Cells are the building blocks of life, from single-celled microbes through to multi-cellular organisms. To understand a multitude of biological processes we need to understand how cells behave, how they interact with each other and how they respond to their environment. The use of new methodologies is changing the way we study cells allowing us to study them on minute scales and in unprecedented detail. These same methods are allowing researchers to begin to sample the vast diversity of microbes that dominate natural environments. The aim of this special issue is to bring together research and perspectives on the application of new approaches to understand the biological properties of cells, including how they interact with other biological entities. This article is part of a discussion meeting issue ‘Single cell ecology’.