Abstract

Skin cancer incidence has increased exponentially over the last three decades. In 2008 skin cancer caused 2280 deaths in the UK, with 2067 due to malignant melanoma. Early diagnosis can prevent mortality, however, conventional treatment requires multiple procedures and increasing treatment times. Second harmonic generation (SHG) imaging could offer diagnosis and demarcation of melanoma borders non-invasively at presentation thereby short-cutting the excision biopsy stage. To test the efficacy and accuracy of SHG imaging of collagen in skin and to delineate the borders of skin cancers, unstained human melanoma biopsy sections were imaged using SHG microscopy. Comparisons with sister sections, stained with H&E or Melan-A were made for correlation of invasion borders. Fresh ex vivo normal human and rat skin was imaged through its whole thickness using SHG to demonstrate this technique is transferable to in vivo tissues. SHG imaging demonstrated detailed collagen distribution in normal skin, with total absence of SHG signal (fibrillar collagen) within the melanoma-invaded tissue. The presence or absence of signal changes dramatically at the borders of the melanoma, accurately demarcating the edges that strongly correlated with H&E and Melan-A defined borders (p<0.002). SHG imaging of ex vivo human and rat skin demonstrated collagen architecture could be imaged through the full thickness of the skin. We propose that SHG imaging could be used for diagnosis and accurate demarcation of melanoma borders on presentation and therefore potentially reduce mortality rates.

© 2011 OSA

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2010 (2)

M. Lohela and Z. Werb, “Intravital imaging of stromal cell dynamics in tumors,” Curr. Opin. Genet. Dev. 20(1), 72–78 (2010).
[CrossRef] [PubMed]

D. S. Rigel, J. Russak, and R. Friedman, “The evolution of melanoma diagnosis: 25 years beyond the ABCDs,” CA Cancer J. Clin. 60(5), 301–316 (2010).
[CrossRef] [PubMed]

2009 (4)

J. Paoli, M. Smedh, and M. B. Ericson, “Multiphoton laser scanning microscopy--a novel diagnostic method for superficial skin cancers,” Semin. Cutan. Med. Surg. 28(3), 190–195 (2009).
[CrossRef] [PubMed]

M. H. Lien and V. K. Sondak, “Diagnostic techniques for primary cutaneous melanoma,” G. Ital. Dermatol. Venereol. 144(2), 187–194 (2009).
[PubMed]

P. P. Provenzano, K. W. Eliceiri, and P. J. Keely, “Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment,” Clin. Exp. Metastasis 26(4), 357–370 (2009).
[CrossRef] [PubMed]

S. Morris, B. Cox, and N. Bosanquet, “Cost of skin cancer in England,” Eur. J. Health Econ. 10(3), 267–273 (2009).
[CrossRef] [PubMed]

2008 (1)

T. Hompland, A. Erikson, M. Lindgren, T. Lindmo, and C. de Lange Davies, “Second-harmonic generation in collagen as a potential cancer diagnostic parameter,” J. Biomed. Opt. 13(5), 054050 (2008).
[CrossRef] [PubMed]

2006 (5)

V. de Giorgi, D. Massi, C. Salvini, S. Sestini, and P. Carli, “Features of regression in dermoscopic diagnosis: a confounding factor? Two clinical, dermoscopic-pathologic case studies,” Dermatol. Surg. 32(2), 282–286 (2006).
[CrossRef] [PubMed]

T. Fikrle and K. Pizinger, “Dermatoscopic differences between atypical melanocytic naevi and thin malignant melanomas,” Melanoma Res. 16(1), 45–50 (2006).
[CrossRef] [PubMed]

D. L. Cummins, J. M. Cummins, H. Pantle, M. A. Silverman, A. L. Leonard, and A. Chanmugam, “Cutaneous malignant melanoma,” Mayo Clin. Proc. 81(4), 500–507 (2006).
[CrossRef] [PubMed]

M. A. Weinstock, “Progress and prospects on melanoma: the way forward for early detection and reduced mortality,” Clin. Cancer Res. 12(7), 2297s–2300s (2006).
[CrossRef] [PubMed]

T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

2005 (5)

A. Gerger, S. Koller, T. Kern, C. Massone, K. Steiger, E. Richtig, H. Kerl, and J. Smolle, “Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors,” J. Invest. Dermatol. 124(3), 493–498 (2005).
[CrossRef] [PubMed]

D. M. Parkin, F. Bray, J. Ferlay, and P. Pisani, “Global cancer statistics, 2002,” CA Cancer J. Clin. 55(2), 74–108 (2005).
[CrossRef] [PubMed]

N. Monhian, B. S. Jewett, S. R. Baker, and J. Varani, “Matrix metalloproteinase expression in normal skin associated with basal cell carcinoma and in distal skin from the same patients,” Arch. Facial Plast. Surg. 7(4), 238–243 (2005).
[CrossRef] [PubMed]

S. Kuphal, R. Bauer, and A. K. Bosserhoff, “Integrin signaling in malignant melanoma,” Cancer Metastasis Rev. 24(2), 195–222 (2005).
[CrossRef] [PubMed]

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005).
[CrossRef] [PubMed]

2003 (6)

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

C. K. Sun, C. C. Chen, S. W. Chu, T. H. Tsai, Y. C. Chen, and B. L. Lin, “Multiharmonic-generation biopsy of skin,” Opt. Lett. 28(24), 2488–2490 (2003).
[CrossRef] [PubMed]

