Gallardo-Escárate Cristian, Alvarez-Borrego Josué, Von Brand Elisabeth, Dupré Enrique, Del Río-Portilla Miguel Angel
Departamento de Oceanografía, Facultad de Ciencias Naturales y Oceanógraficas, Universidad de Concepción, Concepción, Chile.
Biol Res. 2007;40(1):29-40. doi: 10.4067/s0716-97602007000100004. Epub 2007 Jul 19.
In observations by confocal or conventional fluorescence microscopy, important factors should be considered in order to obtain accurate images. One of them, such as the fluorescence bleaching from highest intensity to lowest signal of fluorescence is a common problem with several DNA fluorochromes and especially for DAPI stain. The fluorescence of DAPI fades rapidly when it is exposed to UV light, under optimal conditions of observation. Although the fading process can be retarded using a mounting medium with antifading reagents, the photochemical process underlying the fluorescence decay has not yet been fully explained. In addition, no relationship between fluorescence fading and nuclear DNA content has been tested. In order to test this relationship, we measured by means of image analysis the DAPI-fluorescence intensity in several cellular types (spermatozoa, erythrocytes and haemocytes) during their fluorescence bleaching. An algorithm specifically built in MATLAB software was used for this approach. The correlation coefficient between nuclear DNA content and DAPI-fluorescence fading was found equal to 99%. This study demonstrates the feasibility to measure nuclear DNA content by fluorescence fading quantification, as an alternative method concurrently with image analysis procedures.
在通过共聚焦或传统荧光显微镜进行观察时，为了获得准确的图像，应考虑一些重要因素。其中之一，例如荧光从最高强度漂白至最低荧光信号，这是几种DNA荧光染料常见的问题，尤其是对于DAPI染色而言。在最佳观察条件下，当DAPI暴露于紫外光时，其荧光会迅速褪色。尽管使用含有抗褪色试剂的封片剂可以减缓褪色过程，但荧光衰减背后的光化学过程尚未得到充分解释。此外，尚未测试荧光褪色与核DNA含量之间的关系。为了测试这种关系，我们在几种细胞类型（精子、红细胞和血细胞）的荧光漂白过程中，通过图像分析测量了DAPI荧光强度。为此使用了专门内置于MATLAB软件中的一种算法。发现核DNA含量与DAPI荧光褪色之间的相关系数等于99%。本研究证明了通过荧光褪色定量来测量核DNA含量的可行性，作为一种与图像分析程序同时使用的替代方法。
