Biomedical Engineering Letters

, Volume 9, Issue 3, pp 293–310 | Cite as

Recent trends in two-photon auto-fluorescence lifetime imaging (2P-FLIM) and its biomedical applications

  • Harsh Ranawat
  • Sagnik Pal
  • Nirmal MazumderEmail author
Review Article


Two photon fluorescence microscopy and the numerous technical advances to it have served as valuable tools in biomedical research. The fluorophores (exogenous or endogenous) absorb light and emit lower energy photons than the absorption energy and the emission (fluorescence) signal is measured using a fluorescence decay graph. Additionally, high spatial resolution images can be acquired in two photon fluorescence lifetime imaging (2P-FLIM) with improved penetration depth which helps in detection of fluorescence signal in vivo. 2P-FLIM is a non-invasive imaging technique in order to visualize cellular metabolic, by tracking intrinsic fluorophores present in it, such as nicotinamide adenine dinucleotide, flavin adenine dinucleotide and tryptophan etc. 2P-FLIM of these molecules enable the visualization of metabolic alterations, non-invasively. This comprehensive review discusses the numerous applications of 2P-FLIM towards cancer, neuro-degenerative, infectious diseases, and wound healing.


Two-photon excitation Fluorescence lifetime imaging microscopy (FLIM) Nicotinamide adenine dinucleotide hydrogen (NADH) Flavin adenine dinucleotide (FAD) 



We thank Dr. K. Satyamoorthy, Manipal School of Life Sciences (MSLS), MAHE for his encouragement and Manipal Academy of Higher Education, Manipal for providing the infrastructure and facilities. We also thank Dr. K K Mahato, Department of Biophysics, MSLS, MAHE, Manipal for discussion.

Compliance with ethical standards

Conflict of interest

All authors declare to have no conflict of interests.

Human and animals rights

This review article does not contain any studies with human participants performed by any of the authors. Institutional ethical clearance was taken by the authors of the respective published papers.

Informed consent

Not applicable.


