Abstract

We present a multiphoton microscopy instrument specially designed for in vivo dermatological use that is capable of imaging human skin at 27framespersecond with 256pixels×256pixels resolution without the use of exogenous contrast agents. Imaging at fast frame rates is critical to reducing image blurring due to patient motion and to providing practically short clinical measurement times. Second harmonic generation and two-photon fluorescence images and videos acquired at optimized wavelengths are presented showing cellular and tissue structures from the skin surface down to the reticular dermis.

© 2011 Optical Society of America

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  1. B. R. Masters, P. T. So, and E. Gratton, Biophys. J. 72, 2405 (1997).
    [CrossRef] [PubMed]
  2. K. Konig, J. Biophotonics 1, 13 (2008).
    [CrossRef]
  3. T.-H. Tsai, S.-H. Jee, C.-Y. Dong, and S.-J. Lin, J. Dermatol. Sci. 56, 1 (2009).
    [CrossRef] [PubMed]
  4. F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
    [CrossRef] [PubMed]
  5. S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, J. Biomed. Opt. 14, 060505 (2009).
    [CrossRef]
  6. T. Yasui, Y. Takahashi, M. Ito, S. Fukushima, and T. Araki, Appl. Opt. 48, D88 (2009).
    [CrossRef] [PubMed]
  7. A. N. Bader, A.-M. Pena, C. J. van Voskuilen, J. A. Palero, F. Leroy, A. Colonna, and H. C. Gerritsen, Biomed. Opt. Express 2, 365 (2011).
    [CrossRef] [PubMed]
  8. E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. Konig, P. Elsner, and M. Kaatz, J. Invest. Dermatol. 129, 1752 (2009).
    [CrossRef] [PubMed]
  9. M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
    [CrossRef] [PubMed]
  10. K. Konig, A. Ehlers, F. Stracke, and I. Riemann, Skin Pharmacol. Physiol. 19, 78 (2006).
    [CrossRef] [PubMed]
  11. K. Konig, M. Speicher, M. J. Kohler, R. Scharenberg, and M. Kaatz, J. Biophotonics 3, 759 (2010).
    [CrossRef] [PubMed]
  12. G. Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, R. Y. Tsien, and M. H. Ellisman, Biophys. J. 76, 2412 (1999).
    [CrossRef] [PubMed]
  13. B. Yu, K. Hean Kim, P. T. C. So, D. Blankschtein, and R. Langer, J. Invest. Dermatol. 118, 1085 (2002).
    [CrossRef] [PubMed]

2011 (1)

2010 (1)

K. Konig, M. Speicher, M. J. Kohler, R. Scharenberg, and M. Kaatz, J. Biophotonics 3, 759 (2010).
[CrossRef] [PubMed]

2009 (4)

T. Yasui, Y. Takahashi, M. Ito, S. Fukushima, and T. Araki, Appl. Opt. 48, D88 (2009).
[CrossRef] [PubMed]

T.-H. Tsai, S.-H. Jee, C.-Y. Dong, and S.-J. Lin, J. Dermatol. Sci. 56, 1 (2009).
[CrossRef] [PubMed]

S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, J. Biomed. Opt. 14, 060505 (2009).
[CrossRef]

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. Konig, P. Elsner, and M. Kaatz, J. Invest. Dermatol. 129, 1752 (2009).
[CrossRef] [PubMed]

2008 (2)

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

K. Konig, J. Biophotonics 1, 13 (2008).
[CrossRef]

2006 (1)

K. Konig, A. Ehlers, F. Stracke, and I. Riemann, Skin Pharmacol. Physiol. 19, 78 (2006).
[CrossRef] [PubMed]

2005 (1)

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef] [PubMed]

2002 (1)

B. Yu, K. Hean Kim, P. T. C. So, D. Blankschtein, and R. Langer, J. Invest. Dermatol. 118, 1085 (2002).
[CrossRef] [PubMed]

1999 (1)

G. Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, R. Y. Tsien, and M. H. Ellisman, Biophys. J. 76, 2412 (1999).
[CrossRef] [PubMed]

1997 (1)

B. R. Masters, P. T. So, and E. Gratton, Biophys. J. 72, 2405 (1997).
[CrossRef] [PubMed]

Araki, T.