C. Ntayi, W. Hornebeck, and P. Bernard, “Influence of cultured dermal fibroblasts on human melanoma cell proliferation, matrix metalloproteinase-2 (MMP-2) expression and invasion in vitro,” Arch. Dermatol. Res. 295(6), 236–241 (2003).
[CrossRef] [PubMed]

P. Eves, E. Katerinaki, C. Simpson, C. Layton, R. Dawson, G. Evans, and S. Mac Neil, “Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes,” Clin. Exp. Metastasis 20(8), 685–700 (2003).
[CrossRef] [PubMed]

J. Y. Scoazec, “Tissue and cell imaging in situ: potential for applications in pathology and endoscopy,” Gut 52(90004Suppl 4), 1iv–1iv (2003).
[CrossRef] [PubMed]

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

2002 (1)

E. Wandel, A. Raschke, G. Hildebrandt, J. Eberle, R. Dummer, U. Anderegg, and A. Saalbach, “Fibroblasts enhance the invasive capacity of melanoma cells in vitro,” Arch. Dermatol. Res. 293(12), 601–608 (2002).
[CrossRef] [PubMed]

2001 (1)

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6(3), 277–286 (2001).
[CrossRef] [PubMed]

2000 (1)

J. Varani, Y. Hattori, Y. Chi, T. Schmidt, P. Perone, M. E. Zeigler, D. J. Fader, and T. M. Johnson, “Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin,” Br. J. Cancer 82(3), 657–665 (2000).
[CrossRef] [PubMed]

1996 (1)

F. Wach, A. M. Eyrich, T. Wustrow, T. Krieg, and R. Hein, “Comparison of migration and invasiveness of epithelial tumor and melanoma cells in vitro,” J. Dermatol. Sci. 12(2), 118–126 (1996).
[CrossRef] [PubMed]

1979 (1)

S. Roth and I. Freund, “Second harmonic-generation in collagen,” J. Chem. Phys. 70(4), 1637–1643 (1979).
[CrossRef]

1977 (1)

C. J. R. Sheppard, J. N. Gannaway, R. Kompfner, and D. Walsh, “The scanning harmonic optical microscope,” IEEE J. Quantum Electron. 13(9), 912 (1977).
[CrossRef]

1971 (1)

Anderegg, U.

E. Wandel, A. Raschke, G. Hildebrandt, J. Eberle, R. Dummer, U. Anderegg, and A. Saalbach, “Fibroblasts enhance the invasive capacity of melanoma cells in vitro,” Arch. Dermatol. Res. 293(12), 601–608 (2002).
[CrossRef] [PubMed]

Baker, S. R.

N. Monhian, B. S. Jewett, S. R. Baker, and J. Varani, “Matrix metalloproteinase expression in normal skin associated with basal cell carcinoma and in distal skin from the same patients,” Arch. Facial Plast. Surg. 7(4), 238–243 (2005).
[CrossRef] [PubMed]

Bauer, R.

S. Kuphal, R. Bauer, and A. K. Bosserhoff, “Integrin signaling in malignant melanoma,” Cancer Metastasis Rev. 24(2), 195–222 (2005).
[CrossRef] [PubMed]

Becker, D. L.

T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

Bernard, P.

C. Ntayi, W. Hornebeck, and P. Bernard, “Influence of cultured dermal fibroblasts on human melanoma cell proliferation, matrix metalloproteinase-2 (MMP-2) expression and invasion in vitro,” Arch. Dermatol. Res. 295(6), 236–241 (2003).
[CrossRef] [PubMed]

Bosanquet, N.

S. Morris, B. Cox, and N. Bosanquet, “Cost of skin cancer in England,” Eur. J. Health Econ. 10(3), 267–273 (2009).
[CrossRef] [PubMed]

Bosserhoff, A. K.

S. Kuphal, R. Bauer, and A. K. Bosserhoff, “Integrin signaling in malignant melanoma,” Cancer Metastasis Rev. 24(2), 195–222 (2005).
[CrossRef] [PubMed]

Boucher, Y.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Bray, F.

D. M. Parkin, F. Bray, J. Ferlay, and P. Pisani, “Global cancer statistics, 2002,” CA Cancer J. Clin. 55(2), 74–108 (2005).
[CrossRef] [PubMed]

Brown, E.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Campagnola, P. J.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6(3), 277–286 (2001).
[CrossRef] [PubMed]

Carli, P.

V. de Giorgi, D. Massi, C. Salvini, S. Sestini, and P. Carli, “Features of regression in dermoscopic diagnosis: a confounding factor? Two clinical, dermoscopic-pathologic case studies,” Dermatol. Surg. 32(2), 282–286 (2006).
[CrossRef] [PubMed]

Chanmugam, A.

D. L. Cummins, J. M. Cummins, H. Pantle, M. A. Silverman, A. L. Leonard, and A. Chanmugam, “Cutaneous malignant melanoma,” Mayo Clin. Proc. 81(4), 500–507 (2006).
[CrossRef] [PubMed]

Chen, C. C.

Chen, Y. C.

Chi, Y.

J. Varani, Y. Hattori, Y. Chi, T. Schmidt, P. Perone, M. E. Zeigler, D. J. Fader, and T. M. Johnson, “Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin,” Br. J. Cancer 82(3), 657–665 (2000).
[CrossRef] [PubMed]

Chu, S. W.

Clark, H. A.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6(3), 277–286 (2001).
[CrossRef] [PubMed]

Cox, B.

