Abstract

We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor.

© 2007 Optical Society of America

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2007

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, "In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia," J. Biomed. Opt. 12, 024014-024011-024010 (2007).
[CrossRef] [PubMed]

A. Mayevsky, and G. G. Rogatsky, "Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies," Am. J. Physiol.-Cell Physiol. 292, C615-C640 (2007).
[CrossRef]

J. Martini, V. Andresen, and D. Anselmetti, "Scattering suppression and confocal detection in multifocal multiphoton microscopy," J. Biomed. Opt. 12, 034010-034016 (2007).
[CrossRef] [PubMed]

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 9 (2007).
[CrossRef]

2006

J. L. Qu, L. X. Liu, D. N. Chen, Z. Y. Lin, G. X. Xu, B. P. Guo, and H. B. Niu, "Temporally and spectrally resolved sampling imaging with a specially designed streak camera," Opt. Lett. 31, 368-370 (2006).
[CrossRef] [PubMed]

Y. C. Wu, W. Zheng, and J. N. Y. Qu, "Sensing cell metabolism by time-resolved autofluorescence," Opt. Lett. 31, 3122-3124 (2006).
[CrossRef] [PubMed]

B. Treanor, P. M. P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A. A. Neil, D. N. Burshtyn, P. M. W. French, and D. M. Davis, "Microclusters of inhibitory killer immunoglobulin like receptor signaling at natural killer cell immunological synapses," J. Cell Biol. 174, 153-161 (2006).
[CrossRef] [PubMed]

L. M. Tiede, and M. G. Nichols, "Photobleaching of reduced nicotinamide adenine dinucleotide and the development of highly fluorescent lesions in rat basophilic leukemia cells during multiphoton microscopy," Photochem. Photobiol. 82, 656-664 (2006).
[CrossRef] [PubMed]

L. Liu, J. Qu, Z. Lin, L. Wang, Z. Fu, B. Guo, and H. Niu, "Simultaneous time- and spectrum-resolved multifocal multiphoton microscopy," Appl. Phys. B-Lasers and Optics 84, 379-383 (2006).
[CrossRef]

2005

M. G. Nichols, E. E. Barth, and J. A. Nichols, "Reduction in DNA synthesis during two-photon microscopy of intrinsic reduced nicotinamide adenine dinucleotide fluorescence," Photochem. Photobiol. 81, 259-269 (2005).
[CrossRef] [PubMed]

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, "Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy," J. Biol. Chem. 280, 25119-25126 (2005).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, "Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH," Cancer Res. 65, 8766-8773 (2005).
[CrossRef] [PubMed]

C. Eggeling, A. Volkmer, and C. A. M. Seidel, "Molecular photobleaching kinetics of rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy," Chemphyschem 6, 791-804 (2005).
[CrossRef] [PubMed]

J. A. Conchello, and J. W. Lichtman, "Optical sectioning microscopy," Nature Methods 2, 920-931 (2005).
[CrossRef] [PubMed]

K. Suhling, P. M. W. French, and D. Phillips, "Time-resolved fluorescence microscopy," Photochem. Photobiol. 4, 13-22 (2005).
[CrossRef]

R. K. P. Benninger, O. Hofmann, J. McGinty, J. Requejo-Isidro, I. Munro, M. A. A. Neil, A. J. deMello, and P. M. W. French, "Time-resolved fluorescence imaging of solvent interactions in microfluidic devices," Opt. Express 13, 6275-6285 (2005).
[CrossRef] [PubMed]

2004

S. Leveque-Fort, M. P. Fontaine-Aupart, G. Roger, and P. Georges, "Fluorescence-lifetime imaging with a multifocal two-photon microscope," Opt. Lett. 29, 2884-2886 (2004).
[CrossRef]

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 13 (2004).
[CrossRef]

M. Peter, and S. M. Ameer-Beg, "Imaging molecular interactions by multiphoton FLIM," Biol. Cell 96, 231-236 (2004).
[CrossRef] [PubMed]

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

G. C. Cianci, J. R. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc. Res. Tech. 64, 135-141 (2004).
[CrossRef] [PubMed]

2003

R. V. Krishnan, H. Saitoh, H. Terada, V. E. Centonze, and B. Herman, "Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera," Rev. Sci. Instrum. 74, 2714-2721 (2003).
[CrossRef]

P. D. Borszcz, M. Peterson, L. Standeven, S. Kirwan, M. Sandusky, A. Shaw, E. O. Long, and D. N. Burshtyn, "KIR enrichment at the effector-target cell interface is more sensitive than signaling to the strength of ligand binding," Eur. J. Immunol. 33, 1084-1093 (2003).
[CrossRef] [PubMed]

E. A. Jares-Erijman, and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
[CrossRef] [PubMed]

2002

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D-Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

D. R. Drummond, N. Carter, and R. A. Cross, "Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors," J. Microsc. 206, 161-169 (2002).
[CrossRef] [PubMed]