  1. 1.
    Lakowicz JR. Principles of fluorescence spectroscopy. 3rd ed. New York: Springer; 2006.Google Scholar
  2. 2.
    Periasamy A, Clegg R. FLIM microscopy in biology and medicine. Boca Raton: CRC Press, Taylor & Francis Group; 2010.Google Scholar
  3. 3.
    Diaspro A, editor. Confocal and two-photon microscopy: foundations, applications and advances. Hoboken: Wiley; 2001.Google Scholar
  4. 4.
    Becker W. Fluorescence lifetime imaging—techniques and applications. J Microsc. 2012;247(2):119–36.Google Scholar
  5. 5.
    Becker W. Advanced time-correlated single photon counting techniques. Cham: Springer; 2015.Google Scholar
  6. 6.
    Lin H, Herman P, Lakowicz J. Fluorescence lifetime-resolved pH imaging of living cells. Cytometry. 2003;52A(2):77–89.Google Scholar
  7. 7.
    Williamson D, Lund P, Krebs H. The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J. 1967;103(2):514–27.Google Scholar
  8. 8.
    Chance B, Schoener B, Oshino R, Itshak F, Nakase Y. Oxidation–reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals. J Biol Chem. 1979;254(11):4764–71.Google Scholar
  9. 9.
    Periasamy A, Day R. Molecular imaging. New York: Oxford Univ Press; 2005.Google Scholar
  10. 10.
    Provenzano PP, Eliceiri KW, Keely PJ. Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment. Clin Exp Metastasis. 2009;26(4):357–70.Google Scholar
  11. 11.
    Ghukasyan V, Hsu Y, Kung S, Kao JF. Application of fluorescence resonance energy transfer resolved by fluorescence lifetime imaging microscopy for the detection of enterovirus 71 infection in cells. J Biomed Opt. 2007;12(2):024016.Google Scholar
  12. 12.
    Jyothikumar V, Sun Y, Periasamy A. Investigation of tryptophan–NADH interactions in live human cells using three-photon fluorescence lifetime imaging and Förster resonance energy transfer microscopy. J Biomed Opt. 2013;18(6):060501.Google Scholar
  13. 13.
    Sun Y, Day R, Periasamy A. Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy. Nat Protoc. 2011;6(9):1324–40.Google Scholar
  14. 14.
    Skala M, Riching K, Bird D, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri KW, Keely PJ, Ramanujam N. In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia. J Biomed Opt. 2007;12(2):024014.Google Scholar
  15. 15.
    Kao FJ, Deka G, Mazumder N. Cellular autofluroscence detection through FLIM/FRET microscopy. Curr Trends Opt Photon. 2015;129:471–82.Google Scholar
  16. 16.
    Periasamy A, Mazumder N, Sun Y, Christopher KG, Day RN. FRET microscopy: basics, issues and advantages of FLIM-FRET imaging. In: Becker W, editor. Advanced time-correlated single photon counting applications. Cham: Springer; 2015. p. 249–76.Google Scholar
  17. 17.
    Steiner R. Principles of fluorescence spectroscopy. Anal Biochem. 1984;137(2):539.Google Scholar
  18. 18.
    Bacskai B, Skoch J, Hickey G, Allen R, Hyman B. Fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime imaging microscopy to characterize amyloid-beta plaques. J Biomed Opt. 2003;8(3):368.Google Scholar
  19. 19.
    Bird D, Yan L, Vrotsos K, Eliceiri K, Vaughan E, Keely PJ, White JG, Ramanujam N. Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH. Can Res. 2005;65(19):8766–73.Google Scholar
  20. 20.
    Mazumder N, Lyn R, Singaravelu R, Ridsdale A, Moffatt D, Hu CW, Tsai HR, McLauchlan J, Stolow A, Kao FJ, Pezacki JP. Fluorescence lifetime imaging of alterations to cellular metabolism by domain 2 of the hepatitis C virus core protein. PLoS ONE. 2013;8(6):e66738.Google Scholar
  21. 21.
    Vishwasrao H, Heikal A, Kasischke K, Webb W. Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy. J Biol Chem. 2005;280(26):25119–26.Google Scholar
  22. 22.
    Lakowicz J, Szmacinski H, Nowaczyk K, Johnson M. Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci. 1992;89(4):1271–5.Google Scholar
  23. 23.
