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

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.

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.

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.

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.

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.