Gardella Dean, Hatton William J, Rind Howard B, Rosen Glenn D, von Bartheld Christopher S
Department of Physiology and Cell Biology, Mailstop 352, University of Nevada School of Medicine, Reno 89557, USA.
J Neurosci Methods. 2003 Mar 30;124(1):45-59. doi: 10.1016/s0165-0270(02)00363-1.
The optical disector is among the most efficient cell counting methods, but its accuracy depends on an undistorted particle distribution in the z-axis of tissue sections. Because the optical disector samples particle densities exclusively in the center of sections, it is essential for unbiased estimates of particle numbers that differential shrinkage or compression (and resulting differences in particle densities along the z-axis) are known and corrected. Here we examined, quantified, and compared differential shrinkage and compression of vibratome-, celloidin- and cryosections. Vibratome sections showed a significant z-axis distortion, while celloidin- and cryosections were minimally distorted. Results were directly compared with previous data obtained from paraffin and methacrylate sections. We conclude that z-axis distortion varies significantly between embedding and sectioning methods, and that vibratome-, methacrylate- and paraffin sections can result in grossly biased estimates. We describe a simple method for assessing differential z-axis shrinkage or compression, as well as simple strategies to minimize the bias of the optical disector. Minimal bias can be achieved by either adjusting the placement and extent of counting boxes and guard spaces for sampling, or by applying a correction factor in cases when guard spaces are deemed essential for particle recognition.
光学分选器是最有效的细胞计数方法之一，但其准确性取决于组织切片z轴上未扭曲的颗粒分布。由于光学分选器仅在切片中心对颗粒密度进行采样，因此了解并校正差异收缩或压缩（以及沿z轴的颗粒密度差异）对于无偏估计颗粒数量至关重要。在这里，我们研究、量化并比较了振动切片机切片、火棉胶切片和冰冻切片的差异收缩和压缩情况。振动切片机切片显示出明显的z轴扭曲，而火棉胶切片和冰冻切片的扭曲最小。结果直接与先前从石蜡切片和甲基丙烯酸酯切片获得的数据进行了比较。我们得出结论，z轴扭曲在包埋和切片方法之间存在显著差异，并且振动切片机切片、甲基丙烯酸酯切片和石蜡切片可能导致严重的偏差估计。我们描述了一种评估z轴差异收缩或压缩的简单方法，以及将光学分选器偏差降至最低的简单策略。通过调整计数框的放置和范围以及用于采样的保护空间，或者在保护空间被认为对颗粒识别至关重要的情况下应用校正因子，可以实现最小偏差。
