![]() ![]() Since microglia are finely tuned to neuronal and glial function through continuous cell-cell cross-talk and in vivo motility 4, 5, microglia morphologies may serve as indicators of diverse cell functions and dysfunctions in the brain. Microglia cell ramification is commonly defined as a complex shape having multiple processes and is often reported as the number of endpoints per cell and the length of cell processes. Microglia may also become polarized and rod-shaped 3. Microglia have an immediate and diverse morphologic response to alterations in brain physiology 1 along a continuum of possibilities that range from hyper-ramification and highly complex morphologies to de-ramified and amoeboid morphologies 2. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. ![]() Imagej fiji software#This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. ![]()
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