Research Article| Volume 65, ISSUE 3, P189-195, March 2012

Spatial and temporal analysis of skin glycation by the use of multiphoton microscopy and spectroscopy

  • Author Footnotes
    1 These authors contributed equally to this work.
    Ara A. Ghazaryan
    1 These authors contributed equally to this work.
    Department of Physics, National Taiwan University, Taipei 106, Taiwan
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Po-Sheng Hu
    1 These authors contributed equally to this work.
    Department of Physics, National Taiwan University, Taipei 106, Taiwan
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  • Shean-Jen Chen
    Department of Engineering Science, National Cheng Kung University Medical College, Taipei, Taiwan
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  • Hsin-Yuan Tan
    Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan

    Department of Ophthalmology, Chang Gung Memorial Hospital, Linko, Taiwan
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  • Chen-Yuan Dong
    Corresponding author at: Department of Physics, National Taiwan University, Office: Room 530, Taipei 106, Taiwan. Tel.: +886 975568324; fax: +886 975568324.
    Department of Physics, National Taiwan University, Taipei 106, Taiwan

    Center for Quantum Science and Engineering, National Taiwan University, Taipei 106, Taiwan

    Biomedical Molecular Imaging Core, Division of Genomic Medicine, Research Center for Medical Excellence, National Taiwan University, Taipei, Taiwan
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  • Author Footnotes
    1 These authors contributed equally to this work.



      Tissue glycation, the main cause of many diabetes-related complications, results in the accumulation of advanced glycation endproducts (AGE).


      These AGEs are endogenous fluorophores that can serve as a viable pathological indicator for disease diagnostics. Here we explore the capabilities of multiphoton microscopy to non-invasively localize and quantify the skin glycation.


      In our study, multiphoton microscopy and spectroscopy were used to investigate glycation events-induced changes in the intensities of autofluorescence and second harmonic generation on ex vivo human skin.


      Temporal and spatial dependence of degrees of glycation of the epidermis, collagen and elastin fibers of dermis were evaluated for their relevance to the changes in amplitudes of autofluorescence signals. We found that glycation drastically and linearly increases multiphoton autofluorescence intensity of epidermis and dermal collagen whereas changes in dermal elastin are moderate. We also found decrease in the level of second harmonic generation signal.


      Our study suggests that due to intrinsically weak autofluorescence the dermal collagen is the most sensitive skin tissue to be used for detecting changes in tissue glycation.


      AGE (advanced glycation endproducts), MPAF (multiphoton autofluorescence), SHG (second harmonic generation)


