Abstract

Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been applied in connection to measurements of the point spread function (PSF) for quantitative analysis of sulphorhodamine B (SRB) in excised human skin. The PSF was measured using subresolution fluorescent beads embedded in the skin specimen. The PSF, measured as full width at half maximum (FWHM) was found to be 0.41 ± 0.05 µm in the lateral direction, and 1.2 ± 0.4 μm in the axial direction. The molecular diffusion of SRB inside the skin ranged between 0.5 and 15.0 × 10−8 cm2/s. The diffusion coefficient is not dependent on depths down to 40 µm. The fluorophores were found to accumulate on the upper layers of the skin. This work is the first TPFCS study in human skin. The results show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.

© 2010 OSA

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References

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  9. J. Bender, C. Simonsson, M. Smedh, S. Engström, and M. B. Ericson, “Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy,” J. Control. Release 129(3), 163–169 (2008).
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2010 (1)

D. E. M. Na Ji and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7, ••• (2010).

2009 (2)

K. Samuelsson, C. Simonsson, C. A. Jonsson, G. Westman, M. B. Ericson, and A. T. Karlberg, “Accumulation of FITC near stratum corneum-visualizing epidermal distribution of a strong sensitizer using two-photon microscopy,” Contact Dermat. 61(2), 91–100 (2009).
[CrossRef]

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol. 129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

2008 (2)

J. Bender, C. Simonsson, M. Smedh, S. Engström, and M. B. Ericson, “Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy,” J. Control. Release 129(3), 163–169 (2008).
[CrossRef] [PubMed]

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

2004 (2)

G. Alexandrakis, E. B. Brown, R. T. Tong, T. D. McKee, R. B. Campbell, Y. Boucher, and R. K. Jain, “Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors,” Nat. Med. 10(2), 203–207 (2004).
[CrossRef] [PubMed]

K. K. F. Fischer, S. Puschmann, R. Wepf, I. Riemann, V. Ulrich, and P. Fischer, “Characterization of multiphoton laser scanning device optical parameters for image restoration,” Proc. SPIE 5463, 140–145 (2004).
[CrossRef]

2003 (3)

K. Koenig and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8(3), 432–439 (2003).
[CrossRef]

C. Y. Dong, K. Koenig, and P. So, “Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium,” J. Biomed. Opt. 8(3), 450–459 (2003).
[CrossRef] [PubMed]

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

2002 (1)

J. Kanitakis, “Anatomy, histology and immunohistochemistry of normal human skin,” Eur. J. Dermatol. 12(4), 390–399, quiz 400–401 (2002).
[PubMed]

2001 (2)

B. Yu, C. Y. Dong, P. T. C. So, D. Blankschtein, and R. Langer, “In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy,” J. Invest. Dermatol. 117(1), 16–25 (2001).
[CrossRef] [PubMed]

J. Hadgraft, “Skin, the final frontier,” Int. J. Pharm. 224(1-2), 1–18 (2001).
[CrossRef] [PubMed]

2000 (2)

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(2), 83–104 (2000).
[CrossRef] [PubMed]

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef]

1999 (1)

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

1998 (1)

B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation microscopy of in vivo human skin. Functional and morphological optical biopsy based on three-dimensional imaging, lifetime measurements and fluorescence spectroscopy,” Ann. N. Y. Acad. Sci. 838(1 ADVANCES IN O), 58–67 (1998).
[CrossRef] [PubMed]

1997 (1)

B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72(6), 2405–2412 (1997).
[CrossRef] [PubMed]

1987 (1)

W. J. Addicks, G. L. Flynn, and N. Weiner, “Validation of a flow-through diffusion cell for use in transdermal research,” Pharm. Res. 04(4), 337–341 (1987).
[CrossRef]

1981 (1)

R. R. Anderson and J. A. Parrish, “The optics of human skin,” J. Invest. Dermatol. 77(1), 13–19 (1981).
[CrossRef] [PubMed]

Addicks, W. J.

