Label-Free Metabolic Imaging in Vivo by Two-Photon Fluorescence Lifetime Endomicroscopy

Wenxuan Liang*, Defu Chen, Honghua Guan, Hyeon Cheol Park, Kaiyan Li, Ang Li, Ming Jun Li, Israel Gannot, Xingde Li*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


NADH intensity and fluorescence lifetime characteristics have proved valuable intrinsic biomarkers for profiling the cellular metabolic status of living biological tissues. To fully leverage the potential of NADH fluorescence lifetime imaging microscopy (FLIM) in (pre)clinical studies and translational applications, a compact and flexible endomicroscopic embodiment is essential. Herein we present our newly developed two-photon fluorescence (2PF) lifetime imaging endomicroscope (2p-FLeM) that features an about 2 mm diameter, subcellular resolution, and excellent emission photon utilization efficiency and can extract NADH lifetime parameters of living tissues and organs reliably using a safe excitation power (∼30 mW) and moderate pixel dwelling time (≤10 μs). In vivo experiments showed that the 2p-FLeM system was capable of tracking NADH lifetime dynamics of cultured cancer cells and subcutaneous mouse tumor models subject to induced apoptosis, and of a functioning mouse kidney undergoing acute ischemia-reperfusion perturbation. The complementary structural and metabolic information afforded by the 2p-FLeM system promises functional histological imaging of label-free internal organs in vivo and in situ for practical clinical diagnosis and therapeutics applications.

Original languageEnglish
Pages (from-to)4017-4029
Number of pages13
JournalACS Photonics
Issue number12
StatePublished - 21 Dec 2022
Externally publishedYes


FundersFunder number
National Science FoundationCBET-1430040
National Institutes of HealthR01CA247595, R35CA209960


    • NADH lifetime imaging
    • fluorescence lifetime imaging microscopy (FLIM)
    • label-free imaging
    • metabolic imaging
    • two-photon endomicroscopy


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