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Background  to EM

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Research example: AD and the Tauopathies website

Interesting EM and other scientific links

Compiled by: Julian Thorpe


Morphometric Analysis

This is a method whereby the amounts of particular tissue or subcellular components are quantified. These components may be, for example, particular cell types (at the tissue level) , or the nucleus, mitochondria or secretory vesicles (see example below), for example, at the cellular level. To do this, the area of the particular component of interest and the total (tissue or cellular) area need to be determined. This used to be achieved by what was termed 'point counting', which involved the overlaying of an acetate sheet - with points regularly dotted upon it - over a photographic image. The subsequent counting of points over the component of interest and the whole tissue/cell was rather tedious and time-consuming. Nowadays - in the age of digital imaging - we can determine these areas more quickly and accurately (e.g. by the method outlined below) or by using freeware image analysis software like ImageJ. Once these areas have been determined we can state the actual volume fraction (Vv) within the tissue/cell that a particular component occupies because in a very thin section area/area is equivalent to volume/volume. The results are termed volume fractions or fractional volumes because they are expressed as a fraction of 1, with 1 being the total tissue or cell volume.

Example of the quantification of secretory vesicle accumulation in mutant yeast strains (from the work of John Armstrong's Group).
An initial digital image is taken and - with image analysis software - mapped for suitable contrast (image 1). The image is grey level *'thresholded' for area of interest and the area quantified (image 2). The image is 'thresholded' for the total area and is quantified (image 3).

The results may be displayed in tabulated or graphic form (as below):

* Explanation of 'thresholding': Being a digital image it is composed of pixels (in this case 800 x 1200 pixels make up the complete image). Each pixel has a numeric value associated with it which is equivalent to the 'grey level' value. In this system there are 4056 such grey levels, with 0 equivalent to black and 4056 to white. 'Thresholding' is the procedure whereby pixels within a certain range of grey level values (equivalent to the component of interest) are selectively highlighted on the image.