Abstract

Large-area multiphoton laser scanning microscopy (LMLSM) can be applied in biology and medicine for high sensitivity and resolution tissue imaging. However, factors such as refractive index mismatch induced spherical aberration, emission/excitation absorption and scattering can result in axial intensity attenuation and lateral image heterogeneity, affecting both qualitative and quantitative image analysis. In this work, we describe an image correction algorithm to improve three-dimensional images in LMLSM. The method consists of multiplying the measured nonlinear signal by a three-dimensional correction factor, determined by the use of two-photon images of the appropriate specimens and specimen absorption and scattering properties at the excitation and emission wavelengths. The proposed methodology is demonstrated in correcting multiphoton images of objects imbedded in uniform fluorescent background, lung tissue, and Drosophila larva.

© 2008 Optical Society of America

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2007

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

D. S. dos Santos Jr. and R. F. Aroca, "Selective surface-enhanced fluorescence and dye aggregation with layer-by-layer film substrates," Analyst 132, 450-454 (2007).
[CrossRef]

2006

S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, "Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging," Opt. Lett. 31, 2756-2758 (2006).
[CrossRef] [PubMed]

H. S. Lee, S. W. Teng, H. C. Chen, W. Lo, Y. Sun, T. Y. Lin, L. L. Chiou, C. C. Jiang, and C. Y. Dong, "Imaging human bone marrow stem cell morphogenesis in polyglycolic acid scaffold by multiphoton microscopy," Tissue Eng. 12, 2835-2841 (2006).
[CrossRef]

M. Rueckel, J. A. Mack-Bucher, and W. Denk "Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing," Proc. Nat. Acad. Sci. U.S.A. 103, 17137-17142 (2006).
[CrossRef]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

W. Lo, S. W. Teng, H. Y. Tan, K. H. Kim, H. C. Chen, H. S. Lee, Y. F. Chen, P. T. C. So, and C. Y. Dong, "Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging," Microsc. Res. Tech. 69, 973-975 (2006).
[CrossRef] [PubMed]

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

S. C. Lee and P. Bajcsy, "Intensity correction of fluorescent confocal laser scanning microscope images by mean-weight filtering," J. Microsc. 221, 122-136 (2006).
[CrossRef] [PubMed]

2005

W. Lo, Y. Sun, S. J. Lin, S. H. Jee, and C. Y. Dong, "Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar," J. Biomed. Opt. 10, 034006 (2005).
[CrossRef] [PubMed]

2004

C. Kervrann, D. Legland, and L. Pardini, "Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy," J. Microsc. 214, 297-314 (2004).
[CrossRef] [PubMed]

M. Schwertner, M. J. Booth, and T. Wilson, "Characterizing specimen induced aberrations for high NA adaptive optical microscopy," Opt. Express 12, 6540-6552 (2004).
[CrossRef] [PubMed]

C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
[CrossRef]

2003

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

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

A. Can, O. Al-Kofahi, S. Lasek, D. H. Szaworski, J. N. Turner, and B. Roysam, "Attenuation correction in confocal laser microscopes:a novel two-view approach," J. Microsc. 211, 67-79 (2003).
[CrossRef] [PubMed]

2002

B. Yu, K. H. Kim, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: Determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).
[CrossRef] [PubMed]

2001

J. Markham and J. Conchello, "Artifacts in restored images due to intensity loss in 3D fluorescence microscopy," J. Microsc. 204, 93-98 (2001).
[CrossRef] [PubMed]

2000

1999

1993

J. Roerdink and M. Bakker, "An FFT-based method for attenuation correction in fluorescence confocal microscopy," J. Microsc. 169, 3-14 (1993).
[CrossRef]

1991

T. Visser, F. Groen, and G. Brakenhoff, "Absorption and scattering correction in fluorescence confocal microscopy," J. Microsc. 163, 189-200 (1991).
[CrossRef]

Al-Kofahi, O.

A. Can, O. Al-Kofahi, S. Lasek, D. H. Szaworski, J. N. Turner, and B. Roysam, "Attenuation correction in confocal laser microscopes:a novel two-view approach," J. Microsc. 211, 67-79 (2003).
[CrossRef] [PubMed]

Aroca, R. F.

