Characterization of phototoxic effects in multiphoton FLIM

Shagufta Rehman Alam; Horst Wallrabe; Kathryn G. Christopher; Karsten H. Siller; Ammasi Periasamy

doi:10.1117/12.2618514

3 March 2022 Characterization of phototoxic effects in multiphoton FLIM

Shagufta Rehman Alam, Horst Wallrabe, Kathryn G. Christopher, Karsten H. Siller, Ammasi Periasamy

Proceedings Volume 11965, Multiphoton Microscopy in the Biomedical Sciences XXII; 119650B (2022) https://doi.org/10.1117/12.2618514
Event: SPIE BiOS, 2022, San Francisco, California, United States

Abstract

Mitochondrial dysfunction is increasingly being recognized in many pathologies. Mitochondria, the power houses of cells have central roles to play in energy metabolism and apoptosis. Structure-Function studies designed towards characterizing and understanding defects in mitochondrial metabolism, dynamics and biogenesis in pathologies and response to treatments would provide insight into mitochondrial dysfunction. A 2-step imaging approach was used; (a) Zeiss 880/980 Airyscan Super Resolution microscopy to understand mitochondrial morphological response to treatment and (b) Fluorescence Lifetime Imaging (FLIM) -B&H TCSPC lifetime board coupled to a Zeiss 780 to track metabolic changes in HeLa cells by following the auto-fluorescent metabolic co-enzyme NAD(P)H. FLIM signatures, the lifetimes and the relative fractions of bound and free states of NAD(P)H and FAD are generated with multiphoton excitation by a pulsed femto-second infra-red laser. Publications suggest that FLIM multiphoton laser power requirements for NAD(P)H and FAD may not be well optimized, which could result in injurious effects to cells. We have characterized two photon (2p)- laser induced changes at the cellular level, particularly in mitochondria. Live-cell FLIM measurements were conducted on stage in HeLa cells by gradually increasing the laser average power, followed by the assessment of phototoxic effects. Our results show that NAD(P)H-a2%, the enzyme-bound fraction increases with rising laser average power, inducing cytotoxic damaging effects. As elevated NAD(P)H-a2% is also shown after drug treatment, sub-optimal laser power can be falsely interpreted as drug treatment response. Our study demonstrates how the laser power optimization at the specimen plane is critical in FLIM.

Conference Presentation

Citation Download Citation

Shagufta Rehman Alam, Horst Wallrabe, Kathryn G. Christopher, Karsten H. Siller, and Ammasi Periasamy "Characterization of phototoxic effects in multiphoton FLIM", Proc. SPIE 11965, Multiphoton Microscopy in the Biomedical Sciences XXII, 119650B (3 March 2022); https://doi.org/10.1117/12.2618514

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