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. ? ?
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