Stål O, Hatschek T
Department of Oncology, University Hospital, Linköping, Sweden.
Pathol Res Pract. 1988 Jun;183(3):329-35. doi: 10.1016/S0344-0338(88)80131-6.
A measuring technique enabling the simultaneous determination of nuclear size and DNA content by single cell cytofluorometry is described. The system is based on a Leitz MPV cytophotometer, a Zeiss scanning stage and a microcomputer system. Cells to be measured are not positioned in the ordinary way, but are passed through the excitation light beam. In this way rapid measurements are achieved. Further, an estimate of the object diameter is calculated from the pulse shape obtained as the fluorescence is recorded continuously by using a circular measuring aperture, congruent with the excitation light field. Repeated measurements of nuclei stained with Hoechst 33258 showed a high reproducibility for DNA content with a coefficient of variation (CV) below 1% and an acceptable precision for nuclear area with a mean CV of 7%. Nuclear area estimated by the method was well correlated to area determined from photographs (r = 0.986). Disintegrated samples from eight patients with breast cancer were analyzed and compared to Feulgen-stained samples analyzed by scanning cytophotometry. Scatter diagrams illustrating DNA content and nuclear size showed essentially the same distribution with the two methods. We conclude that the system may be a rapid alternative when fluorescence together with object size are quantified in slide preparations.
本文描述了一种通过单细胞细胞荧光测定法同时测定细胞核大小和DNA含量的测量技术。该系统基于徕卡MPV细胞光度计、蔡司扫描台和微型计算机系统。待测细胞不是以常规方式放置，而是穿过激发光束。通过这种方式实现了快速测量。此外，使用与激发光场一致的圆形测量孔径，在连续记录荧光时从获得的脉冲形状计算出物体直径的估计值。用Hoechst 33258染色的细胞核的重复测量显示，DNA含量具有高重现性，变异系数（CV）低于1%，细胞核面积具有可接受的精度，平均CV为7%。通过该方法估计的细胞核面积与从照片确定的面积高度相关（r = 0.986）。对8例乳腺癌患者的解体样本进行了分析，并与通过扫描细胞光度法分析的福尔根染色样本进行了比较。说明DNA含量和细胞核大小的散点图显示，两种方法的分布基本相同。我们得出结论，当在载玻片制备物中对荧光和物体大小进行量化时，该系统可能是一种快速的替代方法。
