Fluorescence lifetime imaging microscopy (FLIM): a non-traditional approach to study host-microbial symbioses
Pranali Deore A , Iromi Wanigasuriya B , Sarah Jane Tsang Min Ching A , Douglas R. Brumley C , Madeleine J. H. van Oppen A D , Linda L. Blackall A * and Elizabeth Hinde BA School of BioSciences, The University of Melbourne, Vic., Australia.
B School of Physics, The University of Melbourne, Vic., Australia.
C School of Mathematics and Statistics, The University of Melbourne, Vic., Australia.
D Australian Institute of Marine Science, Townsville, Qld, Australia.
Dr Pranali Deore is an environmental microbiologist, and her current research focuses on developing advanced bio-optic tools for visualisation of microalgae-bacteria symbiosis. Her expertise is in microalgal photo-physiology and use of Fourier transform spectroscopy to study microbial interactions. She is currently a post-doctoral research fellow at the University of Melbourne. |
Dr Iromi Wanigasuriya is a molecular biologist and a research fellow at The University of Melbourne. She is currently using advanced microscopy techniques to understand dynamic epigenetic mechanisms. |
Sarah Jane Tsang Min Ching has a Master of Science and is a research associate at The University of Melbourne. She uses advanced molecular biology and microbiology techniques for isolation and identification of microbial symbionts of corals. Her past research involves understanding metabolomic changes in sea anemones (laboratory model of corals) transplanted with heat evolved Symbiodiniaceae strains. |
Dr Douglas Brumley BSc (Hons), PhD (Cantab) is a Senior Lecturer at The University of Melbourne. He leads an interdisciplinary research group which utilises mathematics, microfluidics and microscopy to study a range of dynamic processes in biology including bacterial motility, symbioses, nutrient cycling and flows around coral reefs. |
Professor Madeleine J. H. van Oppen is an ecological geneticist with an interest in microbial symbioses and climate change adaptation of reef corals. Her early career focused on evolutionary and population genetics of algae and fish, and subsequently corals. Currently, her team is using bioengineering approaches aimed at increasing coral climate resilience and the likelihood that coral reefs will survive this century. These interventions include coral host hybridisation and conditioning, directed evolution of microalgal symbionts and bacterial probiotics. |
Professor Linda L. Blackall is an environmental microbial ecologist, who has studied many different complex microbial communities ranging from host associated through to free living in numerous environments. One of her research fields is the microbiota of corals and sponges. The numerous methods she develops and employs in her research allow elucidation of microbial complexity and function in these diverse biomes. |
A/Professor Elizabeth Hinde is a cellular biophysicist with an expertise in fluorescence lifetime imaging microscopy and fluorescence fluctuation spectroscopy. She develops methods to spatially map live cell nuclear architecture and is using this technology to uncover the impact this dynamic structural framework has on DNA target search. |
Microbiology Australia 43(1) 22-27 https://doi.org/10.1071/MA22008
Submitted: 8 March 2022 Accepted: 17 March 2022 Published: 14 April 2022
© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the ASM. This is an open access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND)
Abstract
Corals and their photosynthetic endosymbiotic algae (Symbiodiniaceae) produce a strong autofluorescent signal that spans the visible to near-infrared (NIR) spectrum. However, this broad-spectrum emission hinders the use of fluorescence in situ hybridisation (FISH) for the study of bacterial heterogeneity within the different niches of corals and Symbiodiniaceae, because FISH fluorophores also fluoresce within the visible to NIR spectrum. A solution to this impediment is to use fluorescence lifetime imaging microscopy (FLIM). The ‘lifetime’ property of fluorophores is a feature that enables sample (e.g. coral/Symbiodiniaceae) autofluorescence to be distinguished from FISH-labelled bacteria. In this manner, the location of bacteria around and within Symbiodiniaceae can be quantified along with their identity and spatial distribution. Furthermore, the ‘lifetime’ of the host and associated microbe cellular autofluorescence can be analysed in terms of endogenous fluorophore composition (e.g. metabolic co-factors, aromatic amino acids) and serves as information for symbiotic versus parasitic host-microbe association.
Keywords: autofluorescence, confocal microscopy, fluorescence lifetime imaging microscopy, label-free detection, microbial ecology, microbial symbiosis, phasor analysis, visualisation.
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