S. Morris, B. Cox, and N. Bosanquet, “Cost of skin cancer in England,” Eur. J. Health Econ. 10(3), 267–273 (2009).
[CrossRef] [PubMed]

Cox, G.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Cummins, D. L.

D. L. Cummins, J. M. Cummins, H. Pantle, M. A. Silverman, A. L. Leonard, and A. Chanmugam, “Cutaneous malignant melanoma,” Mayo Clin. Proc. 81(4), 500–507 (2006).
[CrossRef] [PubMed]

Cummins, J. M.

D. L. Cummins, J. M. Cummins, H. Pantle, M. A. Silverman, A. L. Leonard, and A. Chanmugam, “Cutaneous malignant melanoma,” Mayo Clin. Proc. 81(4), 500–507 (2006).
[CrossRef] [PubMed]

Dawson, R.

P. Eves, E. Katerinaki, C. Simpson, C. Layton, R. Dawson, G. Evans, and S. Mac Neil, “Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes,” Clin. Exp. Metastasis 20(8), 685–700 (2003).
[CrossRef] [PubMed]

de Giorgi, V.

V. de Giorgi, D. Massi, C. Salvini, S. Sestini, and P. Carli, “Features of regression in dermoscopic diagnosis: a confounding factor? Two clinical, dermoscopic-pathologic case studies,” Dermatol. Surg. 32(2), 282–286 (2006).
[CrossRef] [PubMed]

de Lange Davies, C.

T. Hompland, A. Erikson, M. Lindgren, T. Lindmo, and C. de Lange Davies, “Second-harmonic generation in collagen as a potential cancer diagnostic parameter,” J. Biomed. Opt. 13(5), 054050 (2008).
[CrossRef] [PubMed]

diTomaso, E.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Dummer, R.

E. Wandel, A. Raschke, G. Hildebrandt, J. Eberle, R. Dummer, U. Anderegg, and A. Saalbach, “Fibroblasts enhance the invasive capacity of melanoma cells in vitro,” Arch. Dermatol. Res. 293(12), 601–608 (2002).
[CrossRef] [PubMed]

Eberle, J.

E. Wandel, A. Raschke, G. Hildebrandt, J. Eberle, R. Dummer, U. Anderegg, and A. Saalbach, “Fibroblasts enhance the invasive capacity of melanoma cells in vitro,” Arch. Dermatol. Res. 293(12), 601–608 (2002).
[CrossRef] [PubMed]

Ekwobi, C.

T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

Eliceiri, K. W.

P. P. Provenzano, K. W. Eliceiri, and P. J. Keely, “Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment,” Clin. Exp. Metastasis 26(4), 357–370 (2009).
[CrossRef] [PubMed]

Ericson, M. B.

J. Paoli, M. Smedh, and M. B. Ericson, “Multiphoton laser scanning microscopy--a novel diagnostic method for superficial skin cancers,” Semin. Cutan. Med. Surg. 28(3), 190–195 (2009).
[CrossRef] [PubMed]

Erikson, A.

T. Hompland, A. Erikson, M. Lindgren, T. Lindmo, and C. de Lange Davies, “Second-harmonic generation in collagen as a potential cancer diagnostic parameter,” J. Biomed. Opt. 13(5), 054050 (2008).
[CrossRef] [PubMed]

Evans, G.

P. Eves, E. Katerinaki, C. Simpson, C. Layton, R. Dawson, G. Evans, and S. Mac Neil, “Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes,” Clin. Exp. Metastasis 20(8), 685–700 (2003).
[CrossRef] [PubMed]

Eves, P.

P. Eves, E. Katerinaki, C. Simpson, C. Layton, R. Dawson, G. Evans, and S. Mac Neil, “Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes,” Clin. Exp. Metastasis 20(8), 685–700 (2003).
[CrossRef] [PubMed]

Eyrich, A. M.

F. Wach, A. M. Eyrich, T. Wustrow, T. Krieg, and R. Hein, “Comparison of migration and invasiveness of epithelial tumor and melanoma cells in vitro,” J. Dermatol. Sci. 12(2), 118–126 (1996).
[CrossRef] [PubMed]

Fader, D. J.

J. Varani, Y. Hattori, Y. Chi, T. Schmidt, P. Perone, M. E. Zeigler, D. J. Fader, and T. M. Johnson, “Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin,” Br. J. Cancer 82(3), 657–665 (2000).
[CrossRef] [PubMed]

Ferlay, J.

D. M. Parkin, F. Bray, J. Ferlay, and P. Pisani, “Global cancer statistics, 2002,” CA Cancer J. Clin. 55(2), 74–108 (2005).
[CrossRef] [PubMed]

Fikrle, T.

T. Fikrle and K. Pizinger, “Dermatoscopic differences between atypical melanocytic naevi and thin malignant melanomas,” Melanoma Res. 16(1), 45–50 (2006).
[CrossRef] [PubMed]

Fine, S.

Fraser, I.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Freund, I.

S. Roth and I. Freund, “Second harmonic-generation in collagen,” J. Chem. Phys. 70(4), 1637–1643 (1979).
[CrossRef]

Friedman, R.

D. S. Rigel, J. Russak, and R. Friedman, “The evolution of melanoma diagnosis: 25 years beyond the ABCDs,” CA Cancer J. Clin. 60(5), 301–316 (2010).
[CrossRef] [PubMed]

Gannaway, J. N.

