I would have to know more about your experimental design and hypothesis in order to advise you. Q: How can I count the vessels? Because we have different orientations. Without proper adherence to both of these principles, the “equal opportunity to be counted once and only once” is not achieved. In addition, stereology uses a 3D disector or counting frame to ensure that each cell in the section thickness is counted only in the area of the section thickness that is not damaged. When you are only examining a single 2D image of a 3D specimen, you cannot identify the unique point, only the number of profiles or cell pieces in the image. Each cell is represented by a unique point – a feature that can only occur in the cell once (like the cell top). Stereology achieves unbiased quantification by providing rules to ensure that each cell has an equal opportunity to be counted once and only once. Q: If I use imagej to count cells in 2d images, is that biased? This can be achieved by a combination of the feature to be quantified, the tissue preparation or the probe having isotropy. To achieve an unbiased result from stereologic analysis, the probe should interact with the feature to be quantified in an isotropic way. Q: Are you saying that, for any analysis to be possible, it needs to be isotropic? But keep in mind you will only be finding out about the biopsy, not about the whole region. But if you need to quantify it, you can use stereology. What are you trying to find out with the biopsy please? If it is something that you can find out about qualitatively, then there is no need for quantification. Q: I just wanted to add, about lung sterology, that we use small human lung biopsies about 2mm to be cut at 2 µ m. Yes, it is relevant for any biological tissue you are viewing under the microscope. It is shown that stereological techniques, based on a sound theoretical basis, are powerful and reliable and have been used successfully.Q: Is the information presented in this webinar relevant for lung stereology? Methods for estimating the volume of the brain and the brain cortex, the total number of endothelial cells in cortical microvessels, the length of cortical microvessels, and the total surface area of cortical microvessel walls are presented step by step in a way understandable for readers with little mathematical background. Stereological methods and their concepts are illustrated using an ongoing study of the dose response of the microvessels in proton-irradiated hemibrain. This presentation reviews the methodology and format of using confocal microscopy and stereological methods to quantify tissue parameters, cell number, tissue volume and surface area, and vessel length using the microvasculature as a model tissue. An important focus is on the vasculature. Before proceeding to new clinical protocols, radiation biologists involved with conformal proton therapy believe it is necessary to quantify the dose response and tolerance of the organs and tissues that will be irradiated. 160, 110–119 (2003).Ĭlinicians involved in conventional radiation therapy are very concerned about the dose–response relationships of normal tissues. Stereology Techniques in Radiation Biology.
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