W. J. Addicks, G. L. Flynn, and N. Weiner, “Validation of a flow-through diffusion cell for use in transdermal research,” Pharm. Res. 04(4), 337–341 (1987).
[CrossRef]

Alexandrakis, G.

G. Alexandrakis, E. B. Brown, R. T. Tong, T. D. McKee, R. B. Campbell, Y. Boucher, and R. K. Jain, “Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors,” Nat. Med. 10(2), 203–207 (2004).
[CrossRef] [PubMed]

Anderson, R. R.

R. R. Anderson and J. A. Parrish, “The optics of human skin,” J. Invest. Dermatol. 77(1), 13–19 (1981).
[CrossRef] [PubMed]

Bender, J.

J. Bender, C. Simonsson, M. Smedh, S. Engström, and M. B. Ericson, “Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy,” J. Control. Release 129(3), 163–169 (2008).
[CrossRef] [PubMed]

Berland, K. M.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef]

Betzig, E.

D. E. M. Na Ji and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7, ••• (2010).

Blankschtein, D.

B. Yu, C. Y. Dong, P. T. C. So, D. Blankschtein, and R. Langer, “In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy,” J. Invest. Dermatol. 117(1), 16–25 (2001).
[CrossRef] [PubMed]

Boucher, Y.

G. Alexandrakis, E. B. Brown, R. T. Tong, T. D. McKee, R. B. Campbell, Y. Boucher, and R. K. Jain, “Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors,” Nat. Med. 10(2), 203–207 (2004).
[CrossRef] [PubMed]

Brown, E. B.

G. Alexandrakis, E. B. Brown, R. T. Tong, T. D. McKee, R. B. Campbell, Y. Boucher, and R. K. Jain, “Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors,” Nat. Med. 10(2), 203–207 (2004).
[CrossRef] [PubMed]

Campbell, R. B.

G. Alexandrakis, E. B. Brown, R. T. Tong, T. D. McKee, R. B. Campbell, Y. Boucher, and R. K. Jain, “Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors,” Nat. Med. 10(2), 203–207 (2004).
[CrossRef] [PubMed]

Dimitrow, E.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol. 129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

Dong, C. Y.

C. Y. Dong, K. Koenig, and P. So, “Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium,” J. Biomed. Opt. 8(3), 450–459 (2003).
[CrossRef] [PubMed]

B. Yu, C. Y. Dong, P. T. C. So, D. Blankschtein, and R. Langer, “In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy,” J. Invest. Dermatol. 117(1), 16–25 (2001).
[CrossRef] [PubMed]

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef]

Elsner, P.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol. 129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

Engström, S.

J. Bender, C. Simonsson, M. Smedh, S. Engström, and M. B. Ericson, “Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy,” J. Control. Release 129(3), 163–169 (2008).
[CrossRef] [PubMed]

Ericson, M. B.

K. Samuelsson, C. Simonsson, C. A. Jonsson, G. Westman, M. B. Ericson, and A. T. Karlberg, “Accumulation of FITC near stratum corneum-visualizing epidermal distribution of a strong sensitizer using two-photon microscopy,” Contact Dermat. 61(2), 91–100 (2009).
[CrossRef]

J. Bender, C. Simonsson, M. Smedh, S. Engström, and M. B. Ericson, “Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy,” J. Control. Release 129(3), 163–169 (2008).
[CrossRef] [PubMed]

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

Fischer, K. K. F.

K. K. F. Fischer, S. Puschmann, R. Wepf, I. Riemann, V. Ulrich, and P. Fischer, “Characterization of multiphoton laser scanning device optical parameters for image restoration,” Proc. SPIE 5463, 140–145 (2004).
[CrossRef]

Fischer, P.

K. K. F. Fischer, S. Puschmann, R. Wepf, I. Riemann, V. Ulrich, and P. Fischer, “Characterization of multiphoton laser scanning device optical parameters for image restoration,” Proc. SPIE 5463, 140–145 (2004).
[CrossRef]

Flynn, G. L.

