The Severity of Osteogenesis Imperfecta and Type I Collagen Pattern in Human Skin as Determined by Nonlinear Microscopy: Proof of Principle of a Diagnostic Method
Background The confirmatory diagnosis of Osteogenesis Imperfecta (OI) requires invasive, commonly bone biopsy, time consuming and destructive methods. This paper proposes an alternative method using a combination of two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) microscopies from easily obtained human skin biopsies. We show that this method can distinguish subtypes of human OI. Methodology/Principal Findings Different aspects of collagen microstructure of skin fresh biopsies and standard H&E-stained sections of normal and OI patients (mild and severe forms) were distinguished by TPEF and SHG images. Moreover, important differences between subtypes of OI were identified using different methods of quantification such as collagen density, ratio between collagen and elastic tissue, and gray-level co-occurrence matrix (GLCM) image-pattern analysis. Collagen density was lower in OI dermis, while the SHG/autofluorescence index of the dermis was significantly higher in OI as compared to that of the normal skin. We also showed that the energy value of GLCM texture analysis is useful to discriminate mild from severe OI and from normal skin. Conclusions/Significance This work demonstrated that nonlinear microscopy techniques in combination with image-analysis approaches represent a powerful tool to investigate the collagen organization in skin dermis in patients with OI and has the potential to distinguish the different types of OI. The procedure outlined in this paper requires a skin biopsy, which is almost painless as compared to the bone biopsy commonly used in conventional methods. The data presented here complement existing clinical diagnostic techniques and can be used as a diagnostic procedure to confirm the disease, evaluate its severity and treatment efficacy.
References
[1]
Sharma A, George L, Erskin K (2001) Osteogenesis imperfecta in pregnancy: two case reports and review of literature. Obstet Gynecol Surv 56: 563–566.
van Dijk FS, Cobben JM, Kariminejad A, Maugeri A, Nikkels PGJ, et al. (2011) Osteogenesis Imperfecta: A Review with Clinical Examples. Mol Syndromol 2: 1–20.
Provenzano PP, Eliceiri KW, Yan L, Ada-Nguema A, Conklin MW, et al. (2008) Nonlinear optical imaging of cellular processes in breast cancer. Microsc Microanal 14: 532–548.
[8]
Cox G, Kable E, Jones A, Fraser I, Manconi F, et al. (2003) 3-dimensional imaging of collagen using second harmonic generation. J Struct Biol 141: 53–62.
[9]
Zipfel WR, Williams RM, Christie R, Nikitin AY, Hyman BT, et al. (2003) Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci USA 100: 7075–7080.
[10]
Zoumi A, Yeh A, Tromberg BJ (2002) Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc Natl Acad Sci USA 99: 11014–11019.
[11]
Williams RM, Zipfel WR, Webb WW (2005) Interpreting second-harmonic generation images of collagen I fibrils. Biophys J 88: 1377–1386.
[12]
Masters BR, So PTC (2001) Confocal microscopy and multi-photon excitation microscopy of human skin in vivo. Opt. Exp. 8: 2–9.
[13]
Konig K, Riemann I (2003) High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed Opt 8: 432–439.
[14]
Konig K, Schenke-Layland K, Riemann I, Stock UA (2005) Multiphoton autofluorescence imaging of intratissue elastic fibers. Biomaterials 26: 495–500.
[15]
Lin SJ, Wu RJ, Tan HY, Lo W, Lin WC, et al. (2005) Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy. Opt Lett 17: 2275–2277.
[16]
Koehler MJ, Konig K, Elsner P, Buckle R, Kaatz M (2006) In vivo assessment of human skin aging by multiphoton laser scanning tomography. Opt Lett 19: 2879–2881.
[17]
Dimitrow E, Riemann I, Ehlers A, Koehler MJ, Norgauer J, et al. (2009) Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis. Exp Dermatol 18: 509–515.
[18]
Dimitrow E, Ziemer M, Koehler MJ, Norgauer J, Konig K, et al. (2009) Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma. Invest Dermatol 129: 1752–1758.
[19]
Cicchi R, Massi D, Sestini S, Carli P, De Giorgi V, et al. (2007) Multidimensional non-linear laser imaging of Basal Cell Carcinoma. Opt Exp 15: 10135–10148.
[20]
Paoli J, Smedh M, Wennberg AM, Ericson MB (2008) Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics. J Invest Dermatol 128: 1248–1255.
[21]
Malone JC, Hood AF, Conley T, Nurnberger J, Baldridge LA, et al. (2002) Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy. Cutan Pathol 29: 453–458.
[22]
Nadiarnykh O, Plotnikov S, Mohler WA, Kalajzic I, Redford-Badwal D, et al. (2007) Second harmonic generation imaging microscopy studies of osteogenesis imperfecta. J Biomed Opt 12: 051805.
[23]
Lacomb R, Nadiarnykh O, Campagnola PJ (2008) Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation. Biophys J 94: 4504–4514.
[24]
Adur J, Pelegati VB, de Thomaz A, D’Souza-Li L, Carmo Assun？？o M, et al. (2012) Quantitative changes in human epithelial cancers and osteogenesis imperfecta disease detected using nonlinear multicontrast microscopy. J Biomed Opt 17: 081407.
[25]
Adur J, Pelegati VB, de Thomaz AA, Baratti MO, Andrade LA, et al. (2012) Second harmonic generation microscopy as a powerful diagnostic imaging modality for human ovarian cancer. J Biophotonics doi:10.1002/jbio.201200108.
[26]
Adur J, Pelegati VB, de Thomaz AA, Baratti MO, Almeida DB, et al. (2012) Optical biomarkers of serous and mucinous human ovarian tumor assessed with nonlinear optics microscopies. PLoS One 7: e47007 doi:10.1371/journal.pone.0047007.
[27]
Dunn AK, Wallace VP, Coleno M, Berns MW, Tromberg BJ (2000) Influence of optical properties on two-photon fluorescence imaging in turbid samples. Appl Opt 39: 1194–1201.
[28]
Torkian BA, Yeh AT, Engel R, Sun CH, Tromberg BJ, et al. (2004) Modeling aberrant wound healing using tissue-engineered skin constructs and multiphoton microscopy. Facial Plast Surg 6: 180–187.
[29]
Zhuo S, Zhu X, Wu G, Chen J, Xiea S (2011) Quantitative biomarkers of colonic dysplasia based on intrinsic second-harmonic generation signal. J Biomed Opt 16: 120501–1.
[30]
Cicchi R, Kapsokalyvas D, De Giorgi V, Maio V, Van Wiechen A, et al. (2010) Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy. J Biophoton 3: 34–43.
[31]
English RS, Shenefelt PD (1999) Keloids and hypertrophic scars. Dermatol Surg 25: 631–638.
[32]
Watson JM, Rice PF, Marion SL, Brewer MA, Davis JR, et al. (2012) Analysis of second-harmonic-generation microscopy in a mouse model of ovarian carcinoma. J Biomed Opt 17: 076002.
[33]
Walker RF, Jackway PT, Longstaff ID (2003) Int. J. Pattern. Recogn. 17, 17–39.
