Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane. This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. The accuracy and performance of the proposed method is demonstrated in several imaging studies important for understanding peripheral nerve regeneration processes. Most importantly, the parallelism of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude.

© 2012 OSA

1. P. I. Bastiaens, P. J. Bonants, F. Müller, and A. J. Visser, “Time-resolved fluorescence spectroscopy of NADPH-cytochrome P-450 reductase: demonstration of energy transfer between the two prosthetic groups,” Biochemistry28(21), 8416–8425 (1989). [CrossRef] [PubMed]
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4. E. Gratton, S. Breusegem, J. Sutin, Q. Ruan, and N. Barry, “Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods,” J. Biomed. Opt.8(3), 381–390 (2003). [CrossRef] [PubMed]
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6. K. König, P. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, “Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress,” J. Microsc.183(Pt 3), 197–204 (1996). [PubMed]
7. J. R. Lakowicz, “Emerging applications of fluorescence spectroscopy to cellular imaging: lifetime imaging, metal-ligand probes, multi-photon excitation and light quenching,” Scanning Microsc. Suppl.10, 213–224 (1996). [PubMed]
8. R. M. Clegg, A. I. Murchie, and D. M. Lilley, “The four-way DNA junction: a fluorescence resonance energy transfer study,” Braz. J. Med. Biol. Res.26(4), 405–416 (1993). [PubMed]
9. A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A.97(10), 5179–5184 (2000). [CrossRef] [PubMed]
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17. E. K. Rofstad, “Microenvironment-induced cancer metastasis,” Int. J. Radiat. Biol.76(5), 589–605 (2000). [CrossRef] [PubMed]
18. S. M. Evans and C. J. Koch, “Prognostic significance of tumor oxygenation in humans,” Cancer Lett.195(1), 1–16 (2003). [CrossRef] [PubMed]
19. M. I. Koukourakis, A. Giatromanolaki, R. A. Brekken, E. Sivridis, K. C. Gatter, A. L. Harris, and E. H. Sage, “Enhanced expression of SPARC/osteonectin in the tumor-associated stroma of non-small cell lung cancer is correlated with markers of hypoxia/acidity and with poor prognosis of patients,” Cancer Res.63(17), 5376–5380 (2003). [PubMed]
20. M. Weinmann, C. Belka, and L. Plasswilm, “Tumour hypoxia: impact on biology, prognosis and treatment of solid malignant tumours,” Onkologie27(1), 83–90 (2004). [CrossRef] [PubMed]
21. J. M. Brown and A. J. Giaccia, “The unique physiology of solid tumors: opportunities (and problems) for cancer therapy,” Cancer Res.58(7), 1408–1416 (1998). [PubMed]
22. I. F. Tannock and D. Rotin, “Acid pH in tumors and its potential for therapeutic exploitation,” Cancer Res.49(16), 4373–4384 (1989). [PubMed]
23. S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods7(9), 755–759 (2010). [CrossRef] [PubMed]
24. Y. Wang, S. Hu, K. Maslov, Y. Zhang, Y. Xia, and L. V. Wang, “In vivo integrated photoacoustic and confocal microscopy of hemoglobin oxygen saturation and oxygen partial pressure,” Opt. Lett.36(7), 1029–1031 (2011). [CrossRef] [PubMed]
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27. K. Suhling, J. Siegel, P. M. Lanigan, S. Lévêque-Fort, S. E. Webb, D. Phillips, D. M. Davis, and P. M. French, “Time-resolved fluorescence anisotropy imaging applied to live cells,” Opt. Lett.29(6), 584–586 (2004). [CrossRef] [PubMed]
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30. J. McGinty, K. B. Tahir, R. Laine, C. B. Talbot, C. Dunsby, M. A. Neil, L. Quintana, J. Swoger, J. Sharpe, and P. M. French, “Fluorescence lifetime optical projection tomography,” J Biophotonics1(5), 390–394 (2008). [CrossRef] [PubMed]