W. J. Addicks, G. L. Flynn, and N. Weiner, “Validation of a flow-through diffusion cell for use in transdermal research,” Pharm. Res. 04(4), 337–341 (1987).
[CrossRef]

Gratton, E.

B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation microscopy of in vivo human skin. Functional and morphological optical biopsy based on three-dimensional imaging, lifetime measurements and fluorescence spectroscopy,” Ann. N. Y. Acad. Sci. 838(1 ADVANCES IN O), 58–67 (1998).
[CrossRef] [PubMed]

B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72(6), 2405–2412 (1997).
[CrossRef] [PubMed]

Hadgraft, J.

J. Hadgraft, “Skin, the final frontier,” Int. J. Pharm. 224(1-2), 1–18 (2001).
[CrossRef] [PubMed]

Haupts, U.

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

Jain, R. K.

G. Alexandrakis, E. B. Brown, R. T. Tong, T. D. McKee, R. B. Campbell, Y. Boucher, and R. K. Jain, “Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors,” Nat. Med. 10(2), 203–207 (2004).
[CrossRef] [PubMed]

Jonsson, C. A.

K. Samuelsson, C. Simonsson, C. A. Jonsson, G. Westman, M. B. Ericson, and A. T. Karlberg, “Accumulation of FITC near stratum corneum-visualizing epidermal distribution of a strong sensitizer using two-photon microscopy,” Contact Dermat. 61(2), 91–100 (2009).
[CrossRef]

Kaatz, M.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol. 129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

Kanitakis, J.

J. Kanitakis, “Anatomy, histology and immunohistochemistry of normal human skin,” Eur. J. Dermatol. 12(4), 390–399, quiz 400–401 (2002).
[PubMed]

Karlberg, A. T.

K. Samuelsson, C. Simonsson, C. A. Jonsson, G. Westman, M. B. Ericson, and A. T. Karlberg, “Accumulation of FITC near stratum corneum-visualizing epidermal distribution of a strong sensitizer using two-photon microscopy,” Contact Dermat. 61(2), 91–100 (2009).
[CrossRef]

Koehler, M. J.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol. 129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

Koenig, K.

C. Y. Dong, K. Koenig, and P. So, “Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium,” J. Biomed. Opt. 8(3), 450–459 (2003).
[CrossRef] [PubMed]

K. Koenig and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8(3), 432–439 (2003).
[CrossRef]

König, K.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol. 129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(2), 83–104 (2000).
[CrossRef] [PubMed]

Langer, R.

B. Yu, C. Y. Dong, P. T. C. So, D. Blankschtein, and R. Langer, “In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy,” J. Invest. Dermatol. 117(1), 16–25 (2001).
[CrossRef] [PubMed]

Maiti, S.

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

Masters, B. R.

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef]

B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation microscopy of in vivo human skin. Functional and morphological optical biopsy based on three-dimensional imaging, lifetime measurements and fluorescence spectroscopy,” Ann. N. Y. Acad. Sci. 838(1 ADVANCES IN O), 58–67 (1998).
[CrossRef] [PubMed]

B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72(6), 2405–2412 (1997).
[CrossRef] [PubMed]

McKee, T. D.

G. Alexandrakis, E. B. Brown, R. T. Tong, T. D. McKee, R. B. Campbell, Y. Boucher, and R. K. Jain, “Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors,” Nat. Med. 10(2), 203–207 (2004).
[CrossRef] [PubMed]

Na Ji, D. E. M.

D. E. M. Na Ji and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7, ••• (2010).

Norgauer, J.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol. 129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

Paoli, J.

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

Parrish, J. A.

R. R. Anderson and J. A. Parrish, “The optics of human skin,” J. Invest. Dermatol. 77(1), 13–19 (1981).
[CrossRef] [PubMed]

Puschmann, S.