Bader, A. N.

Bauer, A.

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

Blankschtein, D.

B. Yu, K. Hean Kim, P. T. C. So, D. Blankschtein, and R. Langer, J. Invest. Dermatol. 118, 1085 (2002).
[CrossRef] [PubMed]

Buckle, R.

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

Chen, S.-Y.

S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, J. Biomed. Opt. 14, 060505 (2009).
[CrossRef]

Colonna, A.

Denk, W.

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef] [PubMed]

Dimitrow, E.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. Konig, P. Elsner, and M. Kaatz, J. Invest. Dermatol. 129, 1752 (2009).
[CrossRef] [PubMed]

Dong, C.-Y.

T.-H. Tsai, S.-H. Jee, C.-Y. Dong, and S.-J. Lin, J. Dermatol. Sci. 56, 1 (2009).
[CrossRef] [PubMed]

Ehlers, A.

K. Konig, A. Ehlers, F. Stracke, and I. Riemann, Skin Pharmacol. Physiol. 19, 78 (2006).
[CrossRef] [PubMed]

Ellisman, M. H.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, R. Y. Tsien, and M. H. Ellisman, Biophys. J. 76, 2412 (1999).
[CrossRef] [PubMed]

Elsner, P.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. Konig, P. Elsner, and M. Kaatz, J. Invest. Dermatol. 129, 1752 (2009).
[CrossRef] [PubMed]

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

Fan, G. Y.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, R. Y. Tsien, and M. H. Ellisman, Biophys. J. 76, 2412 (1999).
[CrossRef] [PubMed]

Fluhr, J. W.

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

Fujisaki, H.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, R. Y. Tsien, and M. H. Ellisman, Biophys. J. 76, 2412 (1999).
[CrossRef] [PubMed]

Fukushima, S.

Gerritsen, H. C.

Gratton, E.

B. R. Masters, P. T. So, and E. Gratton, Biophys. J. 72, 2405 (1997).
[CrossRef] [PubMed]

Hahn, S.

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

Hean Kim, K.

B. Yu, K. Hean Kim, P. T. C. So, D. Blankschtein, and R. Langer, J. Invest. Dermatol. 118, 1085 (2002).
[CrossRef] [PubMed]

Helmchen, F.

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef] [PubMed]

Ito, M.

Jee, S.-H.

T.-H. Tsai, S.-H. Jee, C.-Y. Dong, and S.-J. Lin, J. Dermatol. Sci. 56, 1 (2009).
[CrossRef] [PubMed]

Kaatz, M.

K. Konig, M. Speicher, M. J. Kohler, R. Scharenberg, and M. Kaatz, J. Biophotonics 3, 759 (2010).
[CrossRef] [PubMed]

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. Konig, P. Elsner, and M. Kaatz, J. Invest. Dermatol. 129, 1752 (2009).
[CrossRef] [PubMed]

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

Koehler, M. J.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. Konig, P. Elsner, and M. Kaatz, J. Invest. Dermatol. 129, 1752 (2009).
[CrossRef] [PubMed]

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

Kohler, M. J.

K. Konig, M. Speicher, M. J. Kohler, R. Scharenberg, and M. Kaatz, J. Biophotonics 3, 759 (2010).
[CrossRef] [PubMed]

Konig, K.

K. Konig, M. Speicher, M. J. Kohler, R. Scharenberg, and M. Kaatz, J. Biophotonics 3, 759 (2010).
[CrossRef] [PubMed]

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. Konig, P. Elsner, and M. Kaatz, J. Invest. Dermatol. 129, 1752 (2009).
[CrossRef] [PubMed]

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

K. Konig, J. Biophotonics 1, 13 (2008).
[CrossRef]

K. Konig, A. Ehlers, F. Stracke, and I. Riemann, Skin Pharmacol. Physiol. 19, 78 (2006).
[CrossRef] [PubMed]

Langer, R.

