FLIM and STED Imaging for Investigating Intracellular Lipids

Fluorescence imaging, utilizing molecular fluorophores, often acts as a central tool for the investigation of fundamental biological processes.  It also offers huge future potential for human imaging coupled to therapeutic procedures such as fluorescence guided surgery.¹ Our research has pioneered the BF₂-azadipyrromethene class of near infrared fluorophores from which in vitro and in vivo imaging probes have been developed.² This class has excellent photophysical characteristics such as tuneable emission maxima between 650 and 820 nm, exceptional photostability and high quantum yields. Their suitability for time-lapse live or fixed cell microscopy employing widefield, confocal, STED super-resolution or fluorescence lifetime (FLIM) techniques allows their application to complex chemical-biology investigations. This presentation is concerned with our ongoing development of this emitter class for investigating the chemical-biology of intracellular lipid structures. Non-covalent migratory fluorescence labelling has been introduced to spatially and temporally map intracellular lipid structures throughout a cell division cycle (mitosis). This less-invasive approach utilizes BF₂-azadipyrromethene fluorophores to first non-covalently label known intracellular lipid compartments at cell interphase which then migrate with the lipid components of these structures as they disassemble, redistribute and reassemble prior to daughter cell separation. Through this unique approach to image capture, key prometaphase events such as lipid intrusion into the nucleus and nuclear membrane disassembly are observable, as are the stages of nuclear membrane reassembly in telophase and lipid distribution during cytokinesis (Figure 1).³ Remarkably, the non-covalent BF₂-azadipyrromethene label remains associated with the originating lipid components as they undergo these architectural reorganizations and changes in subcellular localization associated with mitosis. To further address the challenges of lipid structure differentiation during mitosis a novel fluorescence lifetime encoded STED-FLIM approach has been developed. This utilises two spectrally overlapping STED compatible fluorophores, with differing lifetimes, to non-covalently label lipid structures which can be image separated from each other with phasor plot analysis, with corresponding STED images confirming their structural identities.⁴ As lipid-based cell structures are influenced by numerous biological processes, our approaches to their fluorescence imaging could offer novel perspectives into their multifaceted roles.

References

1. A. Cahill et al, British Journal of Surgery 2021, 108, 5.
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3. (a) A. C. Bourgès et al, J. Mater. Chem. B 2026, 14, 161. (b) A. C. Bourgès et al, Membranes 2025, 15, 9.
4. E. Booth et al, ChemRxiv, 2026, https://doi.org/10.26434/chemrxiv.15007988/v1