31. D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A. de Beule, C. Dunsby, M. A. Neil, and P. M. French, “Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source,” Opt. Lett.32(23), 3408–3410 (2007). [CrossRef] [PubMed]
32. S. Kumar, C. Dunsby, P. A. De Beule, D. M. Owen, U. Anand, P. M. Lanigan, R. K. Benninger, D. M. Davis, M. A. Neil, P. Anand, C. Benham, A. Naylor, and P. M. French, “Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging,” Opt. Express15(20), 12548–12561 (2007). [CrossRef] [PubMed]
33. D. Magde, G. E. Rojas, and P. G. Seybold, “Solvent dependence of the fluorescence lifetimes of xanthene dyes,” Photochem. Photobiol.70(5), 737–744 (1999). [CrossRef]
34. G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc.15(5), 805–815 (2005). [CrossRef] [PubMed]
35. M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008). [CrossRef] [PubMed]
36. N. A. A. Rahim, W. McDaniel, K. Bardon, S. Srinivas, V. Vickerman, P. T. C. So, and J. H. Moon, “Conjugated polymer nanoparticles for two-photon imaging of endothelial cells in a tissue model,” Adv. Mater. (Deerfield Beach Fla.)21(34), 3492–3496 (2009). [CrossRef]
37. A. Nagy, J. Wu, and K. M. Berland, “Observation volumes and gamma-factors in two-photon fluorescence fluctuation spectroscopy,” Biophys. J.89(3), 2077–2090 (2005). [CrossRef] [PubMed]
38. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990). [CrossRef] [PubMed]
39. L. C. Cheng, C. Y. Chang, C. Y. Lin, K. C. Cho, W. C. Yen, N. S. Chang, C. Xu, C. Y. Dong, and S. J. Chen, “Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning,” Opt. Express20(8), 8939–8948 (2012). [CrossRef] [PubMed]
40. E. C. Soller, D. S. Tzeranis, K. Miu, P. T. So, and I. V. Yannas, “Common features of optimal collagen scaffolds that disrupt wound contraction and enhance regeneration both in peripheral nerves and in skin,” Biomaterials33(19), 4783–4791 (2012). [CrossRef] [PubMed]
41. L. J. Chamberlain, I. V. Yannas, H. P. Hsu, G. Strichartz, and M. Spector, “Collagen-GAG substrate enhances the quality of nerve regeneration through collagen tubes up to level of autograft,” Exp. Neurol.154(2), 315–329 (1998). [CrossRef] [PubMed]
42. I. V. Yannas, J. F. Burke, D. P. Orgill, and E. M. Skrabut, “Wound tissue can utilize a polymeric template to synthesize a functional extension of skin,” Science215(4529), 174–176 (1982). [CrossRef] [PubMed]
43. I. V. Yannas, Tissue and Organ Regeneration in Adults, (Springer, New York, 2001).
44. S. Pelet, M. J. Previte, L. H. Laiho, and P. T. So, “A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation,” Biophys. J.87(4), 2807–2817 (2004). [CrossRef] [PubMed]
45. P. Walczysko, U. Kuhlicke, S. Knappe, C. Cordes, and T. R. Neu, “In situ activity of suspended and immobilized microbial communities as measured by fluorescence lifetime imaging,” Appl. Environ. Microbiol.74(1), 294–299 (2008). [CrossRef] [PubMed]
46. J. M. Brown, “Tumor microenvironment and the response to anticancer therapy,” Cancer Biol. Ther.1(5), 448–458 (2002). [CrossRef] [PubMed]
47. G. Mehta, K. Mehta, D. Sud, J. W. Song, T. Bersano-Begey, N. Futai, Y. S. Heo, M. A. Mycek, J. J. Linderman, and S. Takayama, “Quantitative measurement and control of oxygen levels in microfluidic poly(dimethylsiloxane) bioreactors during cell culture,” Biomed. Microdevices9(2), 123–134 (2007). [CrossRef] [PubMed]
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49. D. Sud and M. A. Mycek, “Calibration and validation of an optical sensor for intracellular oxygen measurements,” J. Biomed. Opt.14(2), 020506 (2009). [CrossRef] [PubMed]
50. D. Sud, W. Zhong, D. G. Beer, and M. A. Mycek, “Time-resolved optical imaging provides a molecular snapshot of altered metabolic function in living human cancer cell models,” Opt. Express14(10), 4412–4426 (2006). [CrossRef] [PubMed]

