High-speed imaging of transient metabolic dynamics using two-photon fluorescence lifetime imaging microscopy.

Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of autofluorescent metabolic coenzymes has been widely used to investigate energetic perturbations in living cells and tissues in a label-free manner with subcellular resolution. While the currently used state-of-the-art instruments are highly sensitive to local molecular changes associated with these metabolic processes, they are inherently slow and limit the study of dynamic metabolic environments. Here, a sustained video-rate 2P-FLIM imaging system is demonstrated for time-lapse lifetime imaging of reduced nicotinamide adenine dinucleotide, an autofluorescent metabolic coenzyme involved in both aerobic and anaerobic processes. This system is sufficiently sensitive to differences in metabolic activity between aggressive and nonaggressive cancer cell lines and is demonstrated for both wide field-of-view autofluorescence imaging as well as sustained video-rate image acquisition of metabolic dynamics following induction of apoptosis. The unique capabilities ofthis imaging platform provide a powerful technological advance to further explore rapid metabolic dynamics in living cells.

[1]  R. Gillies,et al.  Why do cancers have high aerobic glycolysis? , 2004, Nature Reviews Cancer.

[2]  H. Harada,et al.  Paclitaxel-Induced Apoptosis Is BAK-Dependent, but BAX and BIM-Independent in Breast Tumor , 2013, PloS one.

[3]  Marina Marjanovic,et al.  Longitudinal Label-free Tracking of Cell Death Dynamics in Living Engineered Human Skin Tissue with a Multimodal Microscope References and Links , 2022 .

[4]  N. Ramanujam,et al.  In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia , 2007, Proceedings of the National Academy of Sciences.

[5]  J. Lakowicz,et al.  Fluorescence lifetime imaging of free and protein-bound NADH. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[6]  N. Oleinick,et al.  Staurosporine-induced death of MCF-7 human breast cancer cells: a distinction between caspase-3-dependent steps of apoptosis and the critical lethal lesions. , 2003, Experimental cell research.

[7]  Yau-Huei Wei,et al.  Increase of reduced nicotinamide adenine dinucleotide fluorescence lifetime precedes mitochondrial dysfunction in staurosporine-induced apoptosis of HeLa cells. , 2011, Journal of biomedical optics.

[8]  Alex J Walsh,et al.  Optical metabolic imaging identifies glycolytic levels, subtypes, and early-treatment response in breast cancer. , 2013, Cancer research.

[9]  N. Tamai,et al.  Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy. , 2007, Analytical chemistry.

[10]  G J Brakenhoff,et al.  Analysis of efficiency of two‐photon versus single‐photon absorption for fluorescence generation in biological objects , 1996, Journal of microscopy.

[11]  C. Thompson,et al.  Apoptosis in the pathogenesis and treatment of disease , 1995, Science.

[12]  P. Seybold,et al.  Solvent Dependence of the Fluorescence Lifetimes of Xanthene Dyes , 1999 .

[13]  Karl Münger,et al.  Mapping metabolic changes by noninvasive, multiparametric, high-resolution imaging using endogenous contrast , 2018, Science Advances.

[14]  Michael Unser,et al.  User‐friendly semiautomated assembly of accurate image mosaics in microscopy , 2007, Microscopy research and technique.

[15]  Tom Pfeiffer,et al.  Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate. , 2017, Biomedical optics express.

[16]  Anne E Carpenter,et al.  CellProfiler: image analysis software for identifying and quantifying cell phenotypes , 2006, Genome Biology.

[17]  K. König,et al.  Fluorescence lifetime imaging by time‐correlated single‐photon counting , 2004, Microscopy research and technique.

[18]  Joy Joseph,et al.  Doxorubicin Induces Apoptosis in Normal and Tumor Cells via Distinctly Different Mechanisms , 2004, Journal of Biological Chemistry.

[19]  J. Fujimoto,et al.  Rapid imaging of surgical breast excisions using direct temporal sampling two photon fluorescent lifetime imaging. , 2015, Biomedical optics express.

[20]  Hongki Yoo,et al.  Real-time visualization of two-photon fluorescence lifetime imaging microscopy using a wavelength-tunable femtosecond pulsed laser. , 2018, Biomedical optics express.

[21]  H. Gerritsen,et al.  Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution , 2002, Journal of microscopy.

[22]  Shane Z. Sullivan,et al.  Video-rate two-photon excited fluorescence lifetime imaging system with interleaved digitization. , 2015, Optics letters.

[23]  K. Al-Sakkaf,et al.  Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells , 2002, British Journal of Cancer.

[24]  Haishan Zeng,et al.  In vivo video rate multiphoton microscopy imaging of human skin. , 2011, Optics letters.

[25]  Joanne Li,et al.  A quantitative framework for the analysis of multimodal optical microscopy images. , 2017, Quantitative imaging in medicine and surgery.

[26]  Fu-Jen Kao,et al.  Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells. , 2008, Journal of biomedical optics.

[27]  D. Vaux,et al.  Apoptosis in the development and treatment of cancer. , 2004, Carcinogenesis.

[28]  Laura Marcu,et al.  Fluorescence Lifetime Techniques in Medical Applications , 2012, Annals of Biomedical Engineering.

[29]  Joanne Li,et al.  Label‐free in vivo cellular‐level detection and imaging of apoptosis , 2017, Journal of biophotonics.

[30]  Richard Torres,et al.  Multiphoton fluorescence, second harmonic generation, and fluorescence lifetime imaging of whole cleared mouse organs. , 2011, Journal of biomedical optics.

[31]  G. S. Wilson,et al.  Fluorescence Properties of Fluorescein, Tetramethylrhodamine and Texas Red Linked to a DNA Aptamer¶ , 2005, Photochemistry and photobiology.

[32]  S. Boppart,et al.  Longitudinal in vivo tracking of adverse effects following topical steroid treatment , 2016, Experimental dermatology.