Near‐infrared confocal imaging reveals the spatial organisation of far‐red photosynthesis in Shark Bay microbial mats and Halomicronema sp.

Far-red light (FRL) cyanobacteria occupy spectrally filtered habitats where visible wavelengths are depleted but far-red radiation persists. Shark Bay (Western Australia) stromatolitic microbial mats provide such a habitat, containing subsurface cyanobacterial communities in which chlorophyll d/f (Chl d/f)-containing phototrophs coexist with filamentous Halomicronema-like taxa adapted to far-red photosynthesis. In this Journal of Microscopy issue honouring Associate Professor Guy C. Cox, we used FRL-sensitive confocal imaging to visualise far-red cyanobacteria in intact Shark Bay mat samples, and combined microspectroscopy, lifetime microscopy, HPLC pigment analysis and synchrotron FTIR microspectroscopy to examine pigment organisation and FRL acclimation in cultured Halomicronema sp. 'Clifton'. Confocal imaging with near-infrared-sensitive detection revealed abundant far-red fluorescent cyanobacteria within subsurface mat layers and enabled direct visualisation of Chl d/f-containing phototrophs in situ. In cultured Halomicronema, HPLC confirmed that Chl f accumulation was induced by FRL acclimation and absent under white light, validating assignment of the dominant 730-745 nm emission in FRL-grown cells to Chl f-enriched photosystems. Intracellular excitation-emission mapping showed that FRL acclimation did not produce complete pigment segregation, but generated a pronounced intracellular pigment gradient, with Chl f-associated fluorescence enriched in mid-cell regions and phycobiliprotein- and chlorophyll a (Chl a)-associated fluorescence enriched at cell poles. FLIM and phasor analyses showed that this spatial reorganisation was accompanied by altered excited-state behaviour, including strongest lifetime shortening in Chl a-associated channel, additional shortening in the far-red chlorophyll-enriched channel and increased heterogeneity of PC/APC-associated fluorescence. The absence of a distinct Chl f-only lifetime population indicates that Chl f remains functionally integrated within coupled photosynthetic complexes rather than acting as an isolated fluorescent sink. By linking optical stratification and far-red cyanobacterial diversity within Shark Bay microbial mats to pigment organisation and fluorescence dynamics in living Halomicronema cells, this study extends Guy Cox's microscopy-based exploration of photosynthetic microorganisms and demonstrates the power of modern spectral confocal microscopy for investigating photosynthesis at the far-red edge of oxygenic life.