Zhang X Y, Sun C K, Wheatley A M
Microcirculation Research Laboratory, University of Otago, Dunedin, New Zealand.
Microvasc Res. 2000 Nov;60(3):232-40. doi: 10.1006/mvre.2000.2264.
Hepatic stellate cells (HSC) are nonparenchymal liver cells which reside in the space of Disse within the hepatic microcirculatory unit. HSC can be distinguished using intravital fluorescent microscopy (IVFM) due to the autofluorescence from their intracellular vitamin A. Herein we report on a novel approach for the quantification of video-recorded rat HSC images acquired by IVFM (excitation 360 nm/emission 420 nm) by the combined use of the "Cell counting macro" and the "Measurement macros" in the NIH image software. The approach involved two major steps using (i) the "Cell counting macro" for automatic detection, threshold-setting, and generation of a binary image of the vitamin A autofluorescence in the HSC images and (ii) the "Compute percent black and white" command in the "Measurement macros" to automatically determine the HSC density (%), which was then expressed as percentage of the total area of vitamin A autofluorescence-associated sites per observation area. Comparing the vitamin A autofluorescence areas in the original and the binary fashion HSC images revealed that the "Cell counting macro" was an optimal option for the analysis of the low-magnification (x10 objective) HSC images, whereas this macro was not suitable for the analysis of the higher magnification (40x objective) HSC images unless modifications were made. Our analysis revealed that HSC represent approximately 4-5% of the total area of the liver surface. In analyzing the higher magnification HSC microfluorographs, the use of the original "Cell counting macro" resulted in a significant underestimation of HSC density (60% reduction, P < 0.01) when compared with those analyzed using our modified macro. This study represents the first report of an automatic and reliable approach to the intravital fluorescent microscopic quantification of HSC using a computer-NIH image analysis system.
肝星状细胞（HSC）是位于肝微循环单位狄氏间隙的非实质肝细胞。由于其细胞内维生素A的自发荧光，可通过活体荧光显微镜（IVFM）对HSC进行鉴别。在此，我们报告一种新方法，通过联合使用美国国立卫生研究院（NIH）图像软件中的“细胞计数宏”和“测量宏”，对IVFM（激发波长360nm/发射波长420nm）采集的大鼠HSC视频图像进行定量分析。该方法包括两个主要步骤：一是使用“细胞计数宏”对HSC图像中维生素A自发荧光进行自动检测、阈值设定并生成二值图像；二是使用“测量宏”中的“计算黑白百分比”命令自动确定HSC密度（%），然后将其表示为每个观察区域中与维生素A自发荧光相关位点总面积的百分比。比较原始和二值化HSC图像中的维生素A自发荧光区域发现，“细胞计数宏”是分析低倍（10倍物镜）HSC图像的最佳选择，而该宏不适用于分析高倍（40倍物镜）HSC图像，除非进行修改。我们的分析表明，HSC约占肝脏表面总面积的4 - 5%。在分析高倍HSC显微荧光照片时，与使用我们修改后的宏分析的结果相比，使用原始的“细胞计数宏”会导致HSC密度显著低估（降低60%，P < 0.01）。本研究首次报道了一种使用计算机 - NIH图像分析系统对HSC进行活体荧光显微镜定量分析的自动且可靠的方法。