K. K. F. Fischer, S. Puschmann, R. Wepf, I. Riemann, V. Ulrich, and P. Fischer, “Characterization of multiphoton laser scanning device optical parameters for image restoration,” Proc. SPIE 5463, 140–145 (2004).
[CrossRef]

Riemann, I.

K. K. F. Fischer, S. Puschmann, R. Wepf, I. Riemann, V. Ulrich, and P. Fischer, “Characterization of multiphoton laser scanning device optical parameters for image restoration,” Proc. SPIE 5463, 140–145 (2004).
[CrossRef]

K. Koenig and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8(3), 432–439 (2003).
[CrossRef]

Samuelsson, K.

K. Samuelsson, C. Simonsson, C. A. Jonsson, G. Westman, M. B. Ericson, and A. T. Karlberg, “Accumulation of FITC near stratum corneum-visualizing epidermal distribution of a strong sensitizer using two-photon microscopy,” Contact Dermat. 61(2), 91–100 (2009).
[CrossRef]

Schwille, P.

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

Simonsson, C.

K. Samuelsson, C. Simonsson, C. A. Jonsson, G. Westman, M. B. Ericson, and A. T. Karlberg, “Accumulation of FITC near stratum corneum-visualizing epidermal distribution of a strong sensitizer using two-photon microscopy,” Contact Dermat. 61(2), 91–100 (2009).
[CrossRef]

J. Bender, C. Simonsson, M. Smedh, S. Engström, and M. B. Ericson, “Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy,” J. Control. Release 129(3), 163–169 (2008).
[CrossRef] [PubMed]

Smedh, M.

J. Bender, C. Simonsson, M. Smedh, S. Engström, and M. B. Ericson, “Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy,” J. Control. Release 129(3), 163–169 (2008).
[CrossRef] [PubMed]

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

So, P.

C. Y. Dong, K. Koenig, and P. So, “Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium,” J. Biomed. Opt. 8(3), 450–459 (2003).
[CrossRef] [PubMed]

So, P. T. C.

B. Yu, C. Y. Dong, P. T. C. So, D. Blankschtein, and R. Langer, “In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy,” J. Invest. Dermatol. 117(1), 16–25 (2001).
[CrossRef] [PubMed]

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef]

B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation microscopy of in vivo human skin. Functional and morphological optical biopsy based on three-dimensional imaging, lifetime measurements and fluorescence spectroscopy,” Ann. N. Y. Acad. Sci. 838(1 ADVANCES IN O), 58–67 (1998).
[CrossRef] [PubMed]

B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72(6), 2405–2412 (1997).
[CrossRef] [PubMed]

Tong, R. T.

G. Alexandrakis, E. B. Brown, R. T. Tong, T. D. McKee, R. B. Campbell, Y. Boucher, and R. K. Jain, “Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors,” Nat. Med. 10(2), 203–207 (2004).
[CrossRef] [PubMed]

Ulrich, V.

K. K. F. Fischer, S. Puschmann, R. Wepf, I. Riemann, V. Ulrich, and P. Fischer, “Characterization of multiphoton laser scanning device optical parameters for image restoration,” Proc. SPIE 5463, 140–145 (2004).
[CrossRef]

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

Weiner, N.

W. J. Addicks, G. L. Flynn, and N. Weiner, “Validation of a flow-through diffusion cell for use in transdermal research,” Pharm. Res. 04(4), 337–341 (1987).
[CrossRef]

Wennberg, A. M.

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

Wepf, R.

K. K. F. Fischer, S. Puschmann, R. Wepf, I. Riemann, V. Ulrich, and P. Fischer, “Characterization of multiphoton laser scanning device optical parameters for image restoration,” Proc. SPIE 5463, 140–145 (2004).
[CrossRef]

Westman, G.