C. J. R. Sheppard, J. N. Gannaway, R. Kompfner, and D. Walsh, “The scanning harmonic optical microscope,” IEEE J. Quantum Electron. 13(9), 912 (1977).
[CrossRef]

Gerger, A.

A. Gerger, S. Koller, T. Kern, C. Massone, K. Steiger, E. Richtig, H. Kerl, and J. Smolle, “Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors,” J. Invest. Dermatol. 124(3), 493–498 (2005).
[CrossRef] [PubMed]

Gorrell, M. D.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Hansen, W. P.

Hattori, Y.

J. Varani, Y. Hattori, Y. Chi, T. Schmidt, P. Perone, M. E. Zeigler, D. J. Fader, and T. M. Johnson, “Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin,” Br. J. Cancer 82(3), 657–665 (2000).
[CrossRef] [PubMed]

Hein, R.

F. Wach, A. M. Eyrich, T. Wustrow, T. Krieg, and R. Hein, “Comparison of migration and invasiveness of epithelial tumor and melanoma cells in vitro,” J. Dermatol. Sci. 12(2), 118–126 (1996).
[CrossRef] [PubMed]

Hildebrandt, G.

E. Wandel, A. Raschke, G. Hildebrandt, J. Eberle, R. Dummer, U. Anderegg, and A. Saalbach, “Fibroblasts enhance the invasive capacity of melanoma cells in vitro,” Arch. Dermatol. Res. 293(12), 601–608 (2002).
[CrossRef] [PubMed]

Hompland, T.

T. Hompland, A. Erikson, M. Lindgren, T. Lindmo, and C. de Lange Davies, “Second-harmonic generation in collagen as a potential cancer diagnostic parameter,” J. Biomed. Opt. 13(5), 054050 (2008).
[CrossRef] [PubMed]

Hornebeck, W.

C. Ntayi, W. Hornebeck, and P. Bernard, “Influence of cultured dermal fibroblasts on human melanoma cell proliferation, matrix metalloproteinase-2 (MMP-2) expression and invasion in vitro,” Arch. Dermatol. Res. 295(6), 236–241 (2003).
[CrossRef] [PubMed]

Jain, R. K.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Jewett, B. S.

N. Monhian, B. S. Jewett, S. R. Baker, and J. Varani, “Matrix metalloproteinase expression in normal skin associated with basal cell carcinoma and in distal skin from the same patients,” Arch. Facial Plast. Surg. 7(4), 238–243 (2005).
[CrossRef] [PubMed]

Johnson, T. M.

J. Varani, Y. Hattori, Y. Chi, T. Schmidt, P. Perone, M. E. Zeigler, D. J. Fader, and T. M. Johnson, “Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin,” Br. J. Cancer 82(3), 657–665 (2000).
[CrossRef] [PubMed]

Jones, A.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Kable, E.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Katerinaki, E.

P. Eves, E. Katerinaki, C. Simpson, C. Layton, R. Dawson, G. Evans, and S. Mac Neil, “Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes,” Clin. Exp. Metastasis 20(8), 685–700 (2003).
[CrossRef] [PubMed]

Keely, P. J.

P. P. Provenzano, K. W. Eliceiri, and P. J. Keely, “Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment,” Clin. Exp. Metastasis 26(4), 357–370 (2009).
[CrossRef] [PubMed]

Kerl, H.

A. Gerger, S. Koller, T. Kern, C. Massone, K. Steiger, E. Richtig, H. Kerl, and J. Smolle, “Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors,” J. Invest. Dermatol. 124(3), 493–498 (2005).
[CrossRef] [PubMed]

Kern, T.

A. Gerger, S. Koller, T. Kern, C. Massone, K. Steiger, E. Richtig, H. Kerl, and J. Smolle, “Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors,” J. Invest. Dermatol. 124(3), 493–498 (2005).
[CrossRef] [PubMed]

Koller, S.

A. Gerger, S. Koller, T. Kern, C. Massone, K. Steiger, E. Richtig, H. Kerl, and J. Smolle, “Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors,” J. Invest. Dermatol. 124(3), 493–498 (2005).
[CrossRef] [PubMed]

Kompfner, R.

C. J. R. Sheppard, J. N. Gannaway, R. Kompfner, and D. Walsh, “The scanning harmonic optical microscope,” IEEE J. Quantum Electron. 13(9), 912 (1977).
[CrossRef]

Krieg, T.

F. Wach, A. M. Eyrich, T. Wustrow, T. Krieg, and R. Hein, “Comparison of migration and invasiveness of epithelial tumor and melanoma cells in vitro,” J. Dermatol. Sci. 12(2), 118–126 (1996).
[CrossRef] [PubMed]

Kuphal, S.

S. Kuphal, R. Bauer, and A. K. Bosserhoff, “Integrin signaling in malignant melanoma,” Cancer Metastasis Rev. 24(2), 195–222 (2005).
[CrossRef] [PubMed]

Layton, C.

P. Eves, E. Katerinaki, C. Simpson, C. Layton, R. Dawson, G. Evans, and S. Mac Neil, “Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes,” Clin. Exp. Metastasis 20(8), 685–700 (2003).
[CrossRef] [PubMed]

Leonard, A. L.

D. L. Cummins, J. M. Cummins, H. Pantle, M. A. Silverman, A. L. Leonard, and A. Chanmugam, “Cutaneous malignant melanoma,” Mayo Clin. Proc. 81(4), 500–507 (2006).
[CrossRef] [PubMed]

Lewis, A.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6(3), 277–286 (2001).
[CrossRef] [PubMed]

Lien, M. H.