B. Yu, K. Hean Kim, P. T. C. So, D. Blankschtein, and R. Langer, J. Invest. Dermatol. 118, 1085 (2002).
[CrossRef] [PubMed]

Leroy, F.

Lin, S.-J.

T.-H. Tsai, S.-H. Jee, C.-Y. Dong, and S.-J. Lin, J. Dermatol. Sci. 56, 1 (2009).
[CrossRef] [PubMed]

Masters, B. R.

B. R. Masters, P. T. So, and E. Gratton, Biophys. J. 72, 2405 (1997).
[CrossRef] [PubMed]

Miyawaki, A.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, R. Y. Tsien, and M. H. Ellisman, Biophys. J. 76, 2412 (1999).
[CrossRef] [PubMed]

Norgauer, J.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. Konig, P. Elsner, and M. Kaatz, J. Invest. Dermatol. 129, 1752 (2009).
[CrossRef] [PubMed]

Palero, J. A.

Pena, A.-M.

Preller, A.

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

Riemann, I.

K. Konig, A. Ehlers, F. Stracke, and I. Riemann, Skin Pharmacol. Physiol. 19, 78 (2006).
[CrossRef] [PubMed]

Scharenberg, R.

K. Konig, M. Speicher, M. J. Kohler, R. Scharenberg, and M. Kaatz, J. Biophotonics 3, 759 (2010).
[CrossRef] [PubMed]

So, P. T.

B. R. Masters, P. T. So, and E. Gratton, Biophys. J. 72, 2405 (1997).
[CrossRef] [PubMed]

So, P. T. C.

B. Yu, K. Hean Kim, P. T. C. So, D. Blankschtein, and R. Langer, J. Invest. Dermatol. 118, 1085 (2002).
[CrossRef] [PubMed]

Speicher, M.

K. Konig, M. Speicher, M. J. Kohler, R. Scharenberg, and M. Kaatz, J. Biophotonics 3, 759 (2010).
[CrossRef] [PubMed]

Stracke, F.

K. Konig, A. Ehlers, F. Stracke, and I. Riemann, Skin Pharmacol. Physiol. 19, 78 (2006).
[CrossRef] [PubMed]

Sun, C.-K.

S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, J. Biomed. Opt. 14, 060505 (2009).
[CrossRef]

Takahashi, Y.

Tsai, T.-H.

T.-H. Tsai, S.-H. Jee, C.-Y. Dong, and S.-J. Lin, J. Dermatol. Sci. 56, 1 (2009).
[CrossRef] [PubMed]

Tsay, R.-K.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, R. Y. Tsien, and M. H. Ellisman, Biophys. J. 76, 2412 (1999).
[CrossRef] [PubMed]

Tsien, R. Y.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, R. Y. Tsien, and M. H. Ellisman, Biophys. J. 76, 2412 (1999).
[CrossRef] [PubMed]

van Voskuilen, C. J.

Wu, H.-Y.

S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, J. Biomed. Opt. 14, 060505 (2009).
[CrossRef]

Yasui, T.

Yu, B.

B. Yu, K. Hean Kim, P. T. C. So, D. Blankschtein, and R. Langer, J. Invest. Dermatol. 118, 1085 (2002).
[CrossRef] [PubMed]

Ziemer, M.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. Konig, P. Elsner, and M. Kaatz, J. Invest. Dermatol. 129, 1752 (2009).
[CrossRef] [PubMed]

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biomed. Opt. Express (1)

Biophys. J. (2)

G. Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, R. Y. Tsien, and M. H. Ellisman, Biophys. J. 76, 2412 (1999).
[CrossRef] [PubMed]

B. R. Masters, P. T. So, and E. Gratton, Biophys. J. 72, 2405 (1997).
[CrossRef] [PubMed]

Exp. Dermatol. (1)

M. J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K. Konig, R. Buckle, J. W. Fluhr, and M. Kaatz, Exp. Dermatol. 17, 519 (2008).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