S. H. Huang, A. A. Heikal, and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J. 82, 2811-2825 (2002).
[CrossRef] [PubMed]

H. C. Gerritsen, M. A. H. Asselbergs, A. V. Agronskaia, and W. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
[CrossRef] [PubMed]

2001

T. Nielsen, M. Frick, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

A. Hopt, and E. Neher, "Highly nonlinear photodamage in two-photon fluorescence microscopy," Biophys. J. 80, 2029-2036 (2001).
[CrossRef] [PubMed]

C. J. de Grauw, and H. C. Gerritsen, "Multiple time-gate module for fluorescence lifetime imaging," Appl. Spectrosc. 55, 670-678 (2001).
[CrossRef]

2000

K. Konig, "Multiphoton microscopy in life sciences," J. Microscopy-Oxford. 200, 83-104 (2000).
[CrossRef]

G. H. Patterson, and D. W. Piston, "Photobleaching in two-photon excitation microscopy," Biophys. J. 78, 2159-2162 (2000).
[CrossRef] [PubMed]

A. Egner, and S. W. Hell, "Time multiplexing and parallelization in multifocal multiphoton microscopy," J. Opt. Soc. Am. A-Opt. Image Sci. Vis. 17, 1192-1201 (2000).
[CrossRef] [PubMed]

G. H. Patterson, S. M. Knobel, P. Arkhammar, O. Thastrup, and D. W. Piston, "Separation of the glucose-stimulated cytoplasmic mitochondrial NAD(P)H responses in pancreatic islet beta cells," Proc. Natl. Acad. Sci. U. S. A. 97, 5203-5207 (2000).
[CrossRef] [PubMed]

1999

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, "Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability," Nat. Biotechnol. 17, 763-767 (1999).
[CrossRef] [PubMed]

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, "Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: Signal and photodamage," Biophys. J. 77, 2226-2236 (1999).
[CrossRef] [PubMed]

K. Konig, T. W. Becker, P. Fischer, I. Riemann, and K. J. Halbhuber, "Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes," Opt. Lett. 24, 113-115 (1999).
[CrossRef]

1998

M. Straub, and S. W. Hell, "Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope," Appl. Phys. Lett. 73, 1769-1771 (1998).
[CrossRef]

A. Schonle, and S. W. Hell, "Heating by absorption in the focus of an objective lens," Opt. Lett. 23, 325-327 (1998).
[CrossRef]

J. Bewersdorf, R. Pick, and S. W. Hell, "Multifocal multiphoton microscopy," Opt. Lett. 23, 655-657 (1998).
[CrossRef]

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

1997

B. R. Masters, P. T. C. So, and E. Gratton, "Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin," Biophys. J. 72, 2405-2412 (1997).
[CrossRef] [PubMed]

1995

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, "Steady state and time-resolved fluorescence properties of metastatic and non-metastatic malignant cells from different species," J. Photochem. Photobiol.B-Biol. 31, 101-112 (1995).
[CrossRef]

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence for Localized Cell Heating Induced by Infrared Optical Tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

D. W. Piston, B. R. Masters, and W. W. Webb, "3-Dimensionally Resolved NAD(P)H Cellular Metabolic Redox Imaging of the in-Situ Cornea with 2-Photon Excitation Laser-Scanning Microscopy," J. Microsc. 178, 20-27 (1995).
[CrossRef] [PubMed]

M. Wakita, G. Nishimura, and M. Tamura, "Some Characteristics of the Fluorescence Lifetime of Reduced Pyridine-Nucleotides in Isolated-Mitochondria, Isolated Hepatocytes, and Perfused-Rat-Liver in-Situ," J. Biochem.(Tokyo) 118, 1151-1160 (1995).

1992

H. Schneckenburger, and K. Konig, "Fluorescence Decay Kinetics and Imaging of Nad(P)H and Flavins as Metabolic Indicators," Opt. Eng. 31, 1447-1451 (1992).
[CrossRef]

1990

W. Denk, J. H. Strickler, and W. W. Webb, "2-Photon Laser Scanning Fluorescence Microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

1981

A. J. W. G. Visser, and A. van Hoek, "The fluorescence decay of reduced nicotinamides in aqueous solution after exciation with a UV-mode locked Ar Ion Laser," Photochem. Photobiol. 33, 35-40 (1981).
[CrossRef]

1977

J. C. Brochon, P. Wahl, M. O. Monneuse-Doublet, and A. Olomucki, "Pulse Fluorimetry Study of Octopine Dehydrogenase-Reduced Nicotinamide Adenine Dinucleotide Complexes," Biochemistry 16, 4594-4599 (1977).
[CrossRef] [PubMed]

1976

A. Gafni, and L. Brand, "Fluorescence Decay Studies of Reduced Nicotinamide Adenine Dinucleotide in Solution and Bound to Liver Alcohol Dehydrogenase," Biochemistry 15, 3165-3171 (1976).
[CrossRef] [PubMed]

1962

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, "Intracellular Oxidation-Reduction States in Vivo," Science 137, 499-508 (1962).
[CrossRef] [PubMed]

Agronskaia, A. V.

