Single-cell volume is an important biophysical parameter associated with virtually all aspects of cell physiology, such as cell growth throughout the cell cycle and changes in cell shape. Despite its fundamental importance for cell biology, accurate measurements of single live cell volume based on microscopy are extremely challenging due to the complexity of cell shape and confocal reconstruction artefacts.

To assess single-cell volume, our laboratory adapted the previously described fluorescence exclusion method (FXm). FXm infers cell volume based on the drop in fluorescence intensity created by a cell due to exclusion of an impermeable fluorescent dye present in the culture medium. Intensity measurements are carried out with a single z plane at low magnification and therefore are fast and incur lower phototoxicity compared to 3D confocal reconstruction. This allowed us to track the volume of a large number of individual cells simultaneously with desirable time resolution — from milliseconds (for example, during osmotic shock) to days (to follow cell cycle growth). FXm was successfully used for measuring the volume of mammalian cells with high accuracy, and for smaller cells such as bacteria and fission yeast. Recent modification of FXm enabled simultaneous measurements of the cell and nuclear volume, whereby nuclear volume is calculated based on exclusion of a cytoplasmic fluorescent protein.