Adv. Mater. (Deerfield Beach Fla.)

• N. A. A. Rahim, W. McDaniel, K. Bardon, S. Srinivas, V. Vickerman, P. T. C. So, and J. H. Moon, “Conjugated polymer nanoparticles for two-photon imaging of endothelial cells in a tissue model,” Adv. Mater. (Deerfield Beach Fla.)21(34), 3492–3496 (2009). [CrossRef]

Appl. Environ. Microbiol.

• P. Walczysko, U. Kuhlicke, S. Knappe, C. Cordes, and T. R. Neu, “In situ activity of suspended and immobilized microbial communities as measured by fluorescence lifetime imaging,” Appl. Environ. Microbiol.74(1), 294–299 (2008). [CrossRef] [PubMed]

Biochemistry

• P. I. Bastiaens, P. J. Bonants, F. Müller, and A. J. Visser, “Time-resolved fluorescence spectroscopy of NADPH-cytochrome P-450 reductase: demonstration of energy transfer between the two prosthetic groups,” Biochemistry28(21), 8416–8425 (1989). [CrossRef] [PubMed]

Biomaterials

• E. C. Soller, D. S. Tzeranis, K. Miu, P. T. So, and I. V. Yannas, “Common features of optimal collagen scaffolds that disrupt wound contraction and enhance regeneration both in peripheral nerves and in skin,” Biomaterials33(19), 4783–4791 (2012). [CrossRef] [PubMed]

Biomed. Microdevices

• G. Mehta, K. Mehta, D. Sud, J. W. Song, T. Bersano-Begey, N. Futai, Y. S. Heo, M. A. Mycek, J. J. Linderman, and S. Takayama, “Quantitative measurement and control of oxygen levels in microfluidic poly(dimethylsiloxane) bioreactors during cell culture,” Biomed. Microdevices9(2), 123–134 (2007). [CrossRef] [PubMed]

Biophys. Chem.

• I. Gryczynski, A. Razynska, and J. R. Lakowicz, “Two-photon induced fluorescence of linear alkanes; a possible intrinsic lipid probe,” Biophys. Chem.57(2-3), 291–295 (1996). [CrossRef] [PubMed]

Biophys. J.

• A. Nagy, J. Wu, and K. M. Berland, “Observation volumes and gamma-factors in two-photon fluorescence fluctuation spectroscopy,” Biophys. J.89(3), 2077–2090 (2005). [CrossRef] [PubMed]
• M. A. Digman, V. R. Caiolfa, M. Zamai, and E. Gratton, “The phasor approach to fluorescence lifetime imaging analysis,” Biophys. J.94(2), L14–L16 (2008). [CrossRef] [PubMed]
• S. Pelet, M. J. Previte, L. H. Laiho, and P. T. So, “A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation,” Biophys. J.87(4), 2807–2817 (2004). [CrossRef] [PubMed]

Braz. J. Med. Biol. Res.

• R. M. Clegg, A. I. Murchie, and D. M. Lilley, “The four-way DNA junction: a fluorescence resonance energy transfer study,” Braz. J. Med. Biol. Res.26(4), 405–416 (1993). [PubMed]

Cancer Biol. Ther.

• J. M. Brown, “Tumor microenvironment and the response to anticancer therapy,” Cancer Biol. Ther.1(5), 448–458 (2002). [CrossRef] [PubMed]

Cancer Lett.

• S. M. Evans and C. J. Koch, “Prognostic significance of tumor oxygenation in humans,” Cancer Lett.195(1), 1–16 (2003). [CrossRef] [PubMed]

Cancer Res.

• M. I. Koukourakis, A. Giatromanolaki, R. A. Brekken, E. Sivridis, K. C. Gatter, A. L. Harris, and E. H. Sage, “Enhanced expression of SPARC/osteonectin in the tumor-associated stroma of non-small cell lung cancer is correlated with markers of hypoxia/acidity and with poor prognosis of patients,” Cancer Res.63(17), 5376–5380 (2003). [PubMed]
• J. M. Brown and A. J. Giaccia, “The unique physiology of solid tumors: opportunities (and problems) for cancer therapy,” Cancer Res.58(7), 1408–1416 (1998). [PubMed]
• I. F. Tannock and D. Rotin, “Acid pH in tumors and its potential for therapeutic exploitation,” Cancer Res.49(16), 4373–4384 (1989). [PubMed]

Curr. Opin. Struct. Biol.

• X. Zhuang and M. Rief, “Single-molecule folding,” Curr. Opin. Struct. Biol.13(1), 88–97 (2003). [CrossRef] [PubMed]

Exp. Dermatol.

• E. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, and M. Kaatz, “Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis,” Exp. Dermatol.18(6), 509–515 (2009). [CrossRef] [PubMed]

Exp. Neurol.