K. Samuelsson, C. Simonsson, C. A. Jonsson, G. Westman, M. B. Ericson, and A. T. Karlberg, “Accumulation of FITC near stratum corneum-visualizing epidermal distribution of a strong sensitizer using two-photon microscopy,” Contact Dermat. 61(2), 91–100 (2009).
[CrossRef]

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Yu, B.

B. Yu, C. Y. Dong, P. T. C. So, D. Blankschtein, and R. Langer, “In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy,” J. Invest. Dermatol. 117(1), 16–25 (2001).
[CrossRef] [PubMed]

Ziemer, M.

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol. 129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Ann. N. Y. Acad. Sci. (1)

B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation microscopy of in vivo human skin. Functional and morphological optical biopsy based on three-dimensional imaging, lifetime measurements and fluorescence spectroscopy,” Ann. N. Y. Acad. Sci. 838(1 ADVANCES IN O), 58–67 (1998).
[CrossRef] [PubMed]

Annu. Rev. Biomed. Eng. (1)

P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, “Two-photon excitation fluorescence microscopy,” Annu. Rev. Biomed. Eng. 2(1), 399–429 (2000).
[CrossRef]

Biophys. J. (2)

B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J. 72(6), 2405–2412 (1997).
[CrossRef] [PubMed]

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

Contact Dermat. (1)

K. Samuelsson, C. Simonsson, C. A. Jonsson, G. Westman, M. B. Ericson, and A. T. Karlberg, “Accumulation of FITC near stratum corneum-visualizing epidermal distribution of a strong sensitizer using two-photon microscopy,” Contact Dermat. 61(2), 91–100 (2009).
[CrossRef]

Eur. J. Dermatol. (1)

J. Kanitakis, “Anatomy, histology and immunohistochemistry of normal human skin,” Eur. J. Dermatol. 12(4), 390–399, quiz 400–401 (2002).
[PubMed]

Int. J. Pharm. (1)

J. Hadgraft, “Skin, the final frontier,” Int. J. Pharm. 224(1-2), 1–18 (2001).
[CrossRef] [PubMed]

J. Biomed. Opt. (2)

K. Koenig and I. Riemann, “High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution,” J. Biomed. Opt. 8(3), 432–439 (2003).
[CrossRef]

C. Y. Dong, K. Koenig, and P. So, “Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium,” J. Biomed. Opt. 8(3), 450–459 (2003).
[CrossRef] [PubMed]

J. Control. Release (1)

J. Bender, C. Simonsson, M. Smedh, S. Engström, and M. B. Ericson, “Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy,” J. Control. Release 129(3), 163–169 (2008).
[CrossRef] [PubMed]

J. Invest. Dermatol. (4)

J. Paoli, M. Smedh, A. M. Wennberg, and M. B. Ericson, “Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics,” J. Invest. Dermatol. 128(5), 1248–1255 (2008).
[CrossRef]

E. Dimitrow, M. Ziemer, M. J. Koehler, J. Norgauer, K. König, P. Elsner, and M. Kaatz, “Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma,” J. Invest. Dermatol. 129(7), 1752–1758 (2009).
[CrossRef] [PubMed]

R. R. Anderson and J. A. Parrish, “The optics of human skin,” J. Invest. Dermatol. 77(1), 13–19 (1981).
[CrossRef] [PubMed]

B. Yu, C. Y. Dong, P. T. C. So, D. Blankschtein, and R. Langer, “In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy,” J. Invest. Dermatol. 117(1), 16–25 (2001).
[CrossRef] [PubMed]

J. Microsc. (1)

K. König, “Multiphoton microscopy in life sciences,” J. Microsc. 200(2), 83–104 (2000).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Nat. Med. (1)

G. Alexandrakis, E. B. Brown, R. T. Tong, T. D. McKee, R. B. Campbell, Y. Boucher, and R. K. Jain, “Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumors,” Nat. Med. 10(2), 203–207 (2004).
[CrossRef] [PubMed]

Nat. Methods (1)

