of constructing the size distributions in a step-by-step manner, starting with the preparation of liposomes through the final acquisition of size histograms. Detailed advice is given concerning critical parameters of image acquisition and processing. Size histograms constructed from single-particle measurements provide detailed information on complex distributions that may be easily averaged out in ensemble measurements (e.g., light scattering). In addition, the technique allows accurate measurements of polydisperse samples (e.g., nonextruded liposome preparations).
我们详细描述了一种简单的技术，可通过单对象荧光测量来构建脂质体制剂的尺寸分布。首先将膜上带有荧光标记的脂质体以低密度固定在表面上，然后使用落射荧光显微镜对其进行逐个成像。通过自动化图像处理，使用用户友好的免费软件ImageJ，在几分钟内收集并评估数千个单脂质体的积分强度。然后计算脂质体群体的平均强度，并通过将强度数据与通过动态光散射参考测量获得的平均直径相关联，将其按长度单位（纳米）进行缩放。我们逐步解释构建尺寸分布的过程，从脂质体的制备到最终获得尺寸直方图。针对图像采集和处理的关键参数给出了详细建议。由单颗粒测量构建的尺寸直方图提供了关于复杂分布的详细信息，这些信息在整体测量（例如光散射）中可能很容易被平均掉。此外，该技术允许对多分散样品（例如未挤出的脂质体制剂）进行准确测量。
