Seeing the wood for the trees: towards improved quantification of glial cells in central nervous system tissue

Sinéad Healy; Jill McMahon; Una FitzGerald

doi:10.4103/1673-5374.235222

摘要：The following mini-review attempts to guide researchers in the quantification of fluorescenfly-labelled proteins within cultured thick or chromogenically-stained proteins within thin sections of brain tissue.It follows from our examination of the utility of Fiji ImageJ thresholding and binarization algorithms.Describing how we identified the maximum intensity projection as the best of six tested for two dimensional (2D)-rendering of three-dimensional (3D) images derived from a series of z-stacked micrographs,the review summarises our comparison of 16 global and 9 local algorithms for their ability to accurately quantify the expression of astrocytic glial fibrillary acidic protein (GFAP),microglial ionized calcium binding adapter molecule 1 (IBA1) and oligodendrocyte lineage Olig2 within fixed cultured rat hippocampal brain slices.The application of these algorithms to chromogenically-stained GFAP and IBAI within thin tissue sections,is also described.Fiji's BioVoxxel plugin allowed categorisation of algorithms according to their sensitivity,specificity accuracy and relative quality.The Percentile algorithm was deemed best for quantifying levels of GFAP,the Li algorithm was best when quantifying IBA expression,while the Otsu algorithm was optimum for Olig2 staining,albeit with over-quantification of oligodendrocyte number when compared to a stereological approach.Also,GFAP and IBA expression in 3,3'-diaminobenzidine (DAB)/ haematoxylin-stained cerebellar tissue was best quantified with Default,Isodata and Moments algorithms.The workflow presented in Figure 1 could help to improve the quality of research outcomes that are based on the quantification of protein with brain tissue.