IMPROVED CELL COUNTING USING THE OPTICAL DISSECTOR

描述（申请人提供）：定量形态学在生物医学研究中很重要。为了量化组织中的粒子数，必须对这些组织进行切片。光学解剖计数技术在发育研究中具有特别的优势，因为它不受影响，即使在组之间的颗粒大小变化，不像传统的剖面计数技术。然而，最近发现，当组织切片在z轴上被不同地压缩时，光学解剖器可能会显着偏倚。这一发现引发了一场关于如何正确计数组织切片中的粒子的争论。改进光学解剖粒子计数技术将有助于研究人员获得有效、准确和基本上无偏倚的数据，客观的偏差评估将解决关于细胞计数技术的许多争论。一种简单而有效的技术已经被开发出来，它允许人们定量地评估组织切片z轴上粒子的差异畸变和分布。石蜡切片和塑料切片的数据表明，发生了显著的差异压缩，因此，如果按照目前的建议使用，光学解剖器对大多数（如果不是所有）组织切片是有偏差的。拟议的工作将系统和定量地检查切片受差压影响的程度，包括所有常用的五种组织切片（冷冻切片、石蜡切片、甲基丙烯酸酯塑料切片、纤维素塑料切片和振动切片）。将优化一种简单的新方法来评估差截面压缩。将设计策略，以改善光学解剖计数技术和减少偏差，通过预测偏差和放置计数盒的方式，以尽量减少由此产生的偏差。用光学解剖器从振动体、低温和纤维素塑料切片中获得的细胞计数将通过与3D序列重建的比较来校准，并将与轮廓计数方法的偏差进行比较。最后，也是最重要的一点，我们将为研究人员提供简单易用的细胞计数方案，使用一种改进的光学解剖法，并根据校准分析，明确、实用地指导不同计数技术的精确优缺点。这项提议的工作将允许研究人员在使用光学解剖器时对他们的粒子计数进行简单而简单的校准和偏差评估。为了提高目前在胚胎学、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，迫切需要这种研究方法的进步，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.