H. C. Gerritsen, M. A. H. Asselbergs, A. V. Agronskaia, and W. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
[CrossRef] [PubMed]

Ameer-Beg, S. M.

M. Peter, and S. M. Ameer-Beg, "Imaging molecular interactions by multiphoton FLIM," Biol. Cell 96, 231-236 (2004).
[CrossRef] [PubMed]

Andresen, P.

T. Nielsen, M. Frick, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

Andresen, V.

J. Martini, V. Andresen, and D. Anselmetti, "Scattering suppression and confocal detection in multifocal multiphoton microscopy," J. Biomed. Opt. 12, 034010-034016 (2007).
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Anselmetti, D.

J. Martini, V. Andresen, and D. Anselmetti, "Scattering suppression and confocal detection in multifocal multiphoton microscopy," J. Biomed. Opt. 12, 034010-034016 (2007).
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Arkhammar, P.

G. H. Patterson, S. M. Knobel, P. Arkhammar, O. Thastrup, and D. W. Piston, "Separation of the glucose-stimulated cytoplasmic mitochondrial NAD(P)H responses in pancreatic islet beta cells," Proc. Natl. Acad. Sci. U. S. A. 97, 5203-5207 (2000).
[CrossRef] [PubMed]

Asselbergs, M. A. H.

H. C. Gerritsen, M. A. H. Asselbergs, A. V. Agronskaia, and W. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
[CrossRef] [PubMed]

Auksorius, E.

B. Treanor, P. M. P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A. A. Neil, D. N. Burshtyn, P. M. W. French, and D. M. Davis, "Microclusters of inhibitory killer immunoglobulin like receptor signaling at natural killer cell immunological synapses," J. Cell Biol. 174, 153-161 (2006).
[CrossRef] [PubMed]

Babai, F.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, "Steady state and time-resolved fluorescence properties of metastatic and non-metastatic malignant cells from different species," J. Photochem. Photobiol.B-Biol. 31, 101-112 (1995).
[CrossRef]

Bahlmann, K.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 9 (2007).
[CrossRef]

Balassy, A.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, "Steady state and time-resolved fluorescence properties of metastatic and non-metastatic malignant cells from different species," J. Photochem. Photobiol.B-Biol. 31, 101-112 (1995).
[CrossRef]

Barth, E. E.

M. G. Nichols, E. E. Barth, and J. A. Nichols, "Reduction in DNA synthesis during two-photon microscopy of intrinsic reduced nicotinamide adenine dinucleotide fluorescence," Photochem. Photobiol. 81, 259-269 (2005).
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Baur, D.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, "Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: Signal and photodamage," Biophys. J. 77, 2226-2236 (1999).
[CrossRef] [PubMed]

Bavister, B. D.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, "Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability," Nat. Biotechnol. 17, 763-767 (1999).
[CrossRef] [PubMed]

Becker, T. W.

Becker, W.

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

Benndorf, K.

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

Benninger, R. K. P.

Bergmann, A.

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

Berland, K. M.

G. C. Cianci, J. R. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc. Res. Tech. 64, 135-141 (2004).
[CrossRef] [PubMed]

Berns, M. W.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence for Localized Cell Heating Induced by Infrared Optical Tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Bewersdorf, J.

Bird, D. K.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, "In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia," J. Biomed. Opt. 12, 024014-024011-024010 (2007).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, "Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH," Cancer Res. 65, 8766-8773 (2005).
[CrossRef] [PubMed]

Biskup, C.

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

Blanchard, L.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, "Steady state and time-resolved fluorescence properties of metastatic and non-metastatic malignant cells from different species," J. Photochem. Photobiol.B-Biol. 31, 101-112 (1995).
[CrossRef]

Borszcz, P. D.

P. D. Borszcz, M. Peterson, L. Standeven, S. Kirwan, M. Sandusky, A. Shaw, E. O. Long, and D. N. Burshtyn, "KIR enrichment at the effector-target cell interface is more sensitive than signaling to the strength of ligand binding," Eur. J. Immunol. 33, 1084-1093 (2003).
[CrossRef] [PubMed]

Brakenhoff, G. J.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Brand, L.

A. Gafni, and L. Brand, "Fluorescence Decay Studies of Reduced Nicotinamide Adenine Dinucleotide in Solution and Bound to Liver Alcohol Dehydrogenase," Biochemistry 15, 3165-3171 (1976).
[CrossRef] [PubMed]

Brochon, J. C.

J. C. Brochon, P. Wahl, M. O. Monneuse-Doublet, and A. Olomucki, "Pulse Fluorimetry Study of Octopine Dehydrogenase-Reduced Nicotinamide Adenine Dinucleotide Complexes," Biochemistry 16, 4594-4599 (1977).
[CrossRef] [PubMed]

Buist, A. H.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

Burshtyn, D. N.