M. H. Lien and V. K. Sondak, “Diagnostic techniques for primary cutaneous melanoma,” G. Ital. Dermatol. Venereol. 144(2), 187–194 (2009).
[PubMed]

Lin, B. L.

Lindgren, M.

T. Hompland, A. Erikson, M. Lindgren, T. Lindmo, and C. de Lange Davies, “Second-harmonic generation in collagen as a potential cancer diagnostic parameter,” J. Biomed. Opt. 13(5), 054050 (2008).
[CrossRef] [PubMed]

Lindmo, T.

T. Hompland, A. Erikson, M. Lindgren, T. Lindmo, and C. de Lange Davies, “Second-harmonic generation in collagen as a potential cancer diagnostic parameter,” J. Biomed. Opt. 13(5), 054050 (2008).
[CrossRef] [PubMed]

Loew, L. M.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6(3), 277–286 (2001).
[CrossRef] [PubMed]

Lohela, M.

M. Lohela and Z. Werb, “Intravital imaging of stromal cell dynamics in tumors,” Curr. Opin. Genet. Dev. 20(1), 72–78 (2010).
[CrossRef] [PubMed]

Mac Neil, S.

P. Eves, E. Katerinaki, C. Simpson, C. Layton, R. Dawson, G. Evans, and S. Mac Neil, “Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes,” Clin. Exp. Metastasis 20(8), 685–700 (2003).
[CrossRef] [PubMed]

Manconi, F.

G. Cox, E. Kable, A. Jones, I. Fraser, F. Manconi, and M. D. Gorrell, “3-dimensional imaging of collagen using second harmonic generation,” J. Struct. Biol. 141(1), 53–62 (2003).
[CrossRef] [PubMed]

Massi, D.

V. de Giorgi, D. Massi, C. Salvini, S. Sestini, and P. Carli, “Features of regression in dermoscopic diagnosis: a confounding factor? Two clinical, dermoscopic-pathologic case studies,” Dermatol. Surg. 32(2), 282–286 (2006).
[CrossRef] [PubMed]

Massone, C.

A. Gerger, S. Koller, T. Kern, C. Massone, K. Steiger, E. Richtig, H. Kerl, and J. Smolle, “Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors,” J. Invest. Dermatol. 124(3), 493–498 (2005).
[CrossRef] [PubMed]

McKee, T.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Mohler, W. A.

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6(3), 277–286 (2001).
[CrossRef] [PubMed]

Monhian, N.

N. Monhian, B. S. Jewett, S. R. Baker, and J. Varani, “Matrix metalloproteinase expression in normal skin associated with basal cell carcinoma and in distal skin from the same patients,” Arch. Facial Plast. Surg. 7(4), 238–243 (2005).
[CrossRef] [PubMed]

Morris, S.

S. Morris, B. Cox, and N. Bosanquet, “Cost of skin cancer in England,” Eur. J. Health Econ. 10(3), 267–273 (2009).
[CrossRef] [PubMed]

Ntayi, C.

C. Ntayi, W. Hornebeck, and P. Bernard, “Influence of cultured dermal fibroblasts on human melanoma cell proliferation, matrix metalloproteinase-2 (MMP-2) expression and invasion in vitro,” Arch. Dermatol. Res. 295(6), 236–241 (2003).
[CrossRef] [PubMed]

Pantle, H.

D. L. Cummins, J. M. Cummins, H. Pantle, M. A. Silverman, A. L. Leonard, and A. Chanmugam, “Cutaneous malignant melanoma,” Mayo Clin. Proc. 81(4), 500–507 (2006).
[CrossRef] [PubMed]

Paoli, J.

J. Paoli, M. Smedh, and M. B. Ericson, “Multiphoton laser scanning microscopy--a novel diagnostic method for superficial skin cancers,” Semin. Cutan. Med. Surg. 28(3), 190–195 (2009).
[CrossRef] [PubMed]

Parkin, D. M.

D. M. Parkin, F. Bray, J. Ferlay, and P. Pisani, “Global cancer statistics, 2002,” CA Cancer J. Clin. 55(2), 74–108 (2005).
[CrossRef] [PubMed]

Perone, P.

J. Varani, Y. Hattori, Y. Chi, T. Schmidt, P. Perone, M. E. Zeigler, D. J. Fader, and T. M. Johnson, “Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin,” Br. J. Cancer 82(3), 657–665 (2000).
[CrossRef] [PubMed]

Pisani, P.

D. M. Parkin, F. Bray, J. Ferlay, and P. Pisani, “Global cancer statistics, 2002,” CA Cancer J. Clin. 55(2), 74–108 (2005).
[CrossRef] [PubMed]

Pizinger, K.

T. Fikrle and K. Pizinger, “Dermatoscopic differences between atypical melanocytic naevi and thin malignant melanomas,” Melanoma Res. 16(1), 45–50 (2006).
[CrossRef] [PubMed]

Pluen, A.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Provenzano, P. P.

P. P. Provenzano, K. W. Eliceiri, and P. J. Keely, “Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment,” Clin. Exp. Metastasis 26(4), 357–370 (2009).
[CrossRef] [PubMed]

Raschke, A.

E. Wandel, A. Raschke, G. Hildebrandt, J. Eberle, R. Dummer, U. Anderegg, and A. Saalbach, “Fibroblasts enhance the invasive capacity of melanoma cells in vitro,” Arch. Dermatol. Res. 293(12), 601–608 (2002).
[CrossRef] [PubMed]

Richtig, E.