• L. J. Chamberlain, I. V. Yannas, H. P. Hsu, G. Strichartz, and M. Spector, “Collagen-GAG substrate enhances the quality of nerve regeneration through collagen tubes up to level of autograft,” Exp. Neurol.154(2), 315–329 (1998). [CrossRef] [PubMed]

IEEE Eng. Med. Biol. Mag.

• H. C. Gerritsen, J. M. Vroom, and C. J. de Grauw, “Combining two-photon excitation with fluorescence lifetime imaging,” IEEE Eng. Med. Biol. Mag.18(5), 31–36 (1999). [CrossRef] [PubMed]

Int. J. Radiat. Biol.

• E. K. Rofstad, “Microenvironment-induced cancer metastasis,” Int. J. Radiat. Biol.76(5), 589–605 (2000). [CrossRef] [PubMed]

J Biophotonics

• J. McGinty, K. B. Tahir, R. Laine, C. B. Talbot, C. Dunsby, M. A. Neil, L. Quintana, J. Swoger, J. Sharpe, and P. M. French, “Fluorescence lifetime optical projection tomography,” J Biophotonics1(5), 390–394 (2008). [CrossRef] [PubMed]

J. Appl. Physiol.

• L. S. Ziemer, W. M. Lee, S. A. Vinogradov, C. Sehgal, and D. F. Wilson, “Oxygen distribution in murine tumors: characterization using oxygen-dependent quenching of phosphorescence,” J. Appl. Physiol.98(4), 1503–1510 (2005). [CrossRef] [PubMed]

J. Biomed. Opt.

• E. Gratton, S. Breusegem, J. Sutin, Q. Ruan, and N. Barry, “Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods,” J. Biomed. Opt.8(3), 381–390 (2003). [CrossRef] [PubMed]
• D. Sud, G. Mehta, K. Mehta, J. Linderman, S. Takayama, and M. A. Mycek, “Optical imaging in microfluidic bioreactors enables oxygen monitoring for continuous cell culture,” J. Biomed. Opt.11(5), 050504 (2006). [CrossRef] [PubMed]
• D. Sud and M. A. Mycek, “Calibration and validation of an optical sensor for intracellular oxygen measurements,” J. Biomed. Opt.14(2), 020506 (2009). [CrossRef] [PubMed]

J. Fluoresc.

• G. I. Redford and R. M. Clegg, “Polar plot representation for frequency-domain analysis of fluorescence lifetimes,” J. Fluoresc.15(5), 805–815 (2005). [CrossRef] [PubMed]

J. Microsc.

• K. König, P. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton, “Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress,” J. Microsc.183(Pt 3), 197–204 (1996). [PubMed]

Microsc. Res. Tech.

• W. Becker, A. Bergmann, M. A. Hink, K. König, K. Benndorf, and C. Biskup, “Fluorescence lifetime imaging by time-correlated single-photon counting,” Microsc. Res. Tech.63(1), 58–66 (2004). [CrossRef] [PubMed]
• K. König, A. Ehlers, I. Riemann, S. Schenkl, R. Bückle, and M. Kaatz, “Clinical two-photon microendoscopy,” Microsc. Res. Tech.70(5), 398–402 (2007). [CrossRef] [PubMed]

Nat. Med.

• G. Helmlinger, F. Yuan, M. Dellian, and R. K. Jain, “Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation,” Nat. Med.3(2), 177–182 (1997). [CrossRef] [PubMed]

Nat. Methods

• S. Sakadzić, E. Roussakis, M. A. Yaseen, E. T. Mandeville, V. J. Srinivasan, K. Arai, S. Ruvinskaya, A. Devor, E. H. Lo, S. A. Vinogradov, and D. A. Boas, “Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue,” Nat. Methods7(9), 755–759 (2010). [CrossRef] [PubMed]

Nat. Rev. Cancer

• A. L. Harris, “Hypoxia--a key regulatory factor in tumour growth,” Nat. Rev. Cancer2(1), 38–47 (2002). [CrossRef] [PubMed]

Onkologie

• M. Weinmann, C. Belka, and L. Plasswilm, “Tumour hypoxia: impact on biology, prognosis and treatment of solid malignant tumours,” Onkologie27(1), 83–90 (2004). [CrossRef] [PubMed]