D. S. dos Santos Jr. and R. F. Aroca, "Selective surface-enhanced fluorescence and dye aggregation with layer-by-layer film substrates," Analyst 132, 450-454 (2007).
[CrossRef]

Bahlmann, K.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

Bajcsy, P.

S. C. Lee and P. Bajcsy, "Intensity correction of fluorescent confocal laser scanning microscope images by mean-weight filtering," J. Microsc. 221, 122-136 (2006).
[CrossRef] [PubMed]

Bakker, M.

J. Roerdink and M. Bakker, "An FFT-based method for attenuation correction in fluorescence confocal microscopy," J. Microsc. 169, 3-14 (1993).
[CrossRef]

Berns, M. W.

Blankschtein, D.

B. Yu, K. H. Kim, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: Determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).
[CrossRef] [PubMed]

Booth, M. J.

Brakenhoff, G.

T. Visser, F. Groen, and G. Brakenhoff, "Absorption and scattering correction in fluorescence confocal microscopy," J. Microsc. 163, 189-200 (1991).
[CrossRef]

Can, A.

A. Can, O. Al-Kofahi, S. Lasek, D. H. Szaworski, J. N. Turner, and B. Roysam, "Attenuation correction in confocal laser microscopes:a novel two-view approach," J. Microsc. 211, 67-79 (2003).
[CrossRef] [PubMed]

Chen, H. C.

H. S. Lee, S. W. Teng, H. C. Chen, W. Lo, Y. Sun, T. Y. Lin, L. L. Chiou, C. C. Jiang, and C. Y. Dong, "Imaging human bone marrow stem cell morphogenesis in polyglycolic acid scaffold by multiphoton microscopy," Tissue Eng. 12, 2835-2841 (2006).
[CrossRef]

W. Lo, S. W. Teng, H. Y. Tan, K. H. Kim, H. C. Chen, H. S. Lee, Y. F. Chen, P. T. C. So, and C. Y. Dong, "Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging," Microsc. Res. Tech. 69, 973-975 (2006).
[CrossRef] [PubMed]

Chen, J. S.

Chen, Y. F.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, "Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging," Opt. Lett. 31, 2756-2758 (2006).
[CrossRef] [PubMed]

W. Lo, S. W. Teng, H. Y. Tan, K. H. Kim, H. C. Chen, H. S. Lee, Y. F. Chen, P. T. C. So, and C. Y. Dong, "Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging," Microsc. Res. Tech. 69, 973-975 (2006).
[CrossRef] [PubMed]

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

Chiou, L. L.

H. S. Lee, S. W. Teng, H. C. Chen, W. Lo, Y. Sun, T. Y. Lin, L. L. Chiou, C. C. Jiang, and C. Y. Dong, "Imaging human bone marrow stem cell morphogenesis in polyglycolic acid scaffold by multiphoton microscopy," Tissue Eng. 12, 2835-2841 (2006).
[CrossRef]

Coleno, M.

Conchello, J.

J. Markham and J. Conchello, "Artifacts in restored images due to intensity loss in 3D fluorescence microscopy," J. Microsc. 204, 93-98 (2001).
[CrossRef] [PubMed]

de Grauw, C. J.

H. C. Gerritsen and C. J. de Grauw, "Imaging of optically thick specimen using two-photon excitation microscopy," Microsc. Res. Tech. 47, 206-209 (1999).
[CrossRef] [PubMed]

de Grauw, G. J.

Denk, W.

M. Rueckel, J. A. Mack-Bucher, and W. Denk "Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing," Proc. Nat. Acad. Sci. U.S.A. 103, 17137-17142 (2006).
[CrossRef]

Dong, C. Y.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, "Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging," Opt. Lett. 31, 2756-2758 (2006).
[CrossRef] [PubMed]

H. S. Lee, S. W. Teng, H. C. Chen, W. Lo, Y. Sun, T. Y. Lin, L. L. Chiou, C. C. Jiang, and C. Y. Dong, "Imaging human bone marrow stem cell morphogenesis in polyglycolic acid scaffold by multiphoton microscopy," Tissue Eng. 12, 2835-2841 (2006).
[CrossRef]

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

W. Lo, S. W. Teng, H. Y. Tan, K. H. Kim, H. C. Chen, H. S. Lee, Y. F. Chen, P. T. C. So, and C. Y. Dong, "Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging," Microsc. Res. Tech. 69, 973-975 (2006).
[CrossRef] [PubMed]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

W. Lo, Y. Sun, S. J. Lin, S. H. Jee, and C. Y. Dong, "Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar," J. Biomed. Opt. 10, 034006 (2005).
[CrossRef] [PubMed]

C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
[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, 450-459 (2003).
[CrossRef] [PubMed]

dos Santos, D. S.

