Locations marked with dotted lines have been skipped because they do not contain a large area of non-nuclear beta cell.
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This image has been used to demonstrate how to randomly select regions within 30 different cells for point- counting stereology, by navigating in a snake-like pattern across the islet. However, this magnification provides a useful low resolution 'overview'. Note that it is impossible to see sufficient detail, to distinguish between different types of cells at this resolution. 30 grids x 54 points = 1620, which is already good numbers for an estimate, but since this tissue may not be represented, this was repeated on similar sections from three different animals.įigure 2 - a low resolution micrograph of a 60 nm section cut from a mouse islet, imaged at 140Ã- on the same instrument. An example of this is shown in Figure 2, where 30 different cells (and different grids) were counted on a single thin section. Using a live image, this can be achieved by simply "spinning" the X-Y stage controls to a new area, and then progressing through the entire specimen without doubling back over the same area twice. It is important this image is selected at random from within the large sample of tissue, so as not to be biased by any local differences between the density of organelles. After tallying numbers, a new image is placed under the same grid. many grids) to achieve a reliable statistical estimate. Results from this grid would indicate 5.5% of this area (5/54) is occupied by mature granules, but this clearly isn't a good sample size, hence this process must be repeated over many cells (i.e. Note that any point on the membrane / on the line is counted as in. 3 intersections points fell inside a mature granule (blue arrows) and 5 points fell inside a mitochondria. In this image I recorded that all 54 points fell inside non-nuclear volume in my desired cell type. Points are only counted inside if they are "on the line or in" - similar to tennis! Blue arrows show points inside a mature granule, green arrows show points inside a mitochondria, and the black arrow show a point that is almost inside a mitochondria, but isn't counted. This image is a 2D micrograph of a region of beta cell imaged at 20kÃ- magnification. In this example I was interested in tallying the percent of non-nuclear volume occupied by mature granules (the round things) and mitochondria (the dark sausage things).įigure 1 - an example of point counting stereology using a grid with 54 intersection points (9 colums x 6 rows). For each intersection point, the user then tallies how many points fall within each compartment. The image below ( Figure 1) shows an example of this, where the grid is 9 x 6 lines - meaning a total of 54 intersection points (NOTE: lines and point on the edge of the screen are not counted).
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In point counting stereology an image from your microscope is projected onto a computer screen and your microscope control program and/or special imaging software projects a uniform grid over this image. There are many types of stereology, but on this page I highlight one of the most common and easy forms of stereology: " point counting stereology". I've written this page to show just how quick and easy stereology can be. and yet I've noticed that many are not! Due to its rapid nature (one day of counting is often enough to generate an entire table of results), and the fact that stereology can be done live on a microscope (no saving or storing of images required), or using existing 2D and/or 3D images of cells (images which have already been collected), I believe stereology is a brilliant compliment to slower methods of 3D analysis, such as the use of transmission electron tomography and scanning electron tomography to acquire 3D images. As such, I believe all cell biologists should be familiar and experienced with this technique. In the fields of cell biology and electron microscopy, stereology allows scientists to estimate the volume, surface area, number and the size of cellular compartments by looking at a relatively small number of 2D slices and, unlike 3D reconstruction and segmentation (methods which are notoriously slow) it allows scientists to attain these estimates very very fast. The 3D volume of any object can be determined from the 2D areas of its planar sections. Put another way, stereology exploits the fact that many 3D quantities can be determined without 3D reconstruction. To achieve this, stereology uses a random sampling and systematic approach to provide unbiased quantitative data. Stereology - " the spatial interpretation of sections" - is a technique for 3D interpretation of (2D) planar sections of materials or tissues.
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