    Blinova K, Carroll S, Bose S, Smirnov A, Harvey J, Knutson JR, Balaban RS. Distribution of mitochondrial NADH fluorescence lifetimes: steady-state kinetics of matrix NADH interactions. Biochemistry. 2005;44(7):2585–94.Google Scholar
  24. 24.
    Wakita M, Nishimura G, Tamura M. Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ. J Biochem. 1995;118(6):1151–60.Google Scholar
  25. 25.
    Ramanujan V, Jo J, Cantu G, Herman B. Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells. J Microsc. 2008;230(3):329–38.MathSciNetGoogle Scholar
  26. 26.
    Li D, Zheng W, Qu J. Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence. Opt Lett. 2008;33(20):2365.Google Scholar
  27. 27.
    Chia T, Williamson A, Spencer D, Levene M. Multiphoton fluorescence lifetime imaging of intrinsic fluorescence in human and rat brain tissue reveals spatially distinct NADH binding. Opt Express. 2008;16(6):4237.Google Scholar
  28. 28.
    Stringari C, Cinquin A, Cinquin O, Digman M, Donovan P, Gratton E. Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue. Proc Natl Acad Sci. 2011;108(33):13582–7.Google Scholar
  29. 29.
    Conklin M, Provenzano P, Eliceiri K, Sullivan R, Keely P. Fluorescence lifetime imaging of endogenous fluorophores in histopathology sections reveals differences between normal and tumor epithelium in carcinoma in situ of the breast. Cell Biochem Biophys. 2009;53(3):145–57.Google Scholar
  30. 30.
    Sun Y, Phipps J, Elson D, Stoy H, Tinling S, Meier J, Poirier B, Chuang FS, Farwell DG, Marcu L. Fluorescence lifetime imaging microscopy: in vivo application to diagnosis of oral carcinoma. Opt Lett. 2009;34(13):2081.Google Scholar
  31. 31.
    Thorling C, Liu X, Burczynski F, Fletcher L, Gobe G, Roberts M. Multiphoton microscopy can visualize zonal damage and decreased cellular metabolic activity in hepatic ischemia-reperfusion injury in rats. J Biomed Opt. 2011;16(11):116011.Google Scholar
  32. 32.
    Yaseen M, Sakadžić S, Wu W, Becker W, Kasischke K, Boas D. In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH. Biomed Opt Express. 2013;4(2):307.Google Scholar
  33. 33.
    Yaseen M, Sutin J, Wu W, Fu B, Uhlirova H, Devor A, Boas DA, Skadžić S. Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo. Biomed Opt Express. 2017;8(5):2368.Google Scholar
  34. 34.
    Rmoso C, Forster LS. Tryptophan fluorescence lifetimes in lysozyme. J Biol Chem. 1975;250(10):3738–45.Google Scholar
  35. 35.
    Li C, Pastila R, Pitsillides C, Runnels J, Puoris’haag M, Côté D, Lin CP. Imaging leukocyte trafficking in vivo with two-photon-excited endogenous tryptophan fluorescence. Opt Express. 2010;18(2):988.Google Scholar
  36. 36.
    Yang W, Yuste R. In vivo imaging of neural activity. Nat Methods. 2017;14(4):349–59.Google Scholar
  37. 37.
    Mostany R, Miquelajauregui A, Shtrahman M, Portera-Cailliau C. Two-photon excitation microscopy and its applications in neuroscience. In: Verveer PJ, editor. Advanced fluorescence microscopy. New York: Humana Press; 2015. p. 25–42.Google Scholar
  38. 38.
    Day R, Davidson M. The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev. 2009;38(10):2887.Google Scholar
  39. 39.
    Mostany R, Portera-Cailliau C. A method for 2-photon imaging of blood flow in the neocortex through a cranial window. J Vis Exp. 2008;12:678.Google Scholar
  40. 40.
    Yan P, Bero A, Cirrito J, Xiao Q, Hu X, Wang Y, Gonzales E, Holtzman DM, Lee JM. Characterizing the appearance and growth of amyloid plaques in APP/PS1 mice. J Neurosci. 2009;29(34):10706–14.Google Scholar
  41. 41.
    Klunk W, Bacskai B, Mathis C, Kajdasz S, McLellan M, Frosch MP, Debnath ML, Holt DP, Wang Y, Hyman BT. Imaging Aβ plaques in living transgenic mice with multiphoton microscopy and methoxy-X04, a systemically administered Congo red derivative. J Neuropathol Exp Neurol. 2002;61(9):797–805.Google Scholar
  42. 42.
    Hülsmann S, Hagos L, Heuer H, Schnell C. Limitations of sulforhodamine 101 for brain imaging. Front Cell Neurosci. 2017;11:44.Google Scholar
