Qualitative electron microscopy studies coupled with expert interpretation and analysis play a valid and useful role in the initial stages of many scientific problems. However, it is the use of quantitative methods that is the hallmark of modern scientific research. Acquiring quantitative morphological data (volumes, surface areas, lengths, numbers, etc) about organelles, cells, tissues and organs should be the next step towards elucidating the exact correlation between structure and function [1]. To this end, we have used stereological approaches to investigate mitochondrial integrity/functionality.
To simplify, stereology aims to make quantitative estimates of the “amount” of a geometrical feature within the object of interest. In doing so, it manages to provide 3-D data that makes the interpretation of functional morphology more effective. Although there are more intricate stereological approaches, here we present a relatively quick and easy method to calculate the volume fraction of a component within a reference volume. This is a simple and very widely used parameter to express the proportion of a particular phase or component within the whole structure [2].
In our study, the volume fraction of cristae within the mitochondria, Vv (cristae, mitochondria), was estimated by placing a combined point counting grid (CPCG) over each mitochondrion micrograph. Thirty mitochondria from each experimental group were used for this stereological analysis. The CPCG used for this study was composed of two sets of points of different densities on the same grid; 9 fine points per coarse point. The volume of reference (mitochondria) was estimated by counting the number of coarse points that hit the reference space and multiplied by 9. The volume of the particular phase (cristae) was estimated by counting the number of fine and coarse points that hit the cristae. Thus, the volume fraction of the cristae within the mitochondria was calculated and expressed as a percentage. Student’s t-test was used to assess whether there was a significant difference between the samples at a 99% confidence level.
Our stereological study demonstrates that there is a contrasting mitochondrial morphology between female/male gametes and somatic cells. These differences in morphology are in turn linked to a functional division of labour between sperm and egg as demonstrated by other molecular approaches [3,4]. This analysis is an example that showcases how the application of stereology on Electron microscopy can be a powerful tool allowing quantification of morphological data.
References:[1] Mayhew, T.M (1991).[2] Howard, C.V. and Reed, M.G (2010).[3] Wilson de Paula et al., (2013a).[4] Wilson de Paula et al., (2013b)