Counting dendritic spines in brain tissue slices by image correlation spectroscopy analysis

Summary Growth of new micrometre sized projections called dendritic spines in neurones has been linked to the encoding of long‐term memories in vertebrates. Numerous studies have been carried out at both the light and electron microscopy level to quantify dendritic spine densities in brain tissue in laboratory animals. Currently, such efforts using light microscopy have relied on manual counting of spines in confocal or two‐photon optical slice images of tissue containing fluorescently labelled spines. This manual approach can be slow and tedious, especially for samples with high spine densities. We introduce an alternative way of performing spine counting that uses an applied image intensity threshold followed by spatial image correlation spectroscopy (ICS) analysis. We investigated the effect of particle sizes above the diffraction limit on the autocorrelation analysis as well as the influence of background fluorescence. Our results show that, for well labelled cerebellar tissue samples imaged with a signal‐to‐noise ratio of 5 or greater, ICS‐based spine counts can be conducted with the same 15–20% precision as manual counting, but much more rapidly.