  43. 43.
    Liao Y, Génot V, Audibert J, Pansu R. In situ kinetics study of the formation of organic nanoparticles by fluorescence lifetime imaging microscopy (FLIM) along a microfluidic device. Microfluidics Nanofluidics. 2016;20(4):1–11.Google Scholar
  44. 44.
    Damalakiene L, Karabanovas V, Bagdonas S, Rotomskis R. Fluorescence-lifetime imaging microscopy for visualization of quantum dots’ endocytic pathway. Int J Mol Sci. 2016;17(4):473.Google Scholar
  45. 45.
    Basuki J, Duong H, Macmillan A, Erlich R, Esser L, Akerfeldt MC, Whan RM, Kavallaris M, Boyer C, Davis TP. Using fluorescence lifetime imaging microscopy to monitor theranostic nanoparticle uptake and intracellular doxorubicin release. ACS Nano. 2013;7(11):10175–89.Google Scholar
  46. 46.
    Rossi EA, Rangel-Fonseca P, Parkins K, Fischer W, Latchney LR, Folwell MA, Williams DR, Dubra A, Chung MM. In vivo imaging of retinal pigment epithelium cells in age related macular degeneration. Biomed Opt Express. 2013;4(11):2527–39.Google Scholar
  47. 47.
    Geng Y, Dubra A, Yin L, Merigan W, Sharma R, Libby RT, Williams DR. Adaptive optics retinal imaging in the living mouse eye. Biomed Opt Express. 2012;3(4):715.Google Scholar
  48. 48.
    Wahl D, Jian Y, Bonora S, Zawadzki R, Sarunic M. Wavefront sensorless adaptive optics fluorescence biomicroscope for in vivo retinal imaging in mice. Biomed Opt Express. 2015;7(1):1.Google Scholar
  49. 49.
    Morgan J, Dubra A, Wolfe R, Merigan W, Williams D. In Vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic. Investig Opthalmol Vis Sci. 2009;50(3):1350.Google Scholar
  50. 50.
    Sharma R, Yin L, Geng Y, Merigan W, Williams D, Hunter J. In vivo two-photon imaging of the mouse retina. J Vis. 2012;12(14):51.Google Scholar
  51. 51.
    Hunter J, Masella B, Dubra A, Sharma R, Yin L, Merigan WH, Palczewska G, Palczewski K, Williams DR. Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy. Biomed Opt Express. 2010;2(1):139.Google Scholar
  52. 52.
    Sharma R, Williams D, Palczewska G, Palczewski K, Hunter J. Two-photon autofluorescence imaging reveals cellular structures throughout the retina of the living primate eye. Investig Opthalmol Vis Sci. 2016;57(2):632.Google Scholar
  53. 53.
    Feeks J, Hunter J. Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of exogenous fluorophores in mice. Biomed Opt Express. 2017;8(5):2483.Google Scholar
  54. 54.
    Kapsokalyvas D, Barygina V, Cicchi R, Fiorillo C, Pavone FS. Evaluation of the oxidative stress of psoriatic fibroblasts based on spectral two-photon fluorescence lifetime imaging. In: Multiphoton microscopy in the biomedical sciences XIII. International Society for Optics and Photonics; 2013. Vol. 8588, p. 85882D.Google Scholar
  55. 55.
    Huck V, Gorzelanny C, Thomas K, Getova V, Niemeyer V, Zens K, Unnerstall TR, Feger JS, Fallah MA, Metze D, Stānder S. From morphology to biochemical state—intravital multiphoton fluorescence lifetime imaging of inflamed human skin. Sci Rep. 2016;6(1):1–12.Google Scholar
  56. 56.
    Kantelhardt S, Kalasauskas D, König K, Kim E, Weinigel M, Uchugonova A, Giese A. In vivo multiphoton tomography and fluorescence lifetime imaging of human brain tumor tissue. J Neurooncol. 2016;127(3):473–82.Google Scholar
  57. 57.
    Marcu L, Hartl B. Fluorescence lifetime spectroscopy and imaging in neurosurgery. IEEE J Sel Top Quantum Electron. 2012;18(4):1465–77.Google Scholar
  58. 58.
    Gratton E, Breusegem S, Sutin JD, Ruan Q, Barry NP. Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods. J Biomed Opt. 2003;8(3):381–91.Google Scholar
  59. 59.
    El-Serag H. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology. 2012;142(6):1264–1273.e1.Google Scholar
  60. 60.
    Miyoshi H, Moriya K, Tsutsumi T, Shinzawa S, Fujie H, Shintani Y, Fujinaga H, Goto K, Todoroki T, Suzuki T, Miyamura T. Pathogenesis of lipid metabolism disorder in hepatitis C: polyunsaturated fatty acids counteract lipid alterations induced by the core protein. J Hepatol. 2011;54(3):432–8.Google Scholar