D. S. dos Santos Jr. and R. F. Aroca, "Selective surface-enhanced fluorescence and dye aggregation with layer-by-layer film substrates," Analyst 132, 450-454 (2007).
[CrossRef]

Dunn, A. K.

Gerritsen, H. C.

Groen, F.

T. Visser, F. Groen, and G. Brakenhoff, "Absorption and scattering correction in fluorescence confocal microscopy," J. Microsc. 163, 189-200 (1991).
[CrossRef]

Hsiao, C. H.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

Hsu, C. J.

Huang, H.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

Huang, S. C. M.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

Jee, S. H.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, "Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging," Opt. Lett. 31, 2756-2758 (2006).
[CrossRef] [PubMed]

W. Lo, Y. Sun, S. J. Lin, S. H. Jee, and C. Y. Dong, "Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar," J. Biomed. Opt. 10, 034006 (2005).
[CrossRef] [PubMed]

Jiang, C. C.

H. S. Lee, S. W. Teng, H. C. Chen, W. Lo, Y. Sun, T. Y. Lin, L. L. Chiou, C. C. Jiang, and C. Y. Dong, "Imaging human bone marrow stem cell morphogenesis in polyglycolic acid scaffold by multiphoton microscopy," Tissue Eng. 12, 2835-2841 (2006).
[CrossRef]

Kaplan, P. D.

C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
[CrossRef]

Kervrann, C.

C. Kervrann, D. Legland, and L. Pardini, "Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy," J. Microsc. 214, 297-314 (2004).
[CrossRef] [PubMed]

Kim, K. H.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

W. Lo, S. W. Teng, H. Y. Tan, K. H. Kim, H. C. Chen, H. S. Lee, Y. F. Chen, P. T. C. So, and C. Y. Dong, "Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging," Microsc. Res. Tech. 69, 973-975 (2006).
[CrossRef] [PubMed]

B. Yu, K. H. Kim, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: Determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).
[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, 450-459 (2003).
[CrossRef] [PubMed]

Kuo, C. J.

Langer, R.

B. Yu, K. H. Kim, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: Determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).
[CrossRef] [PubMed]

Lasek, S.

A. Can, O. Al-Kofahi, S. Lasek, D. H. Szaworski, J. N. Turner, and B. Roysam, "Attenuation correction in confocal laser microscopes:a novel two-view approach," J. Microsc. 211, 67-79 (2003).
[CrossRef] [PubMed]

Lee, H. S.

H. S. Lee, S. W. Teng, H. C. Chen, W. Lo, Y. Sun, T. Y. Lin, L. L. Chiou, C. C. Jiang, and C. Y. Dong, "Imaging human bone marrow stem cell morphogenesis in polyglycolic acid scaffold by multiphoton microscopy," Tissue Eng. 12, 2835-2841 (2006).
[CrossRef]

W. Lo, S. W. Teng, H. Y. Tan, K. H. Kim, H. C. Chen, H. S. Lee, Y. F. Chen, P. T. C. So, and C. Y. Dong, "Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging," Microsc. Res. Tech. 69, 973-975 (2006).
[CrossRef] [PubMed]

Lee, R. T.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

Lee, S. C.

S. C. Lee and P. Bajcsy, "Intensity correction of fluorescent confocal laser scanning microscope images by mean-weight filtering," J. Microsc. 221, 122-136 (2006).
[CrossRef] [PubMed]

Legland, D.

C. Kervrann, D. Legland, and L. Pardini, "Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy," J. Microsc. 214, 297-314 (2004).
[CrossRef] [PubMed]

Liao, Y. H.

