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. Author manuscript; available in PMC: 2023 Nov 10.
Published in final edited form as: Nat Rev Methods Primers. 2022 Nov 10;2:89. doi: 10.1038/s43586-022-00168-w

Fig. 4 |. Overcoming tissue motion.

Fig. 4 |

General workflow for motion correction of intravital fluorescence lifetime imaging microscopy (FLIM)–Förster resonance energy transfer (FRET) data using the software Galene. Physiological motion, such as respiration, heartbeat, peristalsis or muscle and vascular tone, can render intravital microscopy (IVM) data unintelligible as shown here for intravital FLIM–FRET in the intestine as an example. After extracting the photon arrival times, individual frames are reconstructed and aligned against a reference frame based on fluorescence intensity information. Following alignment, the individual photon arrival positions are reassigned for each frame to allow quantification of fluorescence lifetimes per pixel. Similarly, Galene can also be used to correct for motion in other IVM data including fluorescence time series or endogenous fluorescence data. ECFP, enhanced cyan fluorescent protein; SEYFP, super enhanced yellow fluorescent protein. Adapted from REF.178, CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/).