B. Treanor, P. M. P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A. A. Neil, D. N. Burshtyn, P. M. W. French, and D. M. Davis, "Microclusters of inhibitory killer immunoglobulin like receptor signaling at natural killer cell immunological synapses," J. Cell Biol. 174, 153-161 (2006).
[CrossRef] [PubMed]

P. D. Borszcz, M. Peterson, L. Standeven, S. Kirwan, M. Sandusky, A. Shaw, E. O. Long, and D. N. Burshtyn, "KIR enrichment at the effector-target cell interface is more sensitive than signaling to the strength of ligand binding," Eur. J. Immunol. 33, 1084-1093 (2003).
[CrossRef] [PubMed]

Carter, N.

D. R. Drummond, N. Carter, and R. A. Cross, "Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors," J. Microsc. 206, 161-169 (2002).
[CrossRef] [PubMed]

Centonze, V. E.

R. V. Krishnan, H. Saitoh, H. Terada, V. E. Centonze, and B. Herman, "Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera," Rev. Sci. Instrum. 74, 2714-2721 (2003).
[CrossRef]

Chance, B.

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, "Intracellular Oxidation-Reduction States in Vivo," Science 137, 499-508 (1962).
[CrossRef] [PubMed]

Chapman, C. F.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence for Localized Cell Heating Induced by Infrared Optical Tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Chen, D. N.

Cheng, D. K.

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence for Localized Cell Heating Induced by Infrared Optical Tweezers," Biophys. J. 68, 2137-2144 (1995).
[CrossRef] [PubMed]

Cianci, G. C.

G. C. Cianci, J. R. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc. Res. Tech. 64, 135-141 (2004).
[CrossRef] [PubMed]

Cohen, P.

B. Chance, P. Cohen, F. Jobsis, and B. Schoener, "Intracellular Oxidation-Reduction States in Vivo," Science 137, 499-508 (1962).
[CrossRef] [PubMed]

Comelli, D.

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D-Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

Conchello, J. A.

J. A. Conchello, and J. W. Lichtman, "Optical sectioning microscopy," Nature Methods 2, 920-931 (2005).
[CrossRef] [PubMed]

Cross, R. A.

D. R. Drummond, N. Carter, and R. A. Cross, "Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors," J. Microsc. 206, 161-169 (2002).
[CrossRef] [PubMed]

Cubeddu, R.

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D-Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

Culley, F. J.

B. Treanor, P. M. P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A. A. Neil, D. N. Burshtyn, P. M. W. French, and D. M. Davis, "Microclusters of inhibitory killer immunoglobulin like receptor signaling at natural killer cell immunological synapses," J. Cell Biol. 174, 153-161 (2006).
[CrossRef] [PubMed]

D'Andrea, C.

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D-Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

Davis, D. M.

B. Treanor, P. M. P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A. A. Neil, D. N. Burshtyn, P. M. W. French, and D. M. Davis, "Microclusters of inhibitory killer immunoglobulin like receptor signaling at natural killer cell immunological synapses," J. Cell Biol. 174, 153-161 (2006).
[CrossRef] [PubMed]

de Grauw, C. J.

deMello, A. J.

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, "2-Photon Laser Scanning Fluorescence Microscopy," Science 248, 73-76 (1990).
[CrossRef] [PubMed]

Drummond, D. R.

D. R. Drummond, N. Carter, and R. A. Cross, "Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors," J. Microsc. 206, 161-169 (2002).
[CrossRef] [PubMed]

Dunsby, C.

B. Treanor, P. M. P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A. A. Neil, D. N. Burshtyn, P. M. W. French, and D. M. Davis, "Microclusters of inhibitory killer immunoglobulin like receptor signaling at natural killer cell immunological synapses," J. Cell Biol. 174, 153-161 (2006).
[CrossRef] [PubMed]

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 13 (2004).
[CrossRef]

Durocher, G.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, "Steady state and time-resolved fluorescence properties of metastatic and non-metastatic malignant cells from different species," J. Photochem. Photobiol.B-Biol. 31, 101-112 (1995).
[CrossRef]

Dymoke-Bradshaw, A.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 13 (2004).
[CrossRef]

Eggeling, C.

C. Eggeling, A. Volkmer, and C. A. M. Seidel, "Molecular photobleaching kinetics of rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy," Chemphyschem 6, 791-804 (2005).
[CrossRef] [PubMed]

Egner, A.

A. Egner, and S. W. Hell, "Time multiplexing and parallelization in multifocal multiphoton microscopy," J. Opt. Soc. Am. A-Opt. Image Sci. Vis. 17, 1192-1201 (2000).
[CrossRef] [PubMed]

Eickhoff, J.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, "In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia," J. Biomed. Opt. 12, 024014-024011-024010 (2007).
[CrossRef] [PubMed]

Eliceiri, K. W.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, "In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia," J. Biomed. Opt. 12, 024014-024011-024010 (2007).
[CrossRef] [PubMed]

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, "Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH," Cancer Res. 65, 8766-8773 (2005).
[CrossRef] [PubMed]

Elson, D. S.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 13 (2004).
[CrossRef]

Fischer, P.