Opt. Express

• S. Kumar, C. Dunsby, P. A. De Beule, D. M. Owen, U. Anand, P. M. Lanigan, R. K. Benninger, D. M. Davis, M. A. Neil, P. Anand, C. Benham, A. Naylor, and P. M. French, “Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging,” Opt. Express15(20), 12548–12561 (2007). [CrossRef] [PubMed]
• L. C. Cheng, C. Y. Chang, C. Y. Lin, K. C. Cho, W. C. Yen, N. S. Chang, C. Xu, C. Y. Dong, and S. J. Chen, “Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning,” Opt. Express20(8), 8939–8948 (2012). [CrossRef] [PubMed]
• D. Sud, W. Zhong, D. G. Beer, and M. A. Mycek, “Time-resolved optical imaging provides a molecular snapshot of altered metabolic function in living human cancer cell models,” Opt. Express14(10), 4412–4426 (2006). [CrossRef] [PubMed]

Opt. Lett.

• Y. Wang, S. Hu, K. Maslov, Y. Zhang, Y. Xia, and L. V. Wang, “In vivo integrated photoacoustic and confocal microscopy of hemoglobin oxygen saturation and oxygen partial pressure,” Opt. Lett.36(7), 1029–1031 (2011). [CrossRef] [PubMed]
• D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A. de Beule, C. Dunsby, M. A. Neil, and P. M. French, “Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source,” Opt. Lett.32(23), 3408–3410 (2007). [CrossRef] [PubMed]
• K. Suhling, J. Siegel, P. M. Lanigan, S. Lévêque-Fort, S. E. Webb, D. Phillips, D. M. Davis, and P. M. French, “Time-resolved fluorescence anisotropy imaging applied to live cells,” Opt. Lett.29(6), 584–586 (2004). [CrossRef] [PubMed]
• J. Requejo-Isidro, J. McGinty, I. Munro, D. S. Elson, N. P. Galletly, M. J. Lever, M. A. Neil, G. W. Stamp, P. M. French, P. A. Kellett, J. D. Hares, and A. K. Dymoke-Bradshaw, “High-speed wide-field time-gated endoscopic fluorescence-lifetime imaging,” Opt. Lett.29(19), 2249–2251 (2004). [CrossRef] [PubMed]
• D. M. Grant, D. S. Elson, D. Schimpf, C. Dunsby, J. Requejo-Isidro, E. Auksorius, I. Munro, M. A. Neil, P. M. French, E. Nye, G. Stamp, and P. Courtney, “Optically sectioned fluorescence lifetime imaging using a Nipkow disk microscope and a tunable ultrafast continuum excitation source,” Opt. Lett.30(24), 3353–3355 (2005). [CrossRef] [PubMed]

Photochem. Photobiol.

• D. Magde, G. E. Rojas, and P. G. Seybold, “Solvent dependence of the fluorescence lifetimes of xanthene dyes,” Photochem. Photobiol.70(5), 737–744 (1999). [CrossRef]

Proc. Natl. Acad. Sci. U.S.A.

• I. P. Torres Filho, M. Leunig, F. Yuan, M. Intaglietta, and R. K. Jain, “Noninvasive measurement of microvascular and interstitial oxygen profiles in a human tumor in SCID mice,” Proc. Natl. Acad. Sci. U.S.A.91(6), 2081–2085 (1994). [CrossRef] [PubMed]
• A. A. Deniz, T. A. Laurence, G. S. Beligere, M. Dahan, A. B. Martin, D. S. Chemla, P. E. Dawson, P. G. Schultz, and S. Weiss, “Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2,” Proc. Natl. Acad. Sci. U.S.A.97(10), 5179–5184 (2000). [CrossRef] [PubMed]

Scanning Microsc. Suppl.

• J. R. Lakowicz, “Emerging applications of fluorescence spectroscopy to cellular imaging: lifetime imaging, metal-ligand probes, multi-photon excitation and light quenching,” Scanning Microsc. Suppl.10, 213–224 (1996). [PubMed]

Science

• W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990). [CrossRef] [PubMed]
• I. V. Yannas, J. F. Burke, D. P. Orgill, and E. M. Skrabut, “Wound tissue can utilize a polymeric template to synthesize a functional extension of skin,” Science215(4529), 174–176 (1982). [CrossRef] [PubMed]

Other

• I. V. Yannas, Tissue and Organ Regeneration in Adults, (Springer, New York, 2001).
• W. Becker, Advanced Time-Correlated Single Photon Counting Techniques, (Springer, Berlin, 2005).
• A. Periasamy and R. M. Clegg, eds., FLIM Microscopy in Biology and Medicine, (Chapman and Hall, Boca Raton, 2009).

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