  61. 61.
    Alvisi G, Madan V, Bartenschlager R. Hepatitis C virus and host cell lipids: an intimate connection. RNA Biol. 2011;8(2):258–69.Google Scholar
  62. 62.
    Tolles WM, Nibler JW, McDonald JR, Harvey AB. A review of the theory and application of coherent anti-Stokes Raman spectroscopy (CARS). Appl Spectrosc. 1977;31(4):253–71.Google Scholar
  63. 63.
    Buryakina T, Su PT, Gukassyan V, Syu WJ, Kao FJ. Monitoring cellular metabolism of 3T3 upon wild type E. coli infection by mapping NADH with FLIM. Chin Opt Lett. 2010;8(10):931–3.Google Scholar
  64. 64.
    Paul RJ, Schneckenburger H. Oxygen concentration and the oxidation-reduction state of yeast: determination of free/bound NADH and flavins by time-resolved spectroscopy. Naturwissenschaften. 1996;83(1):32–5.Google Scholar
  65. 65.
    Szaszák M, Steven P, Shima K, Orzekowsky-Schröder R, Hüttmann G, König IR, Solbach W, Rupp J. Fluorescence lifetime imaging unravels C. trachomatis metabolism and its crosstalk with the host cell. PLoS Pathog. 2011;7(7):e1002108.Google Scholar
  66. 66.
    Hou L, Ning P, Feng Y, Ding Y, Bai L, Li L, Yu H, Meng X. A two-photon fluorescent probe for monitoring autophagy via fluorescence lifetime imaging. Anal Chem. 2018;90(12):7122–6.Google Scholar
  67. 67.
    Gómez CA, Fu B, Sakadžić S, Yaseen MA. Cerebral metabolism in a mouse model of Alzheimer’s disease characterized by two-photon fluorescence lifetime microscopy of intrinsic NADH. Neurophotonics. 2018;4:045008.Google Scholar
  68. 68.
    Le V, Yoo S, Yoon Y, Wang T, Kim B, Lee S, Lee KH, Kim KH, Chung E. Brain tumor delineation enhanced by moxifloxacin-based two-photon/CARS combined microscopy. Biomed Opt Express. 2017;8(4):2148–61.Google Scholar
  69. 69.
    Kasischke K, Lambert E, Panepento B, Sun A, Gelbard H, Burgess RW, Foster TH, Nedergaard M. Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions. J Cereb Blood Flow Metab. 2010;31(1):68–81.Google Scholar
  70. 70.
    Przedborski S, Vila M. MPTP: a review of its mechanisms of neurotoxicity. Clin Neurosci Res. 2001;1(6):407–18.Google Scholar
  71. 71.
    Chakraborty S, Nian F, Tsai J, Karmenyan A, Chiou A. Quantification of the metabolic state in cell-model of Parkinson’s disease by fluorescence lifetime imaging microscopy. Sci Rep. 2016;6(1):1–9.Google Scholar
  72. 72.
    Dauer W, Przedborski S. Parkinson’s disease. Neuron. 2003;39(6):889–909.Google Scholar
  73. 73.
    Schapira A. Evidence for mitochondrial dysfunction in Parkinson’s disease-a critical appraisal. Mov Disord. 2004;9(2):125–38.Google Scholar
  74. 74.
    Chance B, Jamieson D, Coles H. Energy-linked pyridine nucleotide reduction: inhibitory effects of hyperbaric oxygen in vitro and in vivo. Nature. 1965;206(4981):257–63.Google Scholar
  75. 75.
    Rinnenthal J, Börnchen C, Radbruch H, Andresen V, Mossakowski A, Siffrin V, Seelemann T, Spiecker H, Moll I, Herz J, Hauser AE. Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation. PLoS ONE. 2013;8(4):e60100.Google Scholar
  76. 76.
    Yasuda R, Harvey CD, Zhong H, Sobczyk A, van Aelst L, Svoboda K. Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging. Nat Neurosci. 2006;9:283–91.Google Scholar
  77. 77.
    Resendez S, Stuber G. In vivo calcium imaging to illuminate neurocircuit activity dynamics underlying naturalistic behavior. Neuropsychopharmacology. 2014;40(1):238–9.Google Scholar
  78. 78.
    Mayevsky A, Zarchin N, Kaplan H, Haveri J, Haselgroove J, Chance B. Brain metabolic responses to ischemia in the mongolian gerbil: in vivo and freeze trapped redox scanning. Brain Res. 1983;276(1):95–107.Google Scholar
  79. 79.
    Kunz W, Gellerich F. Quantification of the content of fluorescent flavoproteins in mitochondria from liver, kidney cortex, skeletal muscle, and brain. Biochem Med Metab Biol. 1993;50(1):103–10.Google Scholar
