ROICAM · Revisiting the origin of intrinsic cellular autofluorescence in metabolic imaging
Horizon Europe — Marie Skłodowska-Curie Actions
- Duration
- 2023-09-18 → 2025-09-17
- EU contribution
- €222,728
- Participants
- 1
- Scheme
- HORIZON-TMA-MSCA-PF-EF
Lines connect the coordinator with its partners.
Results in brief
Revisiting the origin of intrinsic cellular autofluorescence in metabolic imaging
Metabolic molecules such as NADH and FAD emit light when excited at specific wavelengths by a laser, through a process known as fluorescence. Because it requires no dyes or labels, multiphoton fluorescence lifetime imaging microscopy (MPM-FLIM) can use this autofluorescence to monitor how cells use energy in real time. However, in skin and other complex tissues, structural proteins like keratin are also fluorescent and can confuse the metabolic readout. ROICAM set out to clarify where the autofluorescence signal really comes from, quantify it more reliably, and demonstrate practical imaging workflows that work in realistic biological samples. The project focused on three goals: RO1: Systematically measure how NADH, FAD and keratin fluoresce under different conditions. RO2: Build robust analysis methods and software for quantitative fluorescence lifetime imaging, with particular emphasis on phasor analysis. RO3: Test the approach in relevant skin cell models and under controlled metabolic perturbations. By the end of the project, ROICAM delivered new measurements and analysis tools that improve the interpretation of label free metabolic imaging, upgraded a multiphoton microscope to collect cleaner data, and showed how different skin cell types respond to metabolic challenges in ways that can now be distinguished more confidently. These outcomes support future applications in non invasive diagnostics and basic research on cell metabolism.
Data: CORDIS, © European Union
Project objective
Metabolic dysregulation is a hallmark of numerous diseases. Multiphoton microscopy (MPM) combined with fluorescence lifetime imaging (FLIM) of the metabolic coenzymes NADH and FAD has emerged as a key method for monitoring metabolic processes in live tissue with sub-cellular resolution. However, recent data demonstrates that spectral crosstalk from other cellular components, such as keratin, is substantial. Yet the contributions from endogenous proteins are often neglected, bringing into question the validity of earlier studies. To address this issue, we propose to systematically scrutinize the fluorescence properties of NADH, FAD and keratin in different states and combinations. This will be done by meticulous investigation of the fluorescence lifetime, spectra and related photophysical properties using MPM and time-resolved spectrophotometry under a range of conditions (pH, temperature, viscosity, protein binding), and in combination with computational chemistry. Based on this knowledge, we will develop approaches that can accurately determine the concentration, diffusion and binding state of NADH and FAD even in the presence of spectral crosstalk from keratin. The strategy is to implement phasor-FLIM analysis, fluorescence lifetime correlation spectroscopy (FLCS) and raster lifetime image correlation spectroscopy (RLICS) to separate the fluorescence contributions of bound and unbound NADH and FAD from the protein background and quantify the spatial distribution of each species with unprecedented spatiotemporal resolution. The developed methodologies will be validated by monitoring cellular metabolism in human primary cells and tumour cell lines. This study will solidify our understanding of NADH and FAD autofluorescence in complex cellular environments and substantially expand the analytical toolset by which quantitative information about metabolic dysregulation can be obtained, thereby advancing MPM-FLIM as a research and diagnostic tool for metabolic diseases.
Original text from CORDIS.
Participants
- GOETEBORGS UNIVERSITET · GoeteborgCoordinatorSweden
Links
- View on CORDIS
- DOI: 10.3030/101103885
- https://ec.europa.eu/research/participants/documents/downloadPublic?documentIds=080166e50a98ac48&appId=PPGMS
- https://ec.europa.eu/research/participants/documents/downloadPublic?documentIds=080166e520afc8f7&appId=PPGMS
Data: CORDIS, © European Union