JBO Letters

Quantitative biomarkers of colonic dysplasia based on intrinsic second-harmonic generation signal

[+] Author Affiliations
Shuangmu Zhuo, Xiaoqin Zhu, Jianxin Chen, Shusen Xie

Fujian Normal University, Institute of Laser and Optoelectronics Technology, Fujian Provincial Key Laboratory for Photonics Technology, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fuzhou 350007, China

Guizhu Wu

First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China

J. Biomed. Opt. 16