D. E. M. Na Ji and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods 7, ••• (2010).

Pharm. Res. (1)

W. J. Addicks, G. L. Flynn, and N. Weiner, “Validation of a flow-through diffusion cell for use in transdermal research,” Pharm. Res. 04(4), 337–341 (1987).
[CrossRef]

Proc. SPIE (1)

K. K. F. Fischer, S. Puschmann, R. Wepf, I. Riemann, V. Ulrich, and P. Fischer, “Characterization of multiphoton laser scanning device optical parameters for image restoration,” Proc. SPIE 5463, 140–145 (2004).
[CrossRef]

Other (2)

E. H. P.Schwille, “Fluorescence Correlation Spectroscopy, An introduction to its Concepts and Applications.”

S. Guldbrand, C. Simonsson, M. Smedh, and M. B. Ericson, “Point spread function measured in human skin using two-photon fluorescence microscopy,” Proc. SPIE 7367 (2009).

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Figures (8)

Fig. 1
Fig. 1

Schematic drawing of experimental set up for TPFCS. The insert shows the beam properties of the Ti:Sapphire laser.

Fig. 2
Fig. 2

Simulated Gaussian function with the parameters µ, σ, FWHM. The exp(−1) width is noted as ω and the exp(−2) width is marked as r0 or z0 depending on direction.

Fig. 3
Fig. 3

TPLSM image acquired by a Bio-Rad Radiance 2100 MP microscope using a 40x/0.8 W objective. The green signal corresponds to skin autofluorescence and the red signal to the fluorescent beads. Image size is 126 x 126 μm obtained at a depth of 20 µm. A close up of beads are shown in the insert (size 31.5 x 31.5 µm)

Fig. 4
Fig. 4

Left: Fluorescence images obtained using TPLSM on a fluorescent bead located at a depth of 20 µm in a tape stripped sample of human skin, viewed in lateral (top) and axial (bottom) directions. Right: The corresponding intensity profiles, including the Gaussian fit and the obtained value of FWHM.

Fig. 5
Fig. 5

Measurements of PSF performed on human skin samples. Mean values of FWHM as a function of tissue depth in a) lateral direction, and. b) axial direction. c) The size of the excitation volume as function of depth.

Fig. 6
Fig. 6

TPLSM image of tape-stripped skin exposed to SRB (a). The arrows show the position of the laser, in a cell and outside a cell, during two TPFCS measurements. A selection of four fluorescence intensity curves is shown; two examples from saved runs (b and e) which contributed to give the final correlation curves (d and g) and two examples from rejected runs, (c and f). The values of N and τD are obtained from the FCS fit procedure. The number of molecules is approximately twice as many for (g) than for (d) which agrees with the intensity distribution in the image. The diffusion time is approximately twice as fast for (d) than for (g).

Fig. 7
Fig. 7

The diffusion coefficient calculated from the TPFCS measurements on human skin samples exposed to SRB performed at different tissue depths.

Fig. 8
Fig. 8

The number of molecules detected in the excitation volume based on TPFCS measurements on a skin sample exposed to SRB.

Tables (1)

Tables Icon

Table 1 Mean values of the diffusion time obtained from different fit procedures

Equations (11)

Equations on this page are rendered with MathJax. Learn more.

I = I 0 exp [ ( x μ ) 2 2 σ 2 ]
F W H M = 2 2 ln 2 σ
ω = F W H M 2 ln 2
V T P E = 1.47 π 3 2 ω x y 2 ω z
ω x y = 0.325 λ 2 N A 0.91
ω z = 0.532 λ 2 [ 1 n n 2 N A 2 ]
G ( τ ) = F ( t ) F ( t + τ ) F ( t ) 2
G ( τ ) = 1 N 1 1 + τ τ D 1 1 + ( r 0 z 0 ) 2 τ τ D
r 0 = F W H M x y 2 ln 2
z 0 = F W H M z 2 ln 2
D = r 0 2 8 τ D

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