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Multiphoton Laser Microscopy with Fluorescence Lifetime Imaging and Skin Cancer

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Skin Cancer

Part of the book series: Current Clinical Pathology ((CCPATH))

Abstract

To improve the diagnosis of skin tumours, extensive research on new technologies has been carried out introducing real-time imaging methods as multiphoton laser tomography (MPT) associated to fluorescence lifetime imaging (FLIM). Multiphoton microscopy relies on the simultaneous absorption of two or more photons of low energy in the near-infrared spectrum, avoiding biological tissue damage that occurs with higher laser powers.

With multiphoton microscopy, endogenous fluorophores, including NADH, NADPH and many others, can be efficiently excited. Since the technique is non-invasive and the laser illumination is harmless, in vivo examination by MPT/FLIM can be repeated on the same site without restrictions, enabling long-term studies of skin diseases. Horizontal and vertical optical sections give the possibility to study the tissue sample three-dimensionally with a subcellular spatial resolution. The MPT/FLIM technique has numerous applications in dermatology, being suitable for the study of many physiological and pathological conditions of the skin in vivo, on ex vivo samples and on cell cultures. The application of MPT/FLIM to the field of skin tumours provides imaging at a cellular and at an architectural level, although the field of view is at present limited to the exploration of a square area of 358 × 358 μm.

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References

  1. Garbe C, Blum A. Epidemiology of cutaneous melanoma in Germany and worldwide. Skin Pharmacol Appl Skin Physiol. 2001;14(5):280–90.

    Article  PubMed  CAS  Google Scholar 

  2. Welch H, Woloshin S, Schwartz L. Skin biopsy rates and incidence of melanoma: population based ecological study. BMJ. 2005;331(7515):481.

    Article  PubMed  Google Scholar 

  3. Stang A, Ziegler S, Buchner U, et al. Malignant melanoma and nonmelanoma skin cancers in Northrhine-Westphalia. Germany: a patient-vs. diagnosis-based incidence approach. Int J Dermatol. 2007;46(6):564–70.

    Article  PubMed  Google Scholar 

  4. Roewert-Huber J, Lange-Aschenfeldt B, Stockfleth E, et al. Epidemiology and etiology of basal cell carcinoma. Br J Dermatol. 2007;157(Suppl):47–51.

    Article  PubMed  CAS  Google Scholar 

  5. Leiter U, Garbe C. Epidemiology of melanoma and nonmelanoma skin cancer: the role of sunlight. Adv Exp Med Biol. 2008;624:89–103.

    Article  PubMed  Google Scholar 

  6. Morton CA, Mackie RM. Clinical accuracy of the diagnosis of cutaneous malignant melanoma. Br J Dermatol. 1998;138:283–7.

    Article  PubMed  CAS  Google Scholar 

  7. Dolianitis C, Kelly J, Wolfe R, et al. Comparative performance of 4 dermoscopic algorithms by nonexperts for the diagnosis of melanocytic lesions. Arch Dermatol. 2005;141:1008–14.

    Article  PubMed  Google Scholar 

  8. Annessi G, Bono R, Sampogna F, et al. Sensitivity, specificity, and diagnostic accuracy of three dermoscopic algorithmic methods in the diagnosis of doubtful melanocytic lesions: the importance of light brown structureless areas in differentiating atypical melanocytic nevi from thin melanomas. J Am Acad Dermatol. 2007;56:759–67.

    Article  PubMed  Google Scholar 

  9. Telfer NR, Colver GB, Morton CA. Guidelines for management of basal cell carcinoma. Br J Dermatol. 2008;159:35–48.

    Article  PubMed  CAS  Google Scholar 

  10. Argenziano G, Soyer HP, Chimenti S, et al. Dermoscopy of pigmented skin lesions: results of a consensus meeting via the Internet. J Am Acad Dermatol. 2003;48:679–93.

    Article  PubMed  Google Scholar 

  11. González S, Tannus Z. Real-time in vivo confocal reflectance microscopy of basal cell carcinoma. J Am Acad Dermatol. 2002;47(6):869–74.

    Article  PubMed  Google Scholar 

  12. Pellacani G, Cesinaro AM, Seidenari S. Reflectance-mode confocal microscopy of pigmented skin lesions – improvement in melanoma diagnostic specificity. J Am Acad Dermatol. 2005;53(6):979–85.