A. Gerger, S. Koller, T. Kern, C. Massone, K. Steiger, E. Richtig, H. Kerl, and J. Smolle, “Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors,” J. Invest. Dermatol. 124(3), 493–498 (2005).
[CrossRef] [PubMed]

Rigel, D. S.

D. S. Rigel, J. Russak, and R. Friedman, “The evolution of melanoma diagnosis: 25 years beyond the ABCDs,” CA Cancer J. Clin. 60(5), 301–316 (2010).
[CrossRef] [PubMed]

Roth, S.

S. Roth and I. Freund, “Second harmonic-generation in collagen,” J. Chem. Phys. 70(4), 1637–1643 (1979).
[CrossRef]

Russak, J.

D. S. Rigel, J. Russak, and R. Friedman, “The evolution of melanoma diagnosis: 25 years beyond the ABCDs,” CA Cancer J. Clin. 60(5), 301–316 (2010).
[CrossRef] [PubMed]

Saalbach, A.

E. Wandel, A. Raschke, G. Hildebrandt, J. Eberle, R. Dummer, U. Anderegg, and A. Saalbach, “Fibroblasts enhance the invasive capacity of melanoma cells in vitro,” Arch. Dermatol. Res. 293(12), 601–608 (2002).
[CrossRef] [PubMed]

Salvini, C.

V. de Giorgi, D. Massi, C. Salvini, S. Sestini, and P. Carli, “Features of regression in dermoscopic diagnosis: a confounding factor? Two clinical, dermoscopic-pathologic case studies,” Dermatol. Surg. 32(2), 282–286 (2006).
[CrossRef] [PubMed]

Schmidt, T.

J. Varani, Y. Hattori, Y. Chi, T. Schmidt, P. Perone, M. E. Zeigler, D. J. Fader, and T. M. Johnson, “Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin,” Br. J. Cancer 82(3), 657–665 (2000).
[CrossRef] [PubMed]

Scoazec, J. Y.

J. Y. Scoazec, “Tissue and cell imaging in situ: potential for applications in pathology and endoscopy,” Gut 52(90004Suppl 4), 1iv–1iv (2003).
[CrossRef] [PubMed]

Seed, B.

E. Brown, T. McKee, E. diTomaso, A. Pluen, B. Seed, Y. Boucher, and R. K. Jain, “Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation,” Nat. Med. 9(6), 796–801 (2003).
[CrossRef] [PubMed]

Sestini, S.

V. de Giorgi, D. Massi, C. Salvini, S. Sestini, and P. Carli, “Features of regression in dermoscopic diagnosis: a confounding factor? Two clinical, dermoscopic-pathologic case studies,” Dermatol. Surg. 32(2), 282–286 (2006).
[CrossRef] [PubMed]

Sheppard, C. J. R.

C. J. R. Sheppard, J. N. Gannaway, R. Kompfner, and D. Walsh, “The scanning harmonic optical microscope,” IEEE J. Quantum Electron. 13(9), 912 (1977).
[CrossRef]

Silverman, M. A.

D. L. Cummins, J. M. Cummins, H. Pantle, M. A. Silverman, A. L. Leonard, and A. Chanmugam, “Cutaneous malignant melanoma,” Mayo Clin. Proc. 81(4), 500–507 (2006).
[CrossRef] [PubMed]

Simpson, C.

P. Eves, E. Katerinaki, C. Simpson, C. Layton, R. Dawson, G. Evans, and S. Mac Neil, “Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes,” Clin. Exp. Metastasis 20(8), 685–700 (2003).
[CrossRef] [PubMed]

Smedh, M.

J. Paoli, M. Smedh, and M. B. Ericson, “Multiphoton laser scanning microscopy--a novel diagnostic method for superficial skin cancers,” Semin. Cutan. Med. Surg. 28(3), 190–195 (2009).
[CrossRef] [PubMed]

Smolle, J.

A. Gerger, S. Koller, T. Kern, C. Massone, K. Steiger, E. Richtig, H. Kerl, and J. Smolle, “Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors,” J. Invest. Dermatol. 124(3), 493–498 (2005).
[CrossRef] [PubMed]

Sondak, V. K.

M. H. Lien and V. K. Sondak, “Diagnostic techniques for primary cutaneous melanoma,” G. Ital. Dermatol. Venereol. 144(2), 187–194 (2009).
[PubMed]

Steiger, K.

A. Gerger, S. Koller, T. Kern, C. Massone, K. Steiger, E. Richtig, H. Kerl, and J. Smolle, “Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors,” J. Invest. Dermatol. 124(3), 493–498 (2005).
[CrossRef] [PubMed]

Sun, C. K.

Theodossiou, T. A.

T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

Thrasivoulou, C.

T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

Tsai, T. H.

Varani, J.

N. Monhian, B. S. Jewett, S. R. Baker, and J. Varani, “Matrix metalloproteinase expression in normal skin associated with basal cell carcinoma and in distal skin from the same patients,” Arch. Facial Plast. Surg. 7(4), 238–243 (2005).
[CrossRef] [PubMed]

J. Varani, Y. Hattori, Y. Chi, T. Schmidt, P. Perone, M. E. Zeigler, D. J. Fader, and T. M. Johnson, “Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin,” Br. J. Cancer 82(3), 657–665 (2000).
[CrossRef] [PubMed]

Wach, F.

