Cutting through host autofluorescence: fluorescence lifetime imaging microscopy for visualising intracellular bacteria in Symbiodiniaceae

Abstract

SummaryPhotoperiodicity is key to the synchronization of life stages in Symbiodiniaceae,Breviolum minutumwhich harbors taxonomically diverse epi- and endosymbiotic bacteria. We examined influence of a light dark regime on the spatial association betweenB. minutumand bacteria.We employed a novel approach using combination of fluorescence lifetime imaging microscopy with fluorescencein situhybridisation approach to clearly distinguish labelled intracellular bacteria from broad spectrum (450–800 nm) background autofluorescence ofB. minutum.Bacteria were observed inside, tethered to and burrowing into the cell exterior, and at the furrow of dividing cells inB. minutum. Significant changes in the abundance of intracellular bacteria relative to autofluorescence inB. minutumcells were observed at initiation of light and dark conditions.We suggest that the onset of bacterial endosymbiosis is linked to the photoperiod driven changes inB. minutumlife stages. The re-organisation of thecal plates during cell division ofB.minutumin dark is likely to result in internalisation of bacteria.