Lin, H. C.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

Lin, H. H.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

Lin, S. J.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, "Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging," Opt. Lett. 31, 2756-2758 (2006).
[CrossRef] [PubMed]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

W. Lo, Y. Sun, S. J. Lin, S. H. Jee, and C. Y. Dong, "Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar," J. Biomed. Opt. 10, 034006 (2005).
[CrossRef] [PubMed]

Lin, T. Y.

H. S. Lee, S. W. Teng, H. C. Chen, W. Lo, Y. Sun, T. Y. Lin, L. L. Chiou, C. C. Jiang, and C. Y. Dong, "Imaging human bone marrow stem cell morphogenesis in polyglycolic acid scaffold by multiphoton microscopy," Tissue Eng. 12, 2835-2841 (2006).
[CrossRef]

Lin, W. C.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, "Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging," Opt. Lett. 31, 2756-2758 (2006).
[CrossRef] [PubMed]

Lo, W.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

W. Lo, S. W. Teng, H. Y. Tan, K. H. Kim, H. C. Chen, H. S. Lee, Y. F. Chen, P. T. C. So, and C. Y. Dong, "Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging," Microsc. Res. Tech. 69, 973-975 (2006).
[CrossRef] [PubMed]

H. S. Lee, S. W. Teng, H. C. Chen, W. Lo, Y. Sun, T. Y. Lin, L. L. Chiou, C. C. Jiang, and C. Y. Dong, "Imaging human bone marrow stem cell morphogenesis in polyglycolic acid scaffold by multiphoton microscopy," Tissue Eng. 12, 2835-2841 (2006).
[CrossRef]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

W. Lo, Y. Sun, S. J. Lin, S. H. Jee, and C. Y. Dong, "Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar," J. Biomed. Opt. 10, 034006 (2005).
[CrossRef] [PubMed]

Ma, D. H. K.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

Mack-Bucher, J. A.

M. Rueckel, J. A. Mack-Bucher, and W. Denk "Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing," Proc. Nat. Acad. Sci. U.S.A. 103, 17137-17142 (2006).
[CrossRef]

Markham, J.

J. Markham and J. Conchello, "Artifacts in restored images due to intensity loss in 3D fluorescence microscopy," J. Microsc. 204, 93-98 (2001).
[CrossRef] [PubMed]

Pardini, L.

C. Kervrann, D. Legland, and L. Pardini, "Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy," J. Microsc. 214, 297-314 (2004).
[CrossRef] [PubMed]

Peng, J. L.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

Ragan, T.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

Roerdink, J.

J. Roerdink and M. Bakker, "An FFT-based method for attenuation correction in fluorescence confocal microscopy," J. Microsc. 169, 3-14 (1993).
[CrossRef]

Roysam, B.

A. Can, O. Al-Kofahi, S. Lasek, D. H. Szaworski, J. N. Turner, and B. Roysam, "Attenuation correction in confocal laser microscopes:a novel two-view approach," J. Microsc. 211, 67-79 (2003).
[CrossRef] [PubMed]

Rueckel, M.

M. Rueckel, J. A. Mack-Bucher, and W. Denk "Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing," Proc. Nat. Acad. Sci. U.S.A. 103, 17137-17142 (2006).
[CrossRef]

Schwertner, M.

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, 450-459 (2003).
[CrossRef] [PubMed]

So, P. T. C.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

W. Lo, S. W. Teng, H. Y. Tan, K. H. Kim, H. C. Chen, H. S. Lee, Y. F. Chen, P. T. C. So, and C. Y. Dong, "Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging," Microsc. Res. Tech. 69, 973-975 (2006).
[CrossRef] [PubMed]

C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
[CrossRef]

B. Yu, K. H. Kim, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: Determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).
[CrossRef] [PubMed]

Sun, Y.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

H. S. Lee, S. W. Teng, H. C. Chen, W. Lo, Y. Sun, T. Y. Lin, L. L. Chiou, C. C. Jiang, and C. Y. Dong, "Imaging human bone marrow stem cell morphogenesis in polyglycolic acid scaffold by multiphoton microscopy," Tissue Eng. 12, 2835-2841 (2006).
[CrossRef]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

W. Lo, Y. Sun, S. J. Lin, S. H. Jee, and C. Y. Dong, "Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar," J. Biomed. Opt. 10, 034006 (2005).
[CrossRef] [PubMed]

Sylvan, J. D.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

Szaworski, D. H.

