Speed-Up Phase Resolved Fluorescence Lifetime Imaging Microscopy (SUPER-FLIM) for Real-Time Microenvironmental Sensing

Imaging cell biological processes while simultaneously sensing the microenvironment at high spatial and temporal resolution is a key goal in modern live-cell microscopy. However, adding parameters such as fluorescence lifetime compromises acquisition speed. To address this challenge, we introduce S peed- U p P has E R esolved F luorescence L ifetime I maging M icroscopy ( SUPER-FLIM ), an ultra-fast fluorescence lifetime imaging method that enables 3D live-imaging at unprecedented speed. Our approach combines digital lock-in detection with laser scanning excitation, allowing the simultaneous capture of fluorescence intensity, lifetime, and phasor data in real-time. With an acquisition time of 100 ns, SUPER-FLIM is ten times faster than state of the art FLIM techniques and is, for the first time, integrated with resonant galvo-scanners. SUPER-FLIM is a major step forward in live imaging as it provides high spatial resolution in 3D that cannot be achieved with camera-based frequency domain FLIM. As a proof of concept, we demonstrate its utility in observing lipid-nanoparticle acidification within living cells. Beyond imaging, SUPER-FLIM’s rapid lifetime acquisition unlocks new applications, including multi-lifetime fluorescence correlation spectroscopy (ml-FCS), a novel FCS mode that sorts and correlates fluorescence bursts from diffusing particles based on lifetime characteristics. The technique is compatible with virtually any modulated light source and can be easily integrated into any laser scanning microscope using our open-source software.