F. Wach, A. M. Eyrich, T. Wustrow, T. Krieg, and R. Hein, “Comparison of migration and invasiveness of epithelial tumor and melanoma cells in vitro,” J. Dermatol. Sci. 12(2), 118–126 (1996).
[CrossRef] [PubMed]

Walsh, D.

C. J. R. Sheppard, J. N. Gannaway, R. Kompfner, and D. Walsh, “The scanning harmonic optical microscope,” IEEE J. Quantum Electron. 13(9), 912 (1977).
[CrossRef]

Wandel, E.

E. Wandel, A. Raschke, G. Hildebrandt, J. Eberle, R. Dummer, U. Anderegg, and A. Saalbach, “Fibroblasts enhance the invasive capacity of melanoma cells in vitro,” Arch. Dermatol. Res. 293(12), 601–608 (2002).
[CrossRef] [PubMed]

Webb, W. W.

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005).
[CrossRef] [PubMed]

Weinstock, M. A.

M. A. Weinstock, “Progress and prospects on melanoma: the way forward for early detection and reduced mortality,” Clin. Cancer Res. 12(7), 2297s–2300s (2006).
[CrossRef] [PubMed]

Werb, Z.

M. Lohela and Z. Werb, “Intravital imaging of stromal cell dynamics in tumors,” Curr. Opin. Genet. Dev. 20(1), 72–78 (2010).
[CrossRef] [PubMed]

Williams, R. M.

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005).
[CrossRef] [PubMed]

Wustrow, T.

F. Wach, A. M. Eyrich, T. Wustrow, T. Krieg, and R. Hein, “Comparison of migration and invasiveness of epithelial tumor and melanoma cells in vitro,” J. Dermatol. Sci. 12(2), 118–126 (1996).
[CrossRef] [PubMed]

Zeigler, M. E.

J. Varani, Y. Hattori, Y. Chi, T. Schmidt, P. Perone, M. E. Zeigler, D. J. Fader, and T. M. Johnson, “Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin,” Br. J. Cancer 82(3), 657–665 (2000).
[CrossRef] [PubMed]

Zipfel, W. R.

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005).
[CrossRef] [PubMed]

Appl. Opt. (1)

Arch. Dermatol. Res. (2)

C. Ntayi, W. Hornebeck, and P. Bernard, “Influence of cultured dermal fibroblasts on human melanoma cell proliferation, matrix metalloproteinase-2 (MMP-2) expression and invasion in vitro,” Arch. Dermatol. Res. 295(6), 236–241 (2003).
[CrossRef] [PubMed]

E. Wandel, A. Raschke, G. Hildebrandt, J. Eberle, R. Dummer, U. Anderegg, and A. Saalbach, “Fibroblasts enhance the invasive capacity of melanoma cells in vitro,” Arch. Dermatol. Res. 293(12), 601–608 (2002).
[CrossRef] [PubMed]

Arch. Facial Plast. Surg. (1)

N. Monhian, B. S. Jewett, S. R. Baker, and J. Varani, “Matrix metalloproteinase expression in normal skin associated with basal cell carcinoma and in distal skin from the same patients,” Arch. Facial Plast. Surg. 7(4), 238–243 (2005).
[CrossRef] [PubMed]

Biophys. J. (2)

T. A. Theodossiou, C. Thrasivoulou, C. Ekwobi, and D. L. Becker, “Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections,” Biophys. J. 91(12), 4665–4677 (2006).
[CrossRef] [PubMed]

R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second-harmonic generation images of collagen I fibrils,” Biophys. J. 88(2), 1377–1386 (2005).
[CrossRef] [PubMed]

Br. J. Cancer (1)

J. Varani, Y. Hattori, Y. Chi, T. Schmidt, P. Perone, M. E. Zeigler, D. J. Fader, and T. M. Johnson, “Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin,” Br. J. Cancer 82(3), 657–665 (2000).
[CrossRef] [PubMed]

CA Cancer J. Clin. (2)

D. M. Parkin, F. Bray, J. Ferlay, and P. Pisani, “Global cancer statistics, 2002,” CA Cancer J. Clin. 55(2), 74–108 (2005).
[CrossRef] [PubMed]

D. S. Rigel, J. Russak, and R. Friedman, “The evolution of melanoma diagnosis: 25 years beyond the ABCDs,” CA Cancer J. Clin. 60(5), 301–316 (2010).
[CrossRef] [PubMed]

Cancer Metastasis Rev. (1)

S. Kuphal, R. Bauer, and A. K. Bosserhoff, “Integrin signaling in malignant melanoma,” Cancer Metastasis Rev. 24(2), 195–222 (2005).
[CrossRef] [PubMed]

Clin. Cancer Res. (1)

M. A. Weinstock, “Progress and prospects on melanoma: the way forward for early detection and reduced mortality,” Clin. Cancer Res. 12(7), 2297s–2300s (2006).
[CrossRef] [PubMed]

Clin. Exp. Metastasis (2)

P. P. Provenzano, K. W. Eliceiri, and P. J. Keely, “Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment,” Clin. Exp. Metastasis 26(4), 357–370 (2009).
[CrossRef] [PubMed]

P. Eves, E. Katerinaki, C. Simpson, C. Layton, R. Dawson, G. Evans, and S. Mac Neil, “Melanoma invasion in reconstructed human skin is influenced by skin cells--investigation of the role of proteolytic enzymes,” Clin. Exp. Metastasis 20(8), 685–700 (2003).
[CrossRef] [PubMed]

Curr. Opin. Genet. Dev. (1)

