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

Photoperiodicity is key to the synchronization of life stages in Symbiodiniaceae, Breviolum minutum which harbors taxonomically diverse epi- and endosymbiotic bacteria. We examined influence of a light dark regime on the spatial association between B. minutum and bacteria. We employed a novel approach using combination of fluorescence lifetime imaging microscopy with fluorescence in situ hybridisation approach to clearly distinguish labelled intracellular bacteria from broad spectrum (450–800 nm) background autofluorescence of B. minutum . Bacteria were observed inside, tethered to and burrowing into the cell exterior, and at the furrow of dividing cells in B. minutum . Significant changes in the abundance of intracellular bacteria relative to autofluorescence in B. minutum cells were observed at initiation of light and dark conditions. We suggest that the onset of bacterial endosymbiosis is linked to the photoperiod driven changes in B. minutum life stages. The re-organisation of thecal plates during cell division of B.minutum in dark is likely to result in internalisation of bacteria. ### Competing Interest Statement The authors have declared no competing interest.