S.-Y. Chen, H.-Y. Wu, and C.-K. Sun, J. Biomed. Opt. 14, 060505 (2009).
[CrossRef]

J. Biophotonics (2)

K. Konig, J. Biophotonics 1, 13 (2008).
[CrossRef]

K. Konig, M. Speicher, M. J. Kohler, R. Scharenberg, and M. Kaatz, J. Biophotonics 3, 759 (2010).
[CrossRef] [PubMed]

J. Dermatol. Sci. (1)

T.-H. Tsai, S.-H. Jee, C.-Y. Dong, and S.-J. Lin, J. Dermatol. Sci. 56, 1 (2009).
[CrossRef] [PubMed]

J. Invest. Dermatol. (2)

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. Konig, P. Elsner, and M. Kaatz, J. Invest. Dermatol. 129, 1752 (2009).
[CrossRef] [PubMed]

B. Yu, K. Hean Kim, P. T. C. So, D. Blankschtein, and R. Langer, J. Invest. Dermatol. 118, 1085 (2002).
[CrossRef] [PubMed]

Nat. Methods (1)

F. Helmchen and W. Denk, Nat. Methods 2, 932 (2005).
[CrossRef] [PubMed]

Skin Pharmacol. Physiol. (1)

K. Konig, A. Ehlers, F. Stracke, and I. Riemann, Skin Pharmacol. Physiol. 19, 78 (2006).
[CrossRef] [PubMed]

Supplementary Material (4)

» Media 1: AVI (3904 KB)     
» Media 2: AVI (4038 KB)     
» Media 3: AVI (3910 KB)     
» Media 4: AVI (2285 KB)     

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Figures (4)

Fig. 1
Fig. 1

In vivo video rate MPM setup. The SHG/TPF dichroic and filters preceding PMTs were changed or removed according to the imaging modalities. (a) Example integrated SHG/TPF image extracted from a video ( 27 fps ) of the dorsal forearm of a 24-year-old Asian female (Media 1). Excitation wavelength = 730 nm . Both TPF and SHG photons were directed onto a single PMT. FOV = 200 μm × 200 μm . Resolution = 256 pixels × 256 pixels . Frame scan rate = 27 fps . Frame capture/display rate = 24 fps .

Fig. 2
Fig. 2

TPF images from epidermis (oblique orientation) extracted from an in vivo video from the dorsal forearm of a 25-year-old Asian female increasing in depth from (a) near the surface to (f) near the dermal/epidermal boundary (Media 2). A 405 nm dichroic mirror ensured only TPF signals reached the PMT. Excitation wavelength = 740 nm . FOV = 200 μm × 200 μm . Resolution = 256   pixels × 256   pixels . Frame scan rate = 15 fps . Frame capture/display rate = 12 fps . At this wavelength, the cells of the epidermis are well visualized from the SC down to the SB.

Fig. 3
Fig. 3

Integrated SHG/TPF images of the epidermal ridges and the papillary dermis extracted from an in vivo video of the dorsal forearm of a 28-year-old Asian male (Media 3). The imaging depth increases from (a) to (f) starting from the dermal/epidermal boundary. Excitation wavelength = 900 nm . TPF and SHG photons were directed onto a single PMT. FOV = 200 μm × 200 μm . Resolution = 256   pixels × 256   pixels . Frame scan rate = 15 fps . Frame capture/display rate = 12 fps . At this wavelength, the cells of the SB and collagen and elastin fibers are observed.

Fig. 4
Fig. 4

False color overlay of SHG (green) and TPF (red) images from the reticular dermis extracted from an in vivo video of the dorsal forearm of a 63-year-old Caucasian male (Media 4). A 458 nm dichroic mirror was used to separate the SHG and TPF signals. 440 / 40 nm bandpass and 458 nm long pass filters were placed in front of the SHG and TPF PMTs, respectively. Excitation wavelength = 880 nm . FOV = 200 μm × 200 μm . Resolution = 256 pixels × 256 pixels . Frame scan rate = 15 fps . Frame capture/display rate = 12 fps . Collagen (SHG) and elastin (TPF) fibers are clearly seen.

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