Instant FLIM enables 4D in vivo lifetime imaging of intact and injured zebrafish and mouse brains.

Traditional fluorescence microscopy is blind to molecular microenvironment information that is present in fluorescence lifetime, which can be measured by fluorescence lifetime imaging microscopy (FLIM). However, most existing FLIM techniques are slow to acquire and process lifetime images, difficult to implement, and expensive. Here, we present instant FLIM, an analog signal processing method that allows real-time streaming of fluorescence intensity, lifetime, and phasor imaging data through simultaneous image acquisition and instantaneous data processing. Instant FLIM can be easily implemented by upgrading an existing two-photon microscope using cost-effective components and our open-source software. We further improve the functionality, penetration depth, and resolution of instant FLIM using phasor segmentation, adaptive optics, and super-resolution techniques. We demonstrate through-skull intravital 3D FLIM of mouse brains to depths of 300 μm and present the first in vivo 4D FLIM of microglial dynamics in intact and injured zebrafish and mouse brains up to 12 hours.