Debey P, Renard J P, Coppey-Moisan M, Monnot I, Geze M
Unité en Développement Concerté INSERM-INRA, Institut de Biologie Physico-chimique, Paris, France.
Exp Cell Res. 1989 Aug;183(2):413-33. doi: 10.1016/0014-4827(89)90401-1.
Conditions of minimal dye concentration and minimal irradiation which allow the continuous observation of pronuclei in live unicellular mouse eggs by fluorescence microscopy have been found with the use of Hoechst 33342 as fluorophore and a camera of high sensitivity coupled with an image processing system allowing true integration of weak fluorescent signals and further treatment and analysis. Under these conditions the developmental potential of the embryos is not affected. Using such an approach, which avoids eventual artifacts due to fixation procedure, we describe the changes in the nuclear organization and chromatin structure, from formation of pronuclei to mitosis, with particular attention to the chromatin associated with nucleoli and the timing process of chromatin condensation.
利用Hoechst 33342作为荧光团，并结合高灵敏度相机与图像处理系统，该系统能够对微弱荧光信号进行真正的积分以及进一步处理和分析，现已发现了染料浓度和辐照最小化的条件，这些条件使得能够通过荧光显微镜对活的单细胞小鼠卵中的原核进行连续观察。在这些条件下，胚胎的发育潜能不受影响。使用这种避免了固定程序可能产生的假象的方法，我们描述了从原核形成到有丝分裂过程中核组织和染色质结构的变化，特别关注与核仁相关的染色质以及染色质凝聚的时间进程。