    Article  PubMed  Google Scholar 

  13. Guitera P, Pellacani G, Longo C, et al. In vivo reflectance confocal microscopy enhances secondary evaluation of melanocytic lesions. J Invest Dermatol. 2009;129(1):131–8.

    Article  PubMed  CAS  Google Scholar 

  14. Vestergaard ME, Macaskill P, Holt PE, et al. Dermoscopy compared with naked eye examination for the diagnosis of primary melanoma: a meta-analysis of studies performed in a clinical setting. Br J Dermatol. 2008;159:669–76.

    PubMed  CAS  Google Scholar 

  15. Bauer J, Leinweber B, Metzler G, et al. Correlation with digital dermoscopic images can help dermatopathologists to diagnose equivocal skin tumours. Br J Dermatol. 2006;155(3):546–51.

    Article  PubMed  CAS  Google Scholar 

  16. Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence microscopy. Science. 1990;248:73–6.

    Article  PubMed  CAS  Google Scholar 

  17. König K, Schenke-Layland K, Riemann I, et al. Multiphoton autofluorescence imaging of intratissue elastic fibers. Biomaterials. 2005;26(5):495–500.

    Article  PubMed  Google Scholar 

  18. Lakowicz JR. Principles of fluorescence spectroscopy. New York: Springer; 2006.

    Book  Google Scholar 

  19. Zhao J, Chen J, Yang Y, et al. Jadassohn-Pellizzari anetoderma: study of multiphoton microscopy based on two-photon excited fluorescence and second harmonic generation. Eur J Dermatol. 2009;19(6):570–5.

    PubMed  Google Scholar 

  20. König K, Riemann I. High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picoseconds time resolution. J Biomed Opt. 2003;8(3):432–9.

    Article  PubMed  Google Scholar 

  21. Riemann I, Dimitrow E, Fischer P, Reif A, Kaatz M, Elsner P, et al. High resolution multiphoton tomography of human skin in vivo and in vitro. SPIE-Proceeding. 2004;5312:21–8.

    Article  Google Scholar 

  22. König K. Clinical multiphoton tomography. J Biophotonics. 2008;1(1):13–23. Review.

    Article  PubMed  Google Scholar 

  23. Dimitrow E, Riemann I, Ehlers A, et al. Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis. Exp Dermatol. 2009;18(6):509–15.

    Article  PubMed  Google Scholar 

  24. Elson D, Requejo-Isidro J, Munro I, et al. Time-domain fluorescence lifetime imaging applied to biological tissue. Photochem Photobiol Sci. 2004;3:795–801.

    Article  PubMed  CAS  Google Scholar 

  25. Galletly NP, McGinty J, Dunsby C, et al. Fluorescence lifetime imaging distinguishes basal cell carcinoma from surrounding uninvolved skin. Br J Dermatol. 2008;159:152–61.

    Article  PubMed  CAS  Google Scholar 

  26. Hanson KM, Behne MJ, Barry NP, et al. Two-photon fluorescence imaging of the skin stratum corneum pH gradient. Biophys J. 2002;83:1682–90.

    Article  PubMed  CAS  Google Scholar 

  27. Kaneko H, Putzier I, Frings S, et al. Chloride accumulation in mammalian olfactory sensory neurons. J Neurosci. 2004;24:7931–8.

    Article  PubMed  CAS  Google Scholar 

  28. Lakowicz JR, Szmacinski H. Fluorescence-lifetime based sensing of pH, Ca2þ, and glucose. Sens Actuator Chem. 1993;11:133–4.

    Article  CAS  Google Scholar 

  29. Gerritsen HC, Sanders R, Draaijer A, et al. Fluorescence lifetime imaging of oxygen in cells. J Fluoresc. 1997;7:11–6.

    Article  CAS  Google Scholar 

  30. Schweitzer D, Hammer M, Schweitzer F, et al. In vivo measurement of time-resolved autofluorescence at the human fundus. J Biomed Opt. 2004;9:1214–22.

    Article  PubMed  Google Scholar 

  31. Treanor B, Lanigan PMP, Suhling K, et al. Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse. J Microsc. 2005;217:36–43.

    Article  PubMed  CAS  Google Scholar 

  32. Calleja V, Ameer-Beg S, Vojnovic B, et al. Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy. Biochem J. 2003;372:33–40.

