An automated multiwell plate reading film microscope for live cell autofluorescence lifetime assays

Fluorescence lifetime imaging (FLIM) is increasingly used to read out cellular autofluorescence originating from the coenzyme NADH in the context of investigating cell metabolic state. We present here an automated multiwell plate reading FLIM microscope optimized for UV illumination with the goal of extending high content fluorescence lifetime assays to readouts of metabolism. We demonstrate its application to automated cellular autofluorescence lifetime imaging and discuss the key practical issues associated with its implementation. In particular, we illustrate its capability to read out the NADH-lifetime response of cells to metabolic modulators, thereby illustrating the potential of the instrument for cytotoxicity studies, assays for drug discovery and stratified medicine.

[1]  Fu-Jen Kao,et al.  Reduced nicotinamide adenine dinucleotide fluorescence lifetime separates human mesenchymal stem cells from differentiated progenies. , 2008, Journal of biomedical optics.

[2]  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.

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

[4]  Thomas P. Gonnella,et al.  Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1. , 2013, Chemico-biological interactions.

[5]  W. Webb,et al.  Fluorescent erythrosin B is preferable to trypan blue as a vital exclusion dye for mammalian cells in monolayer culture. , 1984, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[6]  Enrico Gratton,et al.  NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy. , 2012, Biophysical journal.

[7]  J. Elisseeff,et al.  Noninvasive Mitochondrial Imaging in Live Cell Culture , 2005, Photochemistry and photobiology.

[8]  Francois Lacombe,et al.  FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ** , 2011, Chemphyschem : a European journal of chemical physics and physical chemistry.

[9]  Z. Siddik,et al.  Cisplatin: mode of cytotoxic action and molecular basis of resistance , 2003, Oncogene.

[10]  G. King,et al.  Induction and repair of DNA double-strand breaks. , 1993, Radiation research.

[11]  Rodney K. Lyn,et al.  Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein , 2013, PloS one.

[12]  Mark A A Neil,et al.  Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation , 2012, Journal of biophotonics.

[13]  Dusan Chorvat,et al.  Effect of ouabain on metabolic oxidative state in living cardiomyocytes evaluated by time-resolved spectroscopy of endogenous NAD(P)H fluorescence , 2012, Journal of biomedical optics.

[14]  J. Lakowicz Principles of fluorescence spectroscopy , 1983 .

[15]  P. French,et al.  Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging. , 2007, Optics express.

[16]  Chris Allan,et al.  OME Remote Objects (OMERO): a flexible, model-driven data management system for experimental biology , 2012, Nature Methods.

[17]  B. Schoener,et al.  Intracellular Oxidation-Reduction States in Vivo , 1962, Science.

[18]  Dong Li,et al.  Monitoring changes of cellular metabolism and microviscosity in vitro based on time-resolved endogenous fluorescence and its anisotropy decay dynamics. , 2010, Journal of biomedical optics.

[19]  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.

[20]  B. Chance,et al.  Intracellular Oxidation-Reduction States in Vivo , 1962, Science.

[21]  Yves Pommier,et al.  γH2AX and cancer , 2008, Nature Reviews Cancer.

[22]  D. M. Parker,et al.  NADH binding to porcine mitochondrial malate dehydrogenase. , 1979, The Journal of biological chemistry.

[23]  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.

[24]  A. Heikal,et al.  Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level. , 2009, Journal of photochemistry and photobiology. B, Biology.

[25]  A. deMello,et al.  Time-resolved fluorescence imaging of solvent interactions in microfluidic devices. , 2005, Optics express.

[26]  Rainer Pepperkok,et al.  In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays , 2010, Nature Methods.

[27]  T. G. Scott,et al.  Synthetic spectroscopic models related to coenzymes and base pairs. V. Emission properties of NADH. Studies of fluorescence lifetimes and quantum efficiencies of NADH, AcPyADH, [reduced acetylpyridineadenine dinucleotide] and simplified synthetic models , 1970 .

[28]  Artur Bednarkiewicz,et al.  Non-invasive monitoring of cytotoxicity based on kinetic changes of cellular autofluorescence. , 2011, Toxicology in vitro : an international journal published in association with BIBRA.

[29]  M. Torres-Cisneros,et al.  Detection of biological cells in phase-contrast video microscopy , 2006, 2006 Multiconference on Electronics and Photonics.

[30]  Jens Eickhoff,et al.  In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia. , 2007, Journal of biomedical optics.

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

[32]  Karsten König,et al.  Multiphoton fluorescence lifetime imaging of 3D‐stem cell spheroids during differentiation , 2011, Microscopy research and technique.

[33]  E. Gratton,et al.  The phasor approach to fluorescence lifetime imaging analysis. , 2008, Biophysical journal.

[34]  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.

[35]  Michael Zuker,et al.  Delta function convolution method (DFCM) for fluorescence decay experiments , 1985 .

[36]  Stefan Wölfl,et al.  Real-Time Monitoring of Cisplatin-Induced Cell Death , 2011, PloS one.

[37]  Fred S Wouters,et al.  Unsupervised Fluorescence Lifetime Imaging Microscopy for High Content and High Throughput Screening *S , 2007, Molecular & Cellular Proteomics.

[38]  J C Pickup,et al.  Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus. , 2005, Journal of photochemistry and photobiology. B, Biology.

[39]  Sean C. Warren,et al.  Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets , 2013, PloS one.

[40]  P Georges,et al.  Fluorescence-lifetime imaging with a multifocal two-photon microscope. , 2004, Optics letters.

[41]  Irene Georgakoudi,et al.  Optical imaging using endogenous contrast to assess metabolic state. , 2012, Annual review of biomedical engineering.

[42]  Ewan J McGhee,et al.  High speed unsupervised fluorescence lifetime imaging confocal multiwell plate reader for high content analysis , 2008, Journal of biophotonics.

[43]  K. Eliceiri,et al.  Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency , 2012, PloS one.

[44]  B. Vojnovic,et al.  A Multi-Functional Imaging Approach to High-Content Protein Interaction Screening , 2012, PloS one.

[45]  W. Webb,et al.  Conformational Dependence of Intracellular NADH on Metabolic State Revealed by Associated Fluorescence Anisotropy*♦ , 2005, Journal of Biological Chemistry.