Consensus guidelines for cellular label-free optical metabolic imaging: ensuring accuracy and reproducibility in metabolic profiling

Significance: Cellular metabolism plays a central role in health and disease, making its study critical for advancing diagnostics and therapies. Label-free optical metabolic imaging using endogenous fluorescence from reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) provides nondestructive, high-resolution insights into metabolic function and heterogeneity from the sub-cellular to the tissue level. Standardized approaches are essential to ensure reproducibility and comparability across studies. Aim: We aim to establish a consensus framework for the acquisition, calibration, and reporting of microscopic imaging metabolic function assessments based on fluorescence intensity and lifetime measurements of NAD(P)H and FAD. Approach: We present best practices for calibrating, analyzing, and reporting fluorescence intensity-based optical redox ratios and fluorescence lifetime data using multiexponential fitting and phasor analysis. Guidelines for validation experiments and cross-system standardization are provided to improve accuracy and reproducibility. Results: We demonstrate the importance of calibration procedures and normalization strategies for intensity-based optical redox measurements. We highlight needed calibration, signal-to-noise ratio considerations, and the impact of distinct analytical approaches on fluorescence lifetime-based metabolic function metrics. Conclusion: We recommend a consistent, practical framework for reproducible, label-free, optical metabolic imaging, facilitating robust comparisons across studies and supporting the broader adoption of optical metabolic imaging technologies for biomedical research and clinical translation.