PT  - JOURNAL ARTICLE
AU  - Yide Zhang
AU  - Ian H. Guldner
AU  - Evan L. Nichols
AU  - David Benirschke
AU  - Cody J. Smith
AU  - Siyuan Zhang
AU  - Scott S. Howard
TI  - Instant FLIM enables 4D <em>in vivo</em> lifetime imaging of intact brains
AID  - 10.1101/2020.02.05.936039
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.02.05.936039
4099  - http://biorxiv.org/content/early/2020/02/06/2020.02.05.936039.short
4100  - http://biorxiv.org/content/early/2020/02/06/2020.02.05.936039.full
AB  - 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.