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Unbiased Stereology Three Dimensional Measurement in Microscopy C.V

Unbiased Stereology: Three-Dimensional Measurement in Microscopy (Advanced Methods)

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Garland Science - Book: Unbiased Stereology + 2

Principles and Practices of Unbiased Stereology fills a need in the biomedical community as a clear, user-friendly introduction to this area for the increasing number of scientists who need to learn these techniques for their research. The work moves logically from a discussion of the historical background of stereology to full explanations of terms, concepts, and tools, with the latter part of the manuscript devoted to typical stereology designs.

Principles and Practices of Unbiased Stereology fills a need in the biomedical community as a clear, user-friendly introduction to this area for the increasing number of scientists who need to learn these techniques for their research. The work moves logically from a discussion of the historical background of stereology to full explanations of terms, concepts, and tools, with the latter part of the manuscript devoted to typical stereology designs.

Unbiased Stereology: A Concise Guide - SRC

  • Stereologer (ster-ē-äl-əjər): an innovative, user-friendly and state-of-the-art system (hardware-software-microscope-imaging) for quantifying biological structure with unbiased stereology.

UNBIASED STEREOLOGY: DESIGN-BASED VERSUS MODEL-BASED METHODS

Additional technical issues are outlined below: (1) Different batches of the same staining reagents (especially immunofluorescence reagents) might yield inconsistent results. Test the reagents before use for each new lot of reagent purchased. (2) During the immunohistochemistry procedure, dehydrating slices in the same situation and length to keep tissue shrinkage in the comparable range can also be difficult. (3) Tissue structure needs to be identifiable, results are more accurate when 100% of the tissue is available for analysis. Occasionally sections are torn or lost during processing and those animals should be discarded. (4) The measurement of dissector/tissue section thickness in each ROI along the -axis can be manipulated by many factors related to the slice (e.g., irregularity in the section shrinkage) and investigator’s experience with the optics (e.g., thickness of coverslips and type of lens). Performing a careful calibration and pilot study when using a stereological method is useful for avoiding bias related to the practical application. (5) Unbiased stereology methods are tedious and require extensive training. Identifying the top and the bottom of the slices along the -axis is somewhat difficult to detect. Find the first position in the -axis where something comes into focus in the center of the counting frame. Then continue moving in one-direction (up), and when the field of view is completely out of focus, set the current position to 0 (top position). Move down the -axis until nothing is in focus at the opposite end of the slide and push “stop” on the navigator for the bottom position of the slide. The field of view should be unfocused at the top, focused in the middle, and unfocused at the bottom of the slide to get an accurate counting frame volume.

Additional technical issues are outlined below: (1) Different batches of the same staining reagents (especially immunofluorescence reagents) might yield inconsistent results. Test the reagents before use for each new lot of reagent purchased. (2) During the immunohistochemistry procedure, dehydrating slices in the same situation and length to keep tissue shrinkage in the comparable range can also be difficult. (3) Tissue structure needs to be identifiable, results are more accurate when 100% of the tissue is available for analysis. Occasionally sections are torn or lost during processing and those animals should be discarded. (4) The measurement of dissector/tissue section thickness in each ROI along the -axis can be manipulated by many factors related to the slice (e.g., irregularity in the section shrinkage) and investigator’s experience with the optics (e.g., thickness of coverslips and type of lens). Performing a careful calibration and pilot study when using a stereological method is useful for avoiding bias related to the practical application. (5) Unbiased stereology methods are tedious and require extensive training. Identifying the top and the bottom of the slices along the -axis is somewhat difficult to detect. Find the first position in the -axis where something comes into focus in the center of the counting frame. Then continue moving in one-direction (up), and when the field of view is completely out of focus, set the current position to 0 (top position). Move down the -axis until nothing is in focus at the opposite end of the slide and push “stop” on the navigator for the bottom position of the slide. The field of view should be unfocused at the top, focused in the middle, and unfocused at the bottom of the slide to get an accurate counting frame volume.