Labeling fixed brain tissue with fluorescent synaptic and cellular markers can help assess circuit connectivity. Despite the diffraction-limited resolution of light microscopy there are several approaches to identify synaptic contacts onto a cell-of-interest. Understanding which image quantification methods can be applied to estimate cellular and synaptic connectivity at the light microscope level is beneficial to answer a range of questions, from mapping appositions between cellular structures or synaptic proteins to assessing synaptic contact density onto a cell-of-interest. This chapter provides the reader with details of the image analysis methods that can be applied to quantify in situ connectivity patterns at the level of cellular contacts and synaptic appositions.
Keywords: 3D appositions; Correlation analysis; Dot-finding; Immunofluorescence; Synaptic puncta; Volume estimation.