A. Can, O. Al-Kofahi, S. Lasek, D. H. Szaworski, J. N. Turner, and B. Roysam, "Attenuation correction in confocal laser microscopes:a novel two-view approach," J. Microsc. 211, 67-79 (2003).
[CrossRef] [PubMed]

Tan, H. Y.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

W. Lo, S. W. Teng, H. Y. Tan, K. H. Kim, H. C. Chen, H. S. Lee, Y. F. Chen, P. T. C. So, and C. Y. Dong, "Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging," Microsc. Res. Tech. 69, 973-975 (2006).
[CrossRef] [PubMed]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

Teng, S. W.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

W. Lo, S. W. Teng, H. Y. Tan, K. H. Kim, H. C. Chen, H. S. Lee, Y. F. Chen, P. T. C. So, and C. Y. Dong, "Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging," Microsc. Res. Tech. 69, 973-975 (2006).
[CrossRef] [PubMed]

H. S. Lee, S. W. Teng, H. C. Chen, W. Lo, Y. Sun, T. Y. Lin, L. L. Chiou, C. C. Jiang, and C. Y. Dong, "Imaging human bone marrow stem cell morphogenesis in polyglycolic acid scaffold by multiphoton microscopy," Tissue Eng. 12, 2835-2841 (2006).
[CrossRef]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

Tromberg, B. J.

Tsai, T. F.

Turner, J. N.

A. Can, O. Al-Kofahi, S. Lasek, D. H. Szaworski, J. N. Turner, and B. Roysam, "Attenuation correction in confocal laser microscopes:a novel two-view approach," J. Microsc. 211, 67-79 (2003).
[CrossRef] [PubMed]

van der Voort, H. T. M.

Visser, T.

T. Visser, F. Groen, and G. Brakenhoff, "Absorption and scattering correction in fluorescence confocal microscopy," J. Microsc. 163, 189-200 (1991).
[CrossRef]

Vroom, J. M.

Wallace, V. P.

Webb, W. W.

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

Williams, R. M.

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

Wilson, T.

Wu, R. J.

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

S. J. Lin, S. H. Jee, C. J. Kuo, R. J. Wu, W. C. Lin, J. S. Chen, Y. H. Liao, C. J. Hsu, T. F. Tsai, Y. F. Chen, and C. Y. Dong, "Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging," Opt. Lett. 31, 2756-2758 (2006).
[CrossRef] [PubMed]

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C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
[CrossRef]

B. Yu, K. H. Kim, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: Determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).
[CrossRef] [PubMed]

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H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

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

Analyst

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[CrossRef]

Appl. Opt.

Invest. Ophth. Vis. Sci.

H. Y. Tan, Y. Sun, W. Lo, S. J. Lin, C. H. Hsiao, Y. F. Chen, S. C. M. Huang, W. C. Lin, S. H. Jee, H. S. Yu, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo," Invest. Ophth. Vis. Sci. 47, 5251-5259 (2006).
[CrossRef]

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophth. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef]

J. Biomed. Opt.

T. Ragan, J. D. Sylvan, K. H. Kim, H. Huang, K. Bahlmann, R. T. Lee, and P. T. C. So, "High-resolution whole organ imaging using two-photon tissue cytometry," J. Biomed. Opt. 12, 014015 (2007).
[CrossRef] [PubMed]

H. Y. Tan, Y. Sun, W. Lo, S. W. Teng, R. J. Wu, S. H. Jee, W. C. Lin, C. H. Hsiao, H. C. Lin, Y. F. Chen, D. H. K. Ma, S. C. M. Huang, S. J. Lin, and C. Y. Dong, "Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis," J. Biomed. Opt. 12, 024013 (2007).
[CrossRef] [PubMed]

W. Lo, Y. Sun, S. J. Lin, S. H. Jee, and C. Y. Dong, "Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar," J. Biomed. Opt. 10, 034006 (2005).
[CrossRef] [PubMed]

C. Y. Dong, K. Koenig, and P. So, "Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium," J. Biomed. Opt. 8, 450-459 (2003).
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B. Yu, K. H. Kim, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: Determination of representative skin sample sizes," J. Invest. Dermatol. 118, 1085-1088 (2002).
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Microsc. Res. Tech.

