IMPROVED CELL COUNTING USING THE OPTICAL DISSECTOR

描述(申请人提供)：定量形态在生物医学研究中很重要。为了量化组织中的颗粒数量，必须对这种组织进行切片。光学析镜计数技术在发育研究中具有特殊的优势，因为与传统的轮廓计数技术不同，即使在不同组之间的颗粒大小变化时，它也不受影响。然而，最近发现，当组织切片在z轴上进行不同的压缩时，光学分析器可能会显著偏向。这一发现引发了一场关于如何适当计算组织切片中颗粒的争论。改进光学解析器颗粒计数技术将有助于调查人员获得有效、准确和基本无偏的数据，客观的偏差评估将解决关于细胞计数技术的大部分争论。已经开发出一种简单而有效的技术，该技术允许定量地评估颗粒在组织切片的z轴上的差异变形和分布。石蜡切片和塑料切片的数据显示，发生了显著的差异压缩，因此，如果按照目前的推荐使用，光学分析器对大多数组织切片(如果不是所有的组织切片)是有偏差的。拟议的工作将系统和定量地检查所有五种常用组织切片(冰冻切片、石蜡切片、甲基丙烯酸酯塑料切片、火棉胶塑料切片和振动刀切片)受差异压缩影响的程度。将优化一种简单的新方法来评估不同的节段压缩。将设计战略，通过预测偏差并以最大限度地减少由此产生的偏差的方式放置计数框来改进光学析像器计数技术，并将偏差降至最低。用光学分离器从振镜、低温和火棉胶塑料切片中获得的细胞计数将通过与3D序列重建的比较进行校准，并且将与轮廓计数方法的偏差进行比较。最后，也是最重要的是，将向调查人员提供使用改进的光学解剖器法进行细胞计数的简单易用的方案，并将根据校准的分析，就不同计数技术的确切优缺点提供明确、实用的指导。这项拟议的工作将使研究人员能够在使用光学分割器时对他们的颗粒物计数进行容易和简单的校准和偏差评估。迫切需要这种研究方法的进步来提高目前在胚胎学、药物测试和发育生物学研究中获得的数据的有效性和质量，其中许多研究是由NIH，特别是NICHD资助的。

DESCRIPTION (provided by applicant): Quantitative morphology is important in biomedical research. In order to quantify particle numbers in tissues, such tissues have to be sectioned. The optical dissector counting technique has particular advantages in developmental studies, because it is not affected even when particle size changes between groups, unlike conventional profile counting techniques. It was recently discovered, however, that the optical dissector can be significantly biased when tissue sections are differentially compressed in the z-axis. This finding has fueled an already contentious debate about how to appropriately count particles in tissue sections. Improving the optical dissector particle counting technique would help investigators to obtain valid, accurate, and largely unbiased data, and an objective bias assessment would resolve much of the debate on cell counting techniques. A simple and efficient technique has been developed which allows one to assess quantitatively the differential distortion and distribution of particles in the z-axis of tissue sections. Data on paraffin and plastic sections show that significant differential compression occurs, and thus that the optical dissector is biased for most if not all tissue sections if used as currently recommended. The proposed work will examine systematically and quantitatively the extent to which sections are affected by differential compression, for all five types of tissue sections commonly used (cryosections, paraffin sections, methacrylate plastic sections, celloidin plastic sections, and vibratome sections). A simple new methodology will be optimized to assess differential section compression. Strategies will be designed to improve the optical dissector counting technique and to minimize biases, by predicting the bias and placing counting boxes in ways that minimize the resulting bias. Counts of cells obtained with the optical dissector from vibratome, cryo- and celloidin plastic sections will be calibrated by comparison with 3D serial reconstructions, and the biases will be compared with those of profile counting methods. Finally, and most importantly, investigators will be provided with simple and easy-to-use protocols for cell counting using an improved optical dissector method, and clear, practical guidance will be given about the precise advantages and disadvantages of different counting techniques, based on calibrated analyses. The proposed work will allow researchers to perform easy and simple calibration and bias assessments on their particle counts when they use the optical dissector. This advance in research methodology is urgently needed to improve the validity and the quality of data which are currently obtained in studies in embryology, drug testing, and developmental biology, many of which are funded by the NIH, and in particular the NICHD.