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

Area of Science:

• Biomedical Optics
• Cellular Metabolism
• Fluorescence Imaging

Background:

• Cellular metabolism is vital in health and disease, necessitating advanced diagnostic and therapeutic tools.
• Label-free optical metabolic imaging offers nondestructive, high-resolution insights into cellular metabolic function using endogenous fluorescence.
• Reproducibility and comparability across studies are hindered by a lack of standardized methodologies.

Purpose of the Study:

• To establish a consensus framework for the acquisition, calibration, and reporting of microscopic imaging metabolic function.
• To standardize assessments based on fluorescence intensity and lifetime measurements of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD).

Main Methods:

• Presenting best practices for calibrating and analyzing fluorescence intensity-based optical redox ratios.
• Utilizing multiexponential fitting and phasor analysis for fluorescence lifetime data.
• Providing guidelines for validation experiments and cross-system standardization.

Main Results:

• Demonstrating the critical role of calibration and normalization in intensity-based optical redox measurements.
• Highlighting the impact of analytical approaches on fluorescence lifetime-based metabolic metrics.
• Addressing signal-to-noise ratio considerations and calibration needs.

Conclusions:

• Recommending a practical framework for reproducible, label-free optical metabolic imaging.
• Facilitating robust comparisons across studies and promoting wider adoption of these technologies.
• Supporting the translation of optical metabolic imaging for biomedical research and clinical applications.