  80. 80.
    Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74.Google Scholar
  81. 81.
    Skala M, Riching K, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri K, White JG, Ramanujam N. In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Proc Natl Acad Sci. 2007;104(49):19494–9.Google Scholar
  82. 82.
    Zoumi A, Yeh A, Tromberg B. Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc Natl Acad Sci. 2002;99(17):11014–9.Google Scholar
  83. 83.
    Pena A-M., Decencière E., Brizion S., Victorin S., Koudoro S., et al. Multiphoton FLIM in cosmetic clinical research. In: Multiphoton microscopy and fluorescence lifetime imaging: applications in biology and medicine. 2018.Google Scholar
  84. 84.
    Tadrous P, Siegel J, French P, Shousha S, Lalani E, Stamp G. Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer. J Pathol. 2003;199(3):309–17.Google Scholar
  85. 85.
    Taitt HE. Global trends and prostate cancer: a review of incidence, detection, and mortality as influenced by race, ethnicity, and geographic location. Am J Men’s Health. 2018;12(6):1807–23.Google Scholar
  86. 86.
    Alam SR, Wallrabe H, Svindrych Z, Chaudhary AK, Christopher KG, Chandra D, Periasamy A. Investigation of mitochondrial metabolic response to doxorubicin in prostate cancer cells: an NADH, FAD and tryptophan FLIM assay. Sci Rep. 2017;7(1):10451.Google Scholar
  87. 87.
    Deka G, Wu W, Kao F. In vivo wound healing diagnosis with second harmonic and fluorescence lifetime imaging. J Biomed Opt. 2012;18(6):061222.Google Scholar
  88. 88.
    Wang H, Shi L, Qin J, Yousefi S, Li Y, Wang R. Multimodal optical imaging can reveal changes in microcirculation and tissue oxygenation during skin wound healing. Lasers Surg Med. 2014;46(6):470–8.Google Scholar
  89. 89.
    Taylor J, Laity P, Hicks J, Wong S, Norris K, Khunkamchoo P, Johnson AF, Cameron RE. Extent of iron pick-up in deforoxamine-coupled polyurethane materials for therapy of chronic wounds. Biomaterials. 2005;26(30):6024–33.Google Scholar
  90. 90.
    Edwards JV, Howley P, Cohen IK. In vitro inhibition of human neutrophil elastase by oleic acid albumin formulations from derivatized cotton wound dressings. Int J Pharm. 2004;284(1):1–2.Google Scholar
  91. 91.
    Gardner SE, Frantz RA, Troia C, Eastman S, MacDonald M, Buresh K, Healy D. A tool to assess clinical signs and symptoms of localized infection in chronic wounds: development and reliability. Ostomy/Wound Manag. 2001;47(1):40–7.Google Scholar
  92. 92.
    Sanchez WY, Prow TW, Sanchez WH, Grice J, Roberts MS. Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD (P) H by multiphoton tomography and fluorescence lifetime imaging microscopy. J Biomed Opt. 2010;15(4):046008.Google Scholar
  93. 93.
    Wang KH, Majewska A, Schummers J, Farley B, Hu C, Sur M, Tonegawa S. In vivo two-photon imaging reveals a role of arc in enhancing orientation specificity in visual cortex. Cell. 2006;126(2):389–402.Google Scholar
  94. 94.
    Birkner A, Tischbirek CH, Konnerth A. Improved deep two-photon calcium imaging in vivo. Cell Calcium. 2017;64:29–35.Google Scholar
  95. 95.
    Gannaway J, Sheppard CJR. Second-harmonic imaging in the scanning optical microscope. Opt Quant Electron. 1978;10:435.Google Scholar
  96. 96.
    Campagnola P, Loew L. Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat Biotechnol. 2003;21(11):1356–60.Google Scholar
  97. 97.
    Koehler MJ, Speicher M, Lange-Asschenfeldt S, Stockfleth E, Metz S, Elsner P, Kaatz M, König K. Clinical application of multiphoton tomography in combination with confocal laser scanning microscopy for in vivo evaluation of skin diseases. Exp Dermatol. 2011;20(7):589–94.Google Scholar
  98. 98.
    König K, Simon U, Halbhuber JK. 3D-resolved two-photon fluorescence microscopy of living cells using a modified confocal laser scanning microscope. Cell Mol Biol. 1996;42:1181–94.Google Scholar