M. Lohela and Z. Werb, “Intravital imaging of stromal cell dynamics in tumors,” Curr. Opin. Genet. Dev. 20(1), 72–78 (2010).
[CrossRef] [PubMed]

Dermatol. Surg. (1)

V. de Giorgi, D. Massi, C. Salvini, S. Sestini, and P. Carli, “Features of regression in dermoscopic diagnosis: a confounding factor? Two clinical, dermoscopic-pathologic case studies,” Dermatol. Surg. 32(2), 282–286 (2006).
[CrossRef] [PubMed]

Eur. J. Health Econ. (1)

S. Morris, B. Cox, and N. Bosanquet, “Cost of skin cancer in England,” Eur. J. Health Econ. 10(3), 267–273 (2009).
[CrossRef] [PubMed]

G. Ital. Dermatol. Venereol. (1)

M. H. Lien and V. K. Sondak, “Diagnostic techniques for primary cutaneous melanoma,” G. Ital. Dermatol. Venereol. 144(2), 187–194 (2009).
[PubMed]

Gut (1)

J. Y. Scoazec, “Tissue and cell imaging in situ: potential for applications in pathology and endoscopy,” Gut 52(90004Suppl 4), 1iv–1iv (2003).
[CrossRef] [PubMed]

IEEE J. Quantum Electron. (1)

C. J. R. Sheppard, J. N. Gannaway, R. Kompfner, and D. Walsh, “The scanning harmonic optical microscope,” IEEE J. Quantum Electron. 13(9), 912 (1977).
[CrossRef]

J. Biomed. Opt. (2)

P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, “Second-harmonic imaging microscopy of living cells,” J. Biomed. Opt. 6(3), 277–286 (2001).
[CrossRef] [PubMed]

T. Hompland, A. Erikson, M. Lindgren, T. Lindmo, and C. de Lange Davies, “Second-harmonic generation in collagen as a potential cancer diagnostic parameter,” J. Biomed. Opt. 13(5), 054050 (2008).
[CrossRef] [PubMed]

J. Chem. Phys. (1)

S. Roth and I. Freund, “Second harmonic-generation in collagen,” J. Chem. Phys. 70(4), 1637–1643 (1979).
[CrossRef]

J. Dermatol. Sci. (1)

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Figures (6)

Fig. 2
Fig. 2

(A) Enlarged view of Fig. 1D showing regions from which collagen fiber density was quantified. A = Area of normal skin under dermal-epidermal junction, B = Lateral melanoma border under dermal-epidermal junction, C = Area under the middle part of the lesion and under dermal-epidermal junction, areas D, E, F, are all below C but one field vertically deeper to each other, area G is at the same depth as F but away from the lesion (A). (B) Graph of collagen fiber density (Area %, ± SE) of the individual regions of A (n = 5-8). On some samples not all regions could be quantified because the biopsies were cut to shallow, i.e., not enough dermis depth present, therefore the deeper regions were absent and hence there is no data and the n-number vary. Note regression line illustrating a linear correlation of collagen fiber density (R2 = 0.9849) from region C (mid-point of MM lesion) to region G (deep area of non-lesion skin). Scale bar = 1mm

Fig. 1
Fig. 1

Montage of SHG images in the forward propagated (A), backscattered geometry (B), and bright field image (C). Superimposed images of bright-field and SHG images indicate collagen distribution within each section (D). Scale bar = 1mm

Fig. 3
Fig. 3

Cross-reference control comparison of melanoma borders obtained from H&E (A) and Melan-A stained sister sections (B) and comparison with SHG (C) showed a high degree of correlation (p<0.0012). Vertical lines (black lines in A & B; white lines in C) represent lateral borders of MM as identified by two independent histopathologists (A and B) and independent researcher (C). Red box (B-C) represents the areas shown at high power in Figs. 4A and 4B, respectively. Scale bar = 1mm

Fig. 4
Fig. 4

High magnification images of Fig. 3. Melan-A staining (A), SHG/brightfield image (B), corresponding to left hand border (red boxes) in Figs. 3B and 3C, respectively. Note that the SHG signal starts to reappear at the dermo-epidermal junction at the point where MM lesion is absent. Scale bar = 1mm

Fig. 5
Fig. 5

Measured distance from center of each MM sample to left and right borders of lesion for SHG (dotted infill = left border, horizontal line infill = right border) stained section and H&E (diagonal hatch infill = left border, checker hatch infill = right border) imaged sister section (A). Mean difference of distance for left and right MM borders of H&E versus SHG images (B). Statistical analysis shows no significant difference between the two methods with total p-value for both sides p = 0.9339, whilst Kendall-Rank Correlation Analysis Showed a total p-value for both sides p = 0.0012.

Fig. 6
Fig. 6

SHG imaging of normal, ex vivo, live rat skin (A) and human skin sample (B) showing collagen morphology throughout the entire thickness when imaged from the epidermis, in the transmission (red) and back-scattered (green) geometry. Rat skin, SHG transmission images (red) and back-scattered SHG signal (green) were detected through the whole thickness of the skin (A). The blue channel is the two-photon auto fluorescence image of the upper epidermis, whilst the yellow represents the colocalization of the red and green channels. In lightly pigmented human skin samples, we were able to image through the whole thickness of epidermis and dermis to depths of approximately 300µm in the back-scattered geometry (green signal) and over 1000µm in the transmission geometry (red signal) (B). Scale bars = 50 µm for each orthogonal plane.

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