Adaptive fluorescence lifetime imaging with per-pixel signal optimization and flexible scanning

Advances in time-resolved fluorescence lifetime imaging microscopy (FLIM) have significantly enhanced biological imaging compared to steady-state techniques alone. The primary goal of modern FLIM is to acquire high-resolution fluorescence lifetime profiles with a high signal-to-noise ratio (SNR) from heterogeneous samples at high speeds, posing challenges in balancing imaging speed, signal strength, and sample integrity. In this study, we present fluorescence lifetime intensity-inverted imaging microscopy (FLI³M), an adaptive imaging technique based on confocal laser scanning microscopy (CLSM) that dynamically adjusts pixel dwell times using a priori intensity information from a pre-scan and supports flexible scanning patterns. This approach achieves uniform SNR imaging by either providing up to an eight-fold signal enhancement without increasing imaging time or reducing imaging time without compromising SNR. We demonstrate the potential of this technique through imaging studies of biological samples, including Convallaria majalis and human lung tissue. The results show a 56% average improvement in fluorescence lifetime estimation reliability in low-SNR regions and an increase in imaging speed ranging from 27% to 53% across various samples. This enables detailed resolution of optical fingerprints in complex biological environments that are challenging for conventional imaging. Collectively, these results establish our adaptive FLIM system as a powerful tool for high-performance cellular imaging, FLIM-guided diagnostics, and a wide range of biomedical applications.