This second edition volume expands on the previous edition with discussions on the latest approaches used in the spectral cytometry field. Beginning with a brief history of spectral cytometry development, this book continues with a section addressing new protocols in different areas of imaging cytometry based on the unique technology of Imagestream and also introduces FlowCam - cytometer with capabilities of analyzing phytoplankton and zooplankton. Written for the highly successful Methods in Molecular Biology series, chapters include brief introductions to their respective topics, complete lists of the materials and reagents necessary, and reproducible laboratory protocols with tips on troubleshooting and recommendations.Cutting-edge and thorough, Spectral and Imaging Flow Methods and Protocols, Second Edition is a valuable resources for beginners and professionals interested in learning more about this developing field.Chapters 1, 2, and 5 are available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.