  99. 99.
    Williams RM, Zipfel WR, Webb WW. Interpreting second-harmonic generation images of collagen I fibrils. Biophys J. 2005;88(2):1377–86.Google Scholar
  100. 100.
    Torkian B, Yeh A, Engel R, Sun C, Tromberg B, Wong B. Modeling Aberrant Wound healing using tissue-engineered skin constructs and multiphoton microscopy. Arch Facial Plast Surg. 2004;6(3):180.Google Scholar
  101. 101.
    Jones DJ, Ramser EH, Woessner EA, Quinn PK. In vivo multiphoton microscopy detects longitudinal metabolic changes associated with delayed skin wound healing. Commun Biol. 2018;1:198.Google Scholar
  102. 102.
    Cuttle L, Nataatmadja M, Fraser J, Kempf M, Kimble R, Hayes M. Collagen in the scarless fetal skin wound: detection with Picrosirius-polarization. Wound Repair Regen. 2005;13(2):198–204.Google Scholar
  103. 103.
    Gehlsen U, Oetke A, Szaszák M, Koop N, Paulsen F, Gebert A, Huettmann G, Steven P. Two-photon fluorescence lifetime imaging monitors metabolic changes during wound healing of corneal epithelial cells in vitro. Graefe’s Arch Clin Exp Ophthalmol. 2012;250(9):1293–302.Google Scholar
  104. 104.
    Quinn KP, Leal EC, Tellechea A, Kafanas A, Auster ME, Veves A, Georgakoudi I. Diabetic wounds exhibit distinct microstructural and metabolic heterogeneity through label-free multiphoton microscopy. J Investig Dermatol. 2016;136(1):342–4.Google Scholar
  105. 105.
    Liu J. Two-photon microscopy in pre-clinical and clinical cancer research. Front Optoelectron. 2015;8(2):141–51.Google Scholar
  106. 106.
    Cicchi R, Kapsokalyvas D, De Giorgi V, Maio V, Van Wiechen A, Massi D, Lotti T, Pavone FS. Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy. J Biophoton. 2009;3(1–2):34–43.Google Scholar
  107. 107.
    Yasui T, Tohno Y, Araki T. Characterization of collagen orientation in human dermis by two-dimensional second-harmonic-generation polarimetry. J Biomed Opt. 2004;9(2):259.Google Scholar
  108. 108.
    Becker W, Shcheslavkiy V, Frere S, Slutsky I. Spatially resolved recording of transient fluorescence-lifetime effects by line-scanning TCSPC. Microsc Res Tech. 2014;77(3):216–24.Google Scholar
  109. 109.
    Ryu J, Kang U, Kim J, Kim H, Kang HJ, Kim H, Sohn KD, Jeong HJ, Yoo H, Gweon B. Real-time visualization of two-photon fluorescence lifetime imaging microscopy using a wavelength-tunable femtosecond pulsed laser. Biomed Opt Express. 2018;9(7):3449–63.Google Scholar
  110. 110.
    Madden SK, Zettel LM, Majewska KA, Brown BE. Brain tumor imaging: live imaging of glioma by two-photon microscopy. Cold Spring Harb Protoc. 2013;231–236.Google Scholar
  111. 111.
    Cadby A, Dean R, Fox A, Jones R, Lidzey D. Mapping the fluorescence decay lifetime of a conjugated polymer in a phase-separated blend using a scanning near-field optical microscope. Nano Lett. 2005;5(11):2232–7.Google Scholar
  112. 112.
    Vogel SS, Thaler C, Blank PS, Koushik SV. Time resolved fluorescence anisotropy. FLIM Microsc Biol Med. 2009;21(1):245–88.Google Scholar
  113. 113.
    Yu Q, Heikal AA. Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level. J Photochem Photobiol, B. 2009;95(1):46–57.Google Scholar
  114. 114.
    Slepkov A, Ridsdale A, Wan H, Wang M, Pegoraro A, Moffatt DJ, Pezacki JP, Stolow A, Wan HN, Wang MH, Fao KJ. Forward-collected simultaneous fluorescence lifetime imaging and coherent anti-Stokes Raman scattering microscopy. J Biomed Opt. 2011;16(2):021103.Google Scholar
  115. 115.
    Lin P, Lin Y, Chang C, Kao FJ. Fluorescence lifetime imaging microscopy with subdiffraction-limited resolution. Jpn J Appl Phys. 2013;52(2R):028004.Google Scholar

Copyright information

© Korean Society of Medical and Biological Engineering 2019

Authors and Affiliations

  1. 1.Department of Biophysics, Manipal School of Life SciencesManipal Academy of Higher EducationManipalIndia

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