    Article  PubMed  CAS  Google Scholar 

  33. Kelbauskas L, Dietel W. Internalization of aggregated photosensitizers by tumor cells: subcellular time-resolved fluorescence spectroscopy on derivates of pyropheophorbide-a ethers and chlorine6 under femtosecond one- and two-photon excitation. Photochem Photobiol. 2002;76:686–94.

    Article  PubMed  CAS  Google Scholar 

  34. Van Zandvoort MAMJ, de Grauw CJ, Gerritsen HC, et al. Discrimination of DNA and RNA in cells by a vital fluorescent probe: lifetime imaging of SYTO13 in healthy and apoptotic cells. Cytometry. 2002;47:226–35.

    Article  PubMed  Google Scholar 

  35. Skala MC, Riching KM, Gendron-Fitzpatrick A, et al. In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Proc Natl Acad Sci U S A. 2007;104(49):19495.

    Article  Google Scholar 

  36. Lakowicz JR, Szmacinski H, Nowaczyk K, et al. Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci U S A. 1992;89:1271–5.

    Article  PubMed  CAS  Google Scholar 

  37. Becker W, Bergmann A, Hink MA, et al. Fluorescence lifetime imaging by time-correlated single photon counting. Microsc Res Tech. 2004;63:58–66.

    Article  PubMed  CAS  Google Scholar 

  38. Peter M, Ameer-Beg SM, Hughes MKY, et al. Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions. Biophys J. 2005;88:1224–37.

    Article  PubMed  CAS  Google Scholar 

  39. Pitts JD, Sloboda RD, Dragnev KH, et al. Autofluorescence characteristics of immortalized and carcinogen-transformed human bronchial epithelial cells. J Biomed Opt. 2001;6:31–40.

    Article  PubMed  CAS  Google Scholar 

  40. Galeotti T, van Rossum GD, Mayer DH, et al. On the fluorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues. Eur J Biochem. 1970;17:485–96.

    Article  PubMed  CAS  Google Scholar 

  41. Palmer GM, Keely PJ, Breslin TM, et al. Autofluorescence spectroscopy of normal and malignant human breast cell lines. Photochem Photobiol. 2003;78:462–9.

    Article  PubMed  CAS  Google Scholar 

  42. Kollias N, Zonios G, Stamatas GN. Fluorescence spectroscopy of skin. Vib Spectrosc. 2002;28(1):17–23.

    Article  CAS  Google Scholar 

  43. Zipfel WR, Williams RM, Christie R, et al. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci U S A. 2003;100(12):7075–80.

    Article  PubMed  CAS  Google Scholar 

  44. Schenke-Layland K, Riemann I, Damour O, et al. Two-photon microscopes and in vivo multiphoton tomographs-powerful diagnostic tools for tissue engineering and drug delivery. Adv Drug Deliv Rev. 2006;58(7):878–96.

    Article  PubMed  CAS  Google Scholar 

  45. Becker W, Bergmann A, Biskup C. Multispectral fluorescence lifetime imaging by TCSPC. Microsc Res Tech. 2007;70(5):403–9.

    Article  PubMed  CAS  Google Scholar 

  46. Lin SJ et al. Multiphoton microscopy: a new paradigm in dermatological imaging. Eur J Dermatol. 2007;17(5):361–6.

    PubMed  Google Scholar 

  47. Tsai TH, Jee SH, Dong CY, et al. Multiphoton microscopy in dermatological imaging. J Dermatol Sci. 2009;56(1):1–8.

    Article  PubMed  Google Scholar 

  48. Teuchner K, Ehlert J, Freyer W, et al. Fluorescence studies of melanin by stepwise two-photon femtosecond laser excitation. J Fluoresc. 2000;10:275–81.

    Article  CAS  Google Scholar 

  49. Rice WL, Kaplan DL, Georgakoudi I. Two-photon microscopy for non-invasive, quantitative monitoring of stem cell differentiation. PLoS One. 2010;5(4):e10075.

    Article  PubMed  Google Scholar 

  50. Seidenari S, Schianchi S, Azzoni P, et al. High-resolution multiphoton tomography and fluorescence lifetime imaging of UVB-induced cellular damage on cultured fibroblasts producing fibres. Skin Res Technol. 2013. [Epub ahead of print].

    Google Scholar 

  51. Benati E, Bellini V, Borsari S, et al. Quantitative evaluation of healthy epidermis by means of Multiphoton microscopy and FLIM. Skin Res Technol. 2011;17(3): 295–303.