C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
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[CrossRef] [PubMed]

Nat. Biotechnol.

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
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Figures (9)

Fig. 1.
Fig. 1.

A 3×3 TPF image array of the uniform 10 µM SRB sample and axial intensity profiles at the point of intensity maximum (Curve 1) and at image edges (Curves 2-5) acquired using the Plan-Apochromat 63×/NA 1.4 Oil DIC objective. For comparison, the axial intensity profile of the C-Apochromat 40×/NA 1.2W Corr water lens acquired at the image field intensity maximum is shown (Curve 6).

Fig. 2.
Fig. 2.

Depth-dependent lateral intensity profiles along the x (1-3) and y (1′-3′) Region Curve 1 in Fig. 1 (Objective: Plan-Apochromat 63×/NA 1.4 Oil DIC).

Fig. 3.
Fig. 3.

Spectral dependence of optical density. Curve 1: Sample II - aqueous 0.28 mM SRB solution, Curve 2: Sample III- Intrafat-water 20 µM SRB solution. Specimen thickness is 0.49 mm.

Fig. 4.
Fig. 4.

Measured TPF axial intensity profiles for 10µM (Curve 1) and 0.28 mM (Curve 2) SRB samples; calculated (Curve 3) and corrected (Curve 4) TPF intensity for the 0.28 mM SRB sample. Objective- Fluar 40×/NA 1.3 Oil.

Fig. 5.
Fig. 5.

Measured (Curves 1, 4), calculated (Curves 2, 5), and corrected (Curves 3, 6) TPF intensity axial profiles in 0.28 mM aqueous (Curves 1, 2, 3) and 20 µM Intrafat-water emulsion specimen (Curves 4, 5, 6) SRB solutions. Objective: Plan-Apochromat 63×/NA 1.4 Oil DIC.

Fig. 6.
Fig. 6.

(A) Uniform SRB sample TPF intensity X and Y profiles and fitting results using the two-parabola model. (B) x-y image of lateral correction factor, obtained from (A). (Fluar 40×/NA 1.3 Oil objective).

Fig. 7.
Fig. 7.

TPF image array (5×5) and surface plot of the SRB sample before (left) and after (right) lateral intensity correction. Objective- Fluar 40×/NA 1.3 Oil.

Fig. 8.
Fig. 8.

Human lung tissue 7×6 tiles (1.61×1.38 mm) image before (A) and after (B) intensity correction. Scanning depth: 30 µm below the specimen surface. Green - TPE, red - SHG.

Fig. 9.
Fig. 9.

8×4 image array of a Stage 3 Drosophila larva, before (A) and after (B) intensity correction. Scanning depth: 14 µm below the skin surface. Green - TPE, red - SHG.

Tables (1)

Tables Icon

Table 1. Mean value (I m) and standard deviation (StD) in different regions of SRB sample (Fig. 7).

Equations (10)

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

I ( z , λ ) = I 0 exp ( [ ( α exc + α em ) z ] .
I ̂ ( x ¯ ) = C ( x ¯ ) I ( x ¯ ) ,
C ( x ¯ ) = [ ( γ exc ( x ¯ ) γ em ( x ¯ ) ] 1
γ sph ( z ) = exp ( α sph z ) .
γ exc ( x ) = 1 π sin 2 ω 0 ω 0 2 π cos θ sin θ exp ( 0 z α exc ( x ) cos θ dz ) d φ d θ ;
γ em ( x ¯ ) = 1 2 π ( 1 cos ω ) 0 ω 0 2 π sin θ exp ( 0 z α em ( x ) cos θ dz ) d φ d θ ,
I ( z , λ ) = I o 2 exp [ ( 2 α exc + α em ) z ]
C ( x ¯ ) = [ ( γ exc ( x ¯ ) γ em ( x ¯ ) P sph ( z ) L lat ( x ¯ ) ] 1 ·
L lat ( x ¯ )= L x (z) L y (z),
L i ( z ) = C i ( z ) ( ( A i ( z ) i B i ( z ) ) 2 ,

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