Stereology Software

Stereology Stereo Investigator is advanced scientific software for unbiased design-based stereology. Stereo Investigator can analyze data from multiple modalities: using live images from digital or video cameras or stored image sets from confocal microscopes, electron microscopes, and scanning tomographic sources. Stereology Analyzer is a simple to use software tool for reliably estimating quantifications of important 3D structures. This tool is general in its implementation,.

Stereology involves the quantitative assessment of objects in three dimensions. Examples of measurements that can be obtained include total cell count, cell density, cell size, tissue volume, and the length density and/or total length of linear structures such as nerve process or blood vessels.

Current design-based stereological methods have evolved to provide results that are unbiased, efficiently obtained, and repeatable. A key stereological practice that is intended to reduce bias and aid both efficiency and repeatability has been termed systematic random sampling (SRS).

According to this concept, selection of the initial tissue measurement site is randomized, but selection of ensuing measurement sites is regular. One of the most commonly used stereological tools for cell counting, especially in neuropathology, is the optical fractionator approach. This approach employs a virtual three-dimensional geometric probe called a disector that is used to count the number of objects (e.g., cells) in a well-defined, three-dimensional region (e.g., the hippocampal region of the brain). The results of disector counting are unaffected by the size, shape, or orientation of the objects. In anatomic pathology, the disector principle may be used to count cells in paired sets of thin histologic sections (physical disector) or at various focal plane depths in a subsampled series of thick histologic sections (optical disector). A disadvantage of the physical disector approach is the need to align the paired sections based on morphologic landmarks to ensure that a cell is never counted twice; however, the optical disector approach may not be feasible if thick sections cannot be prepared or if the cell type of interest cannot be reliably identified.

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The optical fractionator is a variation of the optical disector approach. The fractionator differs from earlier methods in that it is not necessary to determine the volume of the region in order to estimate the total cell number. This is because unlike prior methods, the entire region of interest is sampled. In terms of obtaining counts and measurements of objects in complex three-dimensional structures, computerized stereological analysis is only the final step in an elaborate and exacting process.

Considerations of tissue collection and preparation are at least equally important. That is why among laboratories performing stereological procedures it is generally considered standard practice and essential that assay optimization be conducted prior to the initiation of experiments intended to provide definitive data. The purpose of this optimization work is to refine various aspects of tissue preparation and evaluation that include but are not limited to dissection technique; tissue preservation, transport and storage; the plane of tissue sectioning and microtomy technique; staining procedures; the verification of consistent microanatomical landmarks, and the establishment of various parameters specific to stereologic cell counting.

It is unlikely that repeatable results can be obtained if inadequate consideration is given to any of these aspects. Advantages of using EPL for your Stereological projects: • EPL technicians have extensive expertise in the creation of thick frozen or paraffin-embedded sections for cell counting purposes • Immunohistochemical staining and evaluation can be performed using either chromogenic (diaminobenzidine, DAB) or fluorescent markers • Stereology projects supervised by an ACVP board-certified pathologist • EPL uses MBF Bioscience’s popular and robust StereoInvestigator software For more information.

Cell counting involves identifying the boundaries of the cells of interest in three dimensions. The following rules should be followed in the execution of rigorous stereological analysis: Figure 1.

Choosing a unique part of the cell to count helps ensure the even application of your counting protocol • Decide on a unique point (the characteristic point) that can be identified for each cell. This can be the top of the cell, i.e., the first part of the cell to come into focus while focusing down through the tissue, or perhaps the top of the nucleus, or even the nucleolus if the cells being examined have only one nucleolus per cell. • The unique point must be visible for each cell to be counted. • At each investigated microscopic field, an unbiased counting frame consisting of two exclusion lines and two inclusion lines is placed on the section. • Select a cell which is a potential candidate for counting and focus on its selected ‘point’ (e.g., ‘top’, nucleolus, etc.) and follow the rules below for counting and see Figure 2 below: 1. Any point that touches the exclusion line, crosses the exclusion line, or is outside of the counting frame is not counted. 2. For any point that is still a valid candidate and that is a) inside the counting frame, b) touching an inclusion line from either inside or outside the counting frame, or c) crossing the an inclusion line, remains a candidate to be counted. 3. If an object touches or crosses both an inclusion line and an exclusion line, it is not counted (as per rule #1 above).