Fontaine-Aupart, M. P.

French, P. M. W.

B. Treanor, P. M. P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A. A. Neil, D. N. Burshtyn, P. M. W. French, and D. M. Davis, "Microclusters of inhibitory killer immunoglobulin like receptor signaling at natural killer cell immunological synapses," J. Cell Biol. 174, 153-161 (2006).
[CrossRef] [PubMed]

R. K. P. Benninger, O. Hofmann, J. McGinty, J. Requejo-Isidro, I. Munro, M. A. A. Neil, A. J. deMello, and P. M. W. French, "Time-resolved fluorescence imaging of solvent interactions in microfluidic devices," Opt. Express 13, 6275-6285 (2005).
[CrossRef] [PubMed]

K. Suhling, P. M. W. French, and D. Phillips, "Time-resolved fluorescence microscopy," Photochem. Photobiol. 4, 13-22 (2005).
[CrossRef]

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 13 (2004).
[CrossRef]

Frick, M.

T. Nielsen, M. Frick, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

Fu, Z.

L. Liu, J. Qu, Z. Lin, L. Wang, Z. Fu, B. Guo, and H. Niu, "Simultaneous time- and spectrum-resolved multifocal multiphoton microscopy," Appl. Phys. B-Lasers and Optics 84, 379-383 (2006).
[CrossRef]

Gaboury, L.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, "Steady state and time-resolved fluorescence properties of metastatic and non-metastatic malignant cells from different species," J. Photochem. Photobiol.B-Biol. 31, 101-112 (1995).
[CrossRef]

Gafni, A.

A. Gafni, and L. Brand, "Fluorescence Decay Studies of Reduced Nicotinamide Adenine Dinucleotide in Solution and Bound to Liver Alcohol Dehydrogenase," Biochemistry 15, 3165-3171 (1976).
[CrossRef] [PubMed]

Galletly, N.

D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 13 (2004).
[CrossRef]

Gendron-Fitzpatrick, A.

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, "In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia," J. Biomed. Opt. 12, 024014-024011-024010 (2007).
[CrossRef] [PubMed]

Georges, P.

Gerritsen, H. C.

H. C. Gerritsen, M. A. H. Asselbergs, A. V. Agronskaia, and W. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
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Qu, J. N. Y.

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M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, "In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia," J. Biomed. Opt. 12, 024014-024011-024010 (2007).
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[CrossRef]

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M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, "In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia," J. Biomed. Opt. 12, 024014-024011-024010 (2007).
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Riemann, I.

Rogatsky, G. G.

A. Mayevsky, and G. G. Rogatsky, "Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies," Am. J. Physiol.-Cell Physiol. 292, C615-C640 (2007).
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Saitoh, H.

R. V. Krishnan, H. Saitoh, H. Terada, V. E. Centonze, and B. Herman, "Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera," Rev. Sci. Instrum. 74, 2714-2721 (2003).
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P. D. Borszcz, M. Peterson, L. Standeven, S. Kirwan, M. Sandusky, A. Shaw, E. O. Long, and D. N. Burshtyn, "KIR enrichment at the effector-target cell interface is more sensitive than signaling to the strength of ligand binding," Eur. J. Immunol. 33, 1084-1093 (2003).
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Seidel, C. A. M.

C. Eggeling, A. Volkmer, and C. A. M. Seidel, "Molecular photobleaching kinetics of rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy," Chemphyschem 6, 791-804 (2005).
[CrossRef] [PubMed]

Shaw, A.

P. D. Borszcz, M. Peterson, L. Standeven, S. Kirwan, M. Sandusky, A. Shaw, E. O. Long, and D. N. Burshtyn, "KIR enrichment at the effector-target cell interface is more sensitive than signaling to the strength of ligand binding," Eur. J. Immunol. 33, 1084-1093 (2003).
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M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, "In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia," J. Biomed. Opt. 12, 024014-024011-024010 (2007).
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T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 9 (2007).
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Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence for Localized Cell Heating Induced by Infrared Optical Tweezers," Biophys. J. 68, 2137-2144 (1995).
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J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, "Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability," Nat. Biotechnol. 17, 763-767 (1999).
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D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. Dunsby, N. Galletly, G. W. Stamp, M. A. A. Neil, M. J. Lever, P. A. Kellett, A. Dymoke-Bradshaw, J. Hares, and P. M. W. French, "Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier," New J. Phys. 6, 13 (2004).
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P. D. Borszcz, M. Peterson, L. Standeven, S. Kirwan, M. Sandusky, A. Shaw, E. O. Long, and D. N. Burshtyn, "KIR enrichment at the effector-target cell interface is more sensitive than signaling to the strength of ligand binding," Eur. J. Immunol. 33, 1084-1093 (2003).
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W. Denk, J. H. Strickler, and W. W. Webb, "2-Photon Laser Scanning Fluorescence Microscopy," Science 248, 73-76 (1990).
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T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 9 (2007).
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R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D-Appl. Phys. 35, R61-R76 (2002).
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R. V. Krishnan, H. Saitoh, H. Terada, V. E. Centonze, and B. Herman, "Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera," Rev. Sci. Instrum. 74, 2714-2721 (2003).
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L. M. Tiede, and M. G. Nichols, "Photobleaching of reduced nicotinamide adenine dinucleotide and the development of highly fluorescent lesions in rat basophilic leukemia cells during multiphoton microscopy," Photochem. Photobiol. 82, 656-664 (2006).
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Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence for Localized Cell Heating Induced by Infrared Optical Tweezers," Biophys. J. 68, 2137-2144 (1995).
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H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, "Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: Signal and photodamage," Biophys. J. 77, 2226-2236 (1999).
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Valentini, G.

