Vamping: Stereology-based automated quantification of fluorescent puncta size and density

The size of dendritic spines and postsynaptic densities (PSDs) is well known to be correlated with molecular and functional characteristics of the synapse. Thus, the development of microscopy methods that allow high throughput quantification and measurement of PSDs is a contemporary need in the field of neurobiology. While the gold standard for measurement of sub-micrometer structures remains electron microscopy (EM), this method is exceedingly laborious and therefore not always feasible. Immunohistochemistry (IHC) is a much faster technique for identifying biological structures such as PSDs, but the fluorescent images resulting from it have traditionally been harder to interpret and quantify. Here, we report on two new image analysis tools that result in accurate size and density measurements of fluorescent puncta. Anti-PSD-95 staining was used to target synapses. The new technique of vamping, using Volume Assisted Measurement of Puncta in 2 and 3 Dimensions (VAMP2D and VAMP3D) respectively, is based on stereological principles. The fully automated image analysis tool was tested on the same subjects for whom we had previously obtained EM measurements of PSD size and/or density. Based on highly consistent results between data obtained by each of these methods, vamping offers an expedient alternative to EM that can nonetheless deliver a high level of accuracy in measuring sub-cellular structures.

► We developed stereology-based automated tools for measurement of fluorescent puncta.
► VAMP2D measures puncta size.
► VAMP3D quantifies puncta density.
► Vamping was validated against electron microscopy on same subjects.