Traditional fluorescence microscopy is blind to molecular microenvironment information that is present in the emission decay lifetime. With fluorescence lifetime imaging microscopy (FLIM), physiological parameters such as pH, refractive index, ion concentration, dissolved gas concentration, and fluorescence resonance energy transfer (FRET) can be measured. Despite these benefits, existing FLIM techniques are typically slow, noisy, and hard to implement due to expensive instrumentation and complex post-processing. To overcome these limitations, we present instant FLIM, a method that allows real-time acquisition and display of two-photon intensity, lifetime, and phasor imaging data. Using analog signal processing, we demonstrate in vivo four-dimensional (4D) FLIM movies by imaging mouse and zebrafish glial cell response to injury over 12 hours through intact skulls. Instant FLIM can be implemented as an upgrade to an existing multiphoton microscope using cost-effective off-the-shelf components, requires no data post-processing, and is demonstrated to be compatible with FD-FLIM super-resolution techniques