    Google Scholar 

  52. Cicchi R, Massi D, Sestini S, et al. Multidimensional non-linear laser imaging of basal cell carcinoma. Opt Express. 2007;15(16):10135–48.

    Article  PubMed  CAS  Google Scholar 

  53. Paoli J, Smedh M, Wennberg AM, et al. Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics. J Invest Dermatol. 2008;128(5):1248–55.

    Article  PubMed  CAS  Google Scholar 

  54. De Giorgi V, Massi D, Sestini S, et al. Combined non-linear laser imaging (two-photon excitation fluorescence microscopy, fluorescence lifetime imaging microscopy, multispectral multiphoton microscopy) in cutaneous tumours: first experiences. J Eur Acad DermatolVenereol. 2009;23(3):314–6.

    Article  Google Scholar 

  55. Paoli J, Smedh M, Ericson MB. Multiphoton laser scanning microscopy-a novel diagnostic method for superficial skin cancers. Semin Cutan Med Surg. 2009;28(3):190–5. Review.

    Article  PubMed  CAS  Google Scholar 

  56. Lin SJ, Jee SH, Kuo CJ, et al. Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging. Opt Lett. 2006;31:2756–8.

    Article  PubMed  Google Scholar 

  57. Brancaleon L, Durkin AJ, Tu JH, et al. In vivo fluorescence spectroscopy of nonmelanoma skin cancer. Photochem Photobiol. 2001;73:178–83.

    Article  PubMed  CAS  Google Scholar 

  58. Seidenari S, Arginelli F, Dunsby C, French P, König K, Magnoni C, Manfredini M, Talbot C, Ponti G. Multiphoton laser tomography and fluorescence lifetime imaging of basal cell carcinoma: morphologic features for non-invasive diagnostics. Exp Dermatol. 2012;21(11):831–6.

    Article  PubMed  Google Scholar 

  59. Skala MC, Squirrell JM, Vrotsos KM, et al. Multiphoton microscopy of endogenous fluorescence differentiates normal, precancerous, and cancerous squamous epithelial tissue. Cancer Res. 2005;65:1180–6.

    Article  PubMed  CAS  Google Scholar 

  60. White FH, Gohari K, Smith CJ. Histological and ultrastructural morphology of 7,12 dimethylbenz(a)-anthracene carcinogenesis in hamster cheek pouch epithelium. Diagn Histopathol. 1981;4:307–33.

    PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Dermatology, University of Modena and Reggio Emilia, Skin Center, via Zattera 130, Modena, 41124, Italy

    Stefania Seidenari, Federica Arginelli & Marco Manfredini

Authors
  1. Stefania Seidenari
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  2. Federica Arginelli
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  3. Marco Manfredini
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Corresponding author

Correspondence to Stefania Seidenari .

Editor information

Editors and Affiliations

  1. and Pharmaceutical Sciences and Technologies, Second University of Naples Department of Environmental, Biological, Naples, Italy

    Alfonso Baldi

  2. Department of Dermatology, Pius Hospital de Valls, Valls, Tarragona, Spain

    Paola Pasquali

  3. S.A.F.U. Department, Regina Elena Cancer Institute, Rome, Italy

    Enrico P Spugnini

Glossary

FAD

Flavin adenine dinucleotide.

FLIM

Fluorescence lifetime imaging.

FRET

Förster resonance energy transfer, is a mechanism describing energy transfer between two chromophores.

MPE

Multiphoton excitation.

MPT

Multiphoton laser tomography

MPT/FLIM

Multiphoton laser microscopy with Fluorescence lifetime imaging.

Multiphoton microscopy

is a specialized optical microscope that relies on the simultaneous absorption of two or more photons of low energy in the near-infrared spectrum, avoiding biological tissue damage that occurs with higher laser powers.

NADH

Nicotinamide adenine dinucleotide.

SHG (second harmonic generation)

is a method for probing interfaces in atomic and molecular systems.

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Seidenari, S., Arginelli, F., Manfredini, M. (2014). Multiphoton Laser Microscopy with Fluorescence Lifetime Imaging and Skin Cancer. In: Baldi, A., Pasquali, P., Spugnini, E. (eds) Skin Cancer. Current Clinical Pathology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4614-7357-2_18

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  • DOI: https://doi.org/10.1007/978-1-4614-7357-2_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4614-7356-5

  • Online ISBN: 978-1-4614-7357-2

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