Tibbe Arjan G J, de Grooth Bart G, Greve Jan, Dolan Gerald J, Terstappen Leon W M M
Faculty of Applied Physics, University of Twente, Enschede, The Netherlands.
Cytometry. 2002 Apr 1;47(4):248-55. doi: 10.1002/cyto.10085.
We developed the CellTracks cell analysis system that, similar to flow cytometry, yields multiparameter information by which the cells can be differentiated. We describe the implementation of a laser scanning imaging method in the system. Image analysis of the cells improves the specificity of cell classification, especially in cases where the particular cells are found relatively infrequently and one has to discriminate between artifacts and real events.
Fluorescent images of immunomagnetically labeled and aligned cells are obtained by passing the cells through a laser focus. The laser focus is smaller than the objects and subsequent frames captured by a regular surveillance CCD camera with a frame grabber board represent different parts of the cells. Complete images of the cells are constructed by shifting each image with respect to each other and adding individual pixel values.
The power of combining a fluorescent image with multiparametric data is demonstrated by imaging fluorescent and magnetically labeled beads and cells. The image gives additional information about the dye distribution across the objects. Changes in dye distribution as a function of time were observed in leukocytes labeled with the red fluorescent label, Oxazine750, which are imaged at different time intervals.
An imaging technique implemented in the CellTracks system provides high-resolution fluorescent images of events previously identified by the system. The images of the fluorescent cells enhance the ability to classify rare events.
我们开发了CellTracks细胞分析系统，该系统与流式细胞术类似，可产生多参数信息，借此能够区分细胞。我们在此描述该系统中激光扫描成像方法的应用。细胞的图像分析提高了细胞分类的特异性，尤其是在特定细胞相对罕见且必须区分假象与真实事件的情况下。
使免疫磁珠标记并排列好的细胞通过激光焦点，从而获得荧光图像。激光焦点小于细胞，由带有图像采集卡的常规监控CCD相机捕获的后续帧代表细胞的不同部分。通过将每个图像相互移位并相加各个像素值，构建细胞的完整图像。
对荧光标记和磁珠标记的珠子及细胞进行成像，证明了将荧光图像与多参数数据相结合的作用。该图像提供了有关染料在物体上分布的额外信息。在用红色荧光标记物恶嗪750标记的白细胞中观察到染料分布随时间的变化，这些白细胞在不同时间间隔成像。
CellTracks系统中采用的成像技术可提供该系统先前识别事件的高分辨率荧光图像。荧光细胞的图像增强了对罕见事件进行分类的能力。
