Rigaut J P, Vassy J
Laboratory of Quantitative Microscopy in Histopathology, Institut National de la Santé et de la Recherche Médicale, University of Paris, France.
Anal Quant Cytol Histol. 1991 Aug;13(4):223-32.
Three-dimensional images can be assembled by piling up consecutive confocal fluorescent images obtained by confocal scanning laser microscopy. The present work was based on three-dimensional (50-microns-deep) images at high (x, y) resolution obtained with an MRC-500 after en bloc staining of thick slices of rat liver by chromomycin A3 for nuclear DNA. The results of studies on bleaching, fluorescence excitation and emission intensities at various depths of histologic preparations are described. These effects could be evaluated separately by acquiring piled-up ("brick-stepping") and non-piled-up ("side-stepping") (x, y) images at consecutive depths and also (x, z) images. Empirical equations allowed the fitting of experimental plots of bleaching versus time, at different laser intensities and at different depths, and of fluorescence emission intensity versus depth. The main conclusions were that under our experimental conditions: (1) there was no attenuation by depth of the fluorochrome penetration, (2) there was no attenuation of the exciting beam intensity up to at least 50 microns deep, (3) there was an attenuation of the fluorescence emission intensity by depth, (4) bleaching happened equally on all planes above and below any confocal plane being studied, and (5) the fluorescence bleaching half-life was independent of depth. A mathematical correction scheme designed to compensate for bleaching and for attenuation of fluorescence emission in depth is presented. This correction is required for obtaining three-dimensional images of better quality, for optimal three-dimensional image segmentation and for any quantitative analysis based upon voxel-discretized emission intensities (gray levels)--e.g., estimating, by confocal image cytometry, textural chromatin parameters and nuclear DNA amounts.
通过堆积共聚焦扫描激光显微镜获得的连续共聚焦荧光图像，可以组装出三维图像。目前的工作基于用放线菌素A3对大鼠肝脏厚切片进行整体染色后，使用MRC - 500获得的高（x，y）分辨率三维（50微米深）图像。描述了对组织学制剂不同深度处的漂白、荧光激发和发射强度的研究结果。通过在连续深度获取堆积（“砖式步进”）和非堆积（“侧步”）（x，y）图像以及（x，z）图像，可以分别评估这些效应。经验方程可以拟合不同激光强度和不同深度下漂白与时间的实验曲线，以及荧光发射强度与深度的实验曲线。主要结论是，在我们的实验条件下：（1）荧光染料穿透深度没有衰减；（2）激发光束强度至少在50微米深度内没有衰减；（3）荧光发射强度随深度衰减；（4）在任何被研究的共聚焦平面上方和下方的所有平面上，漂白情况相同；（5）荧光漂白半衰期与深度无关。本文提出了一种旨在补偿漂白和荧光发射深度衰减的数学校正方案。为了获得更高质量的三维图像、实现最佳的三维图像分割以及基于体素离散发射强度（灰度级）的任何定量分析（例如，通过共聚焦图像细胞术估计纹理染色质参数和核DNA量），都需要进行这种校正。