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D-Appl. Phys. 35, R61-R76 (2002).
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A. J. W. G. Visser, and A. van Hoek, "The fluorescence decay of reduced nicotinamides in aqueous solution after exciation with a UV-mode locked Ar Ion Laser," Photochem. Photobiol. 33, 35-40 (1981).
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H. C. Gerritsen, M. A. H. Asselbergs, A. V. Agronskaia, and W. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
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D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, "Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH," Cancer Res. 65, 8766-8773 (2005).
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A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, "Steady state and time-resolved fluorescence properties of metastatic and non-metastatic malignant cells from different species," J. Photochem. Photobiol.B-Biol. 31, 101-112 (1995).
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H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, "Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy," J. Biol. Chem. 280, 25119-25126 (2005).
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A. J. W. G. Visser, and A. van Hoek, "The fluorescence decay of reduced nicotinamides in aqueous solution after exciation with a UV-mode locked Ar Ion Laser," Photochem. Photobiol. 33, 35-40 (1981).
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C. Eggeling, A. Volkmer, and C. A. M. Seidel, "Molecular photobleaching kinetics of rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy," Chemphyschem 6, 791-804 (2005).
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D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, "Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH," Cancer Res. 65, 8766-8773 (2005).
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J. C. Brochon, P. Wahl, M. O. Monneuse-Doublet, and A. Olomucki, "Pulse Fluorimetry Study of Octopine Dehydrogenase-Reduced Nicotinamide Adenine Dinucleotide Complexes," Biochemistry 16, 4594-4599 (1977).
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M. Wakita, G. Nishimura, and M. Tamura, "Some Characteristics of the Fluorescence Lifetime of Reduced Pyridine-Nucleotides in Isolated-Mitochondria, Isolated Hepatocytes, and Perfused-Rat-Liver in-Situ," J. Biochem.(Tokyo) 118, 1151-1160 (1995).

Wang, L.

L. Liu, J. Qu, Z. Lin, L. Wang, Z. Fu, B. Guo, and H. Niu, "Simultaneous time- and spectrum-resolved multifocal multiphoton microscopy," Appl. Phys. B-Lasers and Optics 84, 379-383 (2006).
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H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, "Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy," J. Biol. Chem. 280, 25119-25126 (2005).
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S. H. Huang, A. A. Heikal, and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J. 82, 2811-2825 (2002).
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D. W. Piston, B. R. Masters, and W. W. Webb, "3-Dimensionally Resolved NAD(P)H Cellular Metabolic Redox Imaging of the in-Situ Cornea with 2-Photon Excitation Laser-Scanning Microscopy," J. Microsc. 178, 20-27 (1995).
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W. Denk, J. H. Strickler, and W. W. Webb, "2-Photon Laser Scanning Fluorescence Microscopy," Science 248, 73-76 (1990).
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D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, "Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH," Cancer Res. 65, 8766-8773 (2005).
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J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, "Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability," Nat. Biotechnol. 17, 763-767 (1999).
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J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, "Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability," Nat. Biotechnol. 17, 763-767 (1999).
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Wu, J. R.

G. C. Cianci, J. R. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc. Res. Tech. 64, 135-141 (2004).
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Wu, Y. C.

Xu, G. X.

Yan, L.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, "Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH," Cancer Res. 65, 8766-8773 (2005).
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Zheng, W.

Am. J. Physiol.-Cell Physiol.

A. Mayevsky, and G. G. Rogatsky, "Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies," Am. J. Physiol.-Cell Physiol. 292, C615-C640 (2007).
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Appl. Phys. B-Lasers and Optics

L. Liu, J. Qu, Z. Lin, L. Wang, Z. Fu, B. Guo, and H. Niu, "Simultaneous time- and spectrum-resolved multifocal multiphoton microscopy," Appl. Phys. B-Lasers and Optics 84, 379-383 (2006).
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Appl. Phys. Lett.