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        • Lee W.K.
        • Akyol M.
        • Shaw S.
        • Dominiczak M.H.
        • Briggs J.D.
        Kidney-transplantation decreases the tissue-level of advanced glycosylation end-products.
        Nephrol Dial Transpl. 1995; 10: 103-107
        • DeGroot J.
        • Verzijl N.
        • Jacobs K.M.G.
        • Budde M.
        • Bank R.A.
        • Bijlsma J.W.J.
        • et al.
        Accumulation of advanced glycation endproducts reduces chondrocyte-mediated extracellular matrix turnover in human articular cartilage.
        Osteoarthr Cartil. 2001; 9: 720-726
        • Smit A.J.
        • Gerrits E.G.
        Skin autofluorescence as a measure of advanced glycation endproduct deposition: a novel risk marker in chronic kidney disease.
        Curr Opin Nephrol Hy. 2010; 19: 527-533
        • Ulrich P.
        • Cerami A.
        Protein glycation, diabetes, and aging.
        Recent Progr Hormone Res. 2001; 56: 1-21
        • Nienhuis H.L.A.
        • Westra J.
        • Smit A.J.
        • Limburg P.C.
        • Kallenberg C.G.M.
        • Bijl M.
        AGE and their receptor RAGE in systemic autoimmune diseases: an inflammation propagating factor contributing to accelerated atherosclerosis.
        Autoimmunity. 2009; 42: 302-304
        • Thomas M.C.
        • Forbes J.M.
        • Cooper M.E.
        Advanced glycation end products and diabetic nephropathy.
        Am J Ther. 2005; 12: 562-572
        • Gerrits E.G.
        • Lutgers H.L.
        • Kleefstra N.
        • Graaff R.
        • Groenier K.H.
        • Smit A.J.
        • et al.
        Skin autofluorescence: a tool to identify type 2 diabetic patients at risk for developing microvascular complications.
        Diabetes Care. 2008; 31: 517-521
        • Brownlee M.
        Biochemistry and molecular cell biology of diabetic complications.
        Nature. 2001; 414: 813-820
        • Ramasamy R.
        • Vannucci S.J.
        • Yan S.S.D.
        • Herold K.
        • Yan S.F.
        • Schmidt A.M.
        Advanced glycation end products and RAGE: a common thread in aging, diabetes, neurodegeneration, and inflammation.
        Glycobiology. 2005; 15: 16r-28r
        • Peppa M.
        Glucose, advanced glycation end products, and diabetes complications: what is new and what works.
        Clin Diabetes. 2003; 2
        • Genuth S.
        • Sun W.J.
        • Cleary P.
        • Sell D.R.
        • Dahms W.
        • Malone J.
        • et al.
        Glycation and carboxymethyllysine levels in skin collagen predict the risk of future 10-year progression of diabetic retinopathy and nephropathy in the diabetes control and complications trial and epidemiology of diabetes interventions and complications participants with type 1 diabetes.
        Diabetes. 2005; 54: 3103-3111
        • Chen W.L.
        • Li T.H.
        • Su P.J.
        • Chou C.K.
        • Fwu P.T.
        • Lin S.J.
        • et al.
        Second harmonic generation chi tensor microscopy for tissue imaging.
        Appl Phys Lett. 2009; : 94
        • Hadley J.
        • Malik N.
        • Meek K.
        Collagen as a model system to investigate the use of aspirin as an inhibitor of protein glycation and crosslinking.
        Micron. 2001; 32: 307-315
        • Tseng J.Y.
        • Ghazaryan A.A.
        • Lo W.
        • Chen Y.F.
        • Hovhannisyan V.
        • Chen S.J.
        • et al.
        Multiphoton spectral microscopy for imaging and quantification of tissue glycation.
        Biomed Opt Express. 2010; 2: 218-230
        • Hovhannisyan V.A.
        • Su P.J.
        • Chen Y.F.
        • Dong C.Y.
        Image heterogeneity correction in large-area, three-dimensional multiphoton microscopy.
        Opt Express. 2008; 16: 5107-5117
        • Lin S.J.
        • Hsiao C.Y.
        • Sun Y.
        • Lo W.
        • Lin W.C.
        • Jan G.J.
        • et al.
        Monitoring the thermally induced structural transitions of collagen by use of second-harmonic generation microscopy.
        Opt Lett. 2005; 30: 622-624
        • Kim B.M.
        • Eichler J.
        • Reiser K.M.
        • Rubenchik A.M.
        • Da Silva L.B.
        Collagen structure and nonlinear susceptibility: effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity.
        Lasers Surg Med. 2000; 27: 329-335
        • Monnier V.M.
        • Kohn R.R.
        • Cerami A.
        Accelerated age-related browning of human collagen in diabetes-mellitus.
        Proc Natl Acad Sci USA: Biol Sci. 1984; 81: 583-587
        • Pena A.
        • Strupler M.
        • Boulesteix T.
        • Schanne-Klein M.
        Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy.
        Opt Express. 2005; 13: 6268-6274
        • Palero J.A.
        • de Bruijn H.S.
        • van den Heuvel A.V.
        • Sterenborg H.J.C.M.
        • Gerritsen H.C.
        Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues.
        Biophys J. 2007; 93: 992-1007
        • Banerjee B.
        • Miedema B.
        • Chandrasekhar H.R.
        Emission spectra of colonic tissue and endogenous fluorophores.
        Am J Med Sci. 1998; 316: 220-226
        • Masters B.R.
        • Piston D.W.
        • Webb W.W.
        3-Dimensional Nad(P)h redox imaging of the insitu cornea with 2 photon excitation laser scanning microscopy.
        Invest Ophthalmol Vis Sci. 1993; 34: 1402
        • Sokolov K.
        • Galvan J.
        • Myakov A.
        • Lacy A.
        • Lotan R.
        • Richards-Kortum R.
        Realistic three-dimensional epithelial tissue phantoms for biomedical optics.
        J Biomed Opt. 2002; 7: 148-156