Birmingham Women's Hospital View Institution's Website 1 article published in JoVE Biology Techniques for Imaging Ca2+ Signaling in Human Sperm Katherine Nash1, Linda Lefievre2, Ruben Peralta-Arias1, Jennifer Morris1, Aduen Morales-Garcia1, Tom Connolly2, Sarah Costello1, Jackson C. Kirkman-Brown3, Stephen J. Publicover1 1School of Biosciences, University of Birmingham, 2School of Medicine, University of Birmingham, 3Centre for Human Reproductive Science, Birmingham Women’s Hospital Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.