M. Straub, and S. W. Hell, "Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope," Appl. Phys. Lett. 73, 1769-1771 (1998).
[CrossRef]

Appl. Spectrosc.

B-Biol.

A. Pradhan, P. Pal, G. Durocher, L. Villeneuve, A. Balassy, F. Babai, L. Gaboury, and L. Blanchard, "Steady state and time-resolved fluorescence properties of metastatic and non-metastatic malignant cells from different species," J. Photochem. Photobiol.B-Biol. 31, 101-112 (1995).
[CrossRef]

Biochemistry

J. C. Brochon, P. Wahl, M. O. Monneuse-Doublet, and A. Olomucki, "Pulse Fluorimetry Study of Octopine Dehydrogenase-Reduced Nicotinamide Adenine Dinucleotide Complexes," Biochemistry 16, 4594-4599 (1977).
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A. Gafni, and L. Brand, "Fluorescence Decay Studies of Reduced Nicotinamide Adenine Dinucleotide in Solution and Bound to Liver Alcohol Dehydrogenase," Biochemistry 15, 3165-3171 (1976).
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Biol. Cell

M. Peter, and S. M. Ameer-Beg, "Imaging molecular interactions by multiphoton FLIM," Biol. Cell 96, 231-236 (2004).
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Biophys. J.

G. H. Patterson, and D. W. Piston, "Photobleaching in two-photon excitation microscopy," Biophys. J. 78, 2159-2162 (2000).
[CrossRef] [PubMed]

Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, "Evidence for Localized Cell Heating Induced by Infrared Optical Tweezers," Biophys. J. 68, 2137-2144 (1995).
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A. Hopt, and E. Neher, "Highly nonlinear photodamage in two-photon fluorescence microscopy," Biophys. J. 80, 2029-2036 (2001).
[CrossRef] [PubMed]

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, "Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: Signal and photodamage," Biophys. J. 77, 2226-2236 (1999).
[CrossRef] [PubMed]

B. R. Masters, P. T. C. So, and E. Gratton, "Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin," Biophys. J. 72, 2405-2412 (1997).
[CrossRef] [PubMed]

S. H. Huang, A. A. Heikal, and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J. 82, 2811-2825 (2002).
[CrossRef] [PubMed]

Cancer Res.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, "Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH," Cancer Res. 65, 8766-8773 (2005).
[CrossRef] [PubMed]

Chemphyschem

C. Eggeling, A. Volkmer, and C. A. M. Seidel, "Molecular photobleaching kinetics of rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy," Chemphyschem 6, 791-804 (2005).
[CrossRef] [PubMed]

Eur. J. Immunol.

P. D. Borszcz, M. Peterson, L. Standeven, S. Kirwan, M. Sandusky, A. Shaw, E. O. Long, and D. N. Burshtyn, "KIR enrichment at the effector-target cell interface is more sensitive than signaling to the strength of ligand binding," Eur. J. Immunol. 33, 1084-1093 (2003).
[CrossRef] [PubMed]

J. Biochem.(Tokyo)

M. Wakita, G. Nishimura, and M. Tamura, "Some Characteristics of the Fluorescence Lifetime of Reduced Pyridine-Nucleotides in Isolated-Mitochondria, Isolated Hepatocytes, and Perfused-Rat-Liver in-Situ," J. Biochem.(Tokyo) 118, 1151-1160 (1995).

J. Biol. Chem.

H. D. Vishwasrao, A. A. Heikal, K. A. Kasischke, and W. W. Webb, "Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy," J. Biol. Chem. 280, 25119-25126 (2005).
[CrossRef] [PubMed]

J. Biomed. Opt.

J. Martini, V. Andresen, and D. Anselmetti, "Scattering suppression and confocal detection in multifocal multiphoton microscopy," J. Biomed. Opt. 12, 034010-034016 (2007).
[CrossRef] [PubMed]

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 9 (2007).
[CrossRef]

M. C. Skala, K. M. Riching, D. K. Bird, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, P. J. Keely, and N. Ramanujam, "In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia," J. Biomed. Opt. 12, 024014-024011-024010 (2007).
[CrossRef] [PubMed]

J. Cell Biol.

B. Treanor, P. M. P. Lanigan, S. Kumar, C. Dunsby, I. Munro, E. Auksorius, F. J. Culley, M. A. Purbhoo, D. Phillips, M. A. A. Neil, D. N. Burshtyn, P. M. W. French, and D. M. Davis, "Microclusters of inhibitory killer immunoglobulin like receptor signaling at natural killer cell immunological synapses," J. Cell Biol. 174, 153-161 (2006).
[CrossRef] [PubMed]

J. Microsc.

D. W. Piston, B. R. Masters, and W. W. Webb, "3-Dimensionally Resolved NAD(P)H Cellular Metabolic Redox Imaging of the in-Situ Cornea with 2-Photon Excitation Laser-Scanning Microscopy," J. Microsc. 178, 20-27 (1995).
[CrossRef] [PubMed]

