DeadEasy neurons: automatic counting of HB9 neuronal nuclei in Drosophila

Research into the genetic basis of nervous system development and neurodegenerative diseases requires counting neurons to find out the extent of neurogenesis or neuronal loss. Drosophila is a widely used model organism for in vivo studies. However, counting neurons throughout the nervous system of the intact animal is humanly unfeasible. Automatic methods for cell counting in intact Drosophila are desirable. Here, we show a method called DeadEasy Neurons to count the number of neurons stained with anti-HB9 antibodies in Drosophila embryos. DeadEasy Neurons employs image filtering and mathematical morphology techniques in 2D and 3D, followed by identification of nuclei in 3D based on minimum volume, to count automatically the number of HB9 neurons in vivo. The resultant method has been validated for Drosophila embryos and we show here how it can be used to address biological questions. Counting neurons with DeadEasy is very fast, extremely accurate, and objective, and it enables analyses otherwise humanly unmanageable. DeadEasy Neurons can be modified by the user for other applications, and it will be freely available as an ImageJ plug-in. DeadEasy Neurons will be of interest to the microscopy, image processing, Drosophila, neurobiology, and biomedical communities.