High-speed multifocal plane fluorescence microscopy for three-dimensional visualisation of beating flagella.

Analysis of flagellum and cilium beating in three dimensions (3D) is important for understanding cell motility, and using fluorescence microscopy to do so would be extremely powerful. Here, high-speed multifocal plane fluorescence microscopy, where the light path is split to visualise multiple focal planes simultaneously, was used to reconstruct and movement in 3D. These species are uniflagellate unicellular parasites for which motility is vital. It was possible to use either a fluorescent stain or a genetically-encoded fluorescent protein to visualise flagellum and cell movement at 200 Hz frame rates. This addressed two open questions regarding and flagellum beating, which contributes to their swimming behaviours: 1) how planar is the flagellum beat, and 2) what is the nature of flagellum beating during 'tumbling'? We showed that has notable deviations from a planar flagellum beat, and that during tumbling the flagellum bends the cell and beats only in the distal portion to achieve cell reorientation. This demonstrates high-speed multifocal plane fluorescence microscopy as a powerful tool for the analysis of beating flagella.