D. R. Drummond, N. Carter, and R. A. Cross, "Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors," J. Microsc. 206, 161-169 (2002).
[CrossRef] [PubMed]

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Microsc. 192, 217-226 (1998).
[CrossRef]

T. Nielsen, M. Frick, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc. 201, 368-376 (2001).
[CrossRef] [PubMed]

H. C. Gerritsen, M. A. H. Asselbergs, A. V. Agronskaia, and W. Van Sark, "Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution," J. Microsc. 206, 218-224 (2002).
[CrossRef] [PubMed]

J. Microscopy-Oxford.

K. Konig, "Multiphoton microscopy in life sciences," J. Microscopy-Oxford. 200, 83-104 (2000).
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J. Opt. Soc. Am. A-Opt. Image Sci. Vis.

A. Egner, and S. W. Hell, "Time multiplexing and parallelization in multifocal multiphoton microscopy," J. Opt. Soc. Am. A-Opt. Image Sci. Vis. 17, 1192-1201 (2000).
[CrossRef] [PubMed]

J. Phys. D-Appl. Phys.

R. Cubeddu, D. Comelli, C. D'Andrea, P. Taroni, and G. Valentini, "Time-resolved fluorescence imaging in biology and medicine," J. Phys. D-Appl. Phys. 35, R61-R76 (2002).
[CrossRef]

Microsc. Res. Tech.

W. Becker, A. Bergmann, M. A. Hink, K. Konig, K. Benndorf, and C. Biskup, "Fluorescence lifetime imaging by time-correlated single-photon counting," Microsc. Res. Tech. 63, 58-66 (2004).
[CrossRef]

G. C. Cianci, J. R. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc. Res. Tech. 64, 135-141 (2004).
[CrossRef] [PubMed]

Nat. Biotechnol.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, "Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability," Nat. Biotechnol. 17, 763-767 (1999).
[CrossRef] [PubMed]

E. A. Jares-Erijman, and T. M. Jovin, "FRET imaging," Nat. Biotechnol. 21, 1387-1395 (2003).
[CrossRef] [PubMed]

Nature Methods

J. A. Conchello, and J. W. Lichtman, "Optical sectioning microscopy," Nature Methods 2, 920-931 (2005).
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Supplementary Material (1)

» Media 1: AVI (1561 KB)     

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Figures (6)

Fig. 1.
Fig. 1.

Experimental setup: P1, P2 are pinholes, λ1,λ2 are half wave plates, L1-L6 are lenses, PR1, PR2 are prisms, MUX is the beam multiplexer, Pol is a rotatable polarizing beamsplitter cube and IP1, IP2 are image planes.

Fig. 2.
Fig. 2.

3-D FLIM image of fluorescently labelled pollen grains. (a) fluorescence intensity and (b) intensity-merged false-colour FLIM map for one slice in the image stack. (c) shows a single movie frame of a 3-D rendering [1561 KB] of the whole FLIM image stack using the same false colour scale as used for (b). λex = 800 nm and the excitation power was set to Pex = 1 mW per beam, which corresponded to a count rate of ~2.4 MHz. Scale bars in (a) and (b) are 2.5 μm and the data volume rendered in (c) is 25.6×40×75 μm3. [Media 1]

Fig. 3.
Fig. 3.

(a-b) fluorescence intensity and (c-d) corresponding intensity-merged false-colour FLIM image of KIR2DL1-GFP expressing YTS cells. A total of 25 optically sectioned images were recorded using an acquisition time of 60 s per slice with λex = 880 nm and an excitation power of 3.8 mW per foci. The field of view for this image was 25.6×40 μm with a z-step of 0.5 μm between consecutively numbered images. Scale bars are 2.5 μm.

Fig. 4.
Fig. 4.

(a). transmitted light image and (b) corresponding autofluorescence intensity image (averaged over 3 frames) of a HEK293 cell; image size is 80x256 pixels and the field of view is 40×25.6 μm Scale bars in both images are 2.5 μm.

Fig. 5.
Fig. 5.

Fluorescence decay profile averaged over an individual HEK293 cell from a single (7.7 s) acquisition frame and fit using a double exponential decay model. The bottom part of the figure shows the residuals of the fit.

Fig. 6.
Fig. 6.

Fitted time-lapse decay parameters for a HEK293a cell stimulated by 5 mM NaCN at frame 60. Each frame corresponds to 7.7 s. The graphs show (from top to bottom) total number of counts over the ROI, the pre-exponential factors (a1 and a2), the fluorescence decay lifetimes (tau1 and tau2), the calculated mean fluorescence lifetime (meantau) and the goodness of fit parameter χ2 (chisqr). The vertical red lines indicate the time points at which HBSS and NaCN were added.

Tables (1)

Tables Icon

Table 1. Summary of changes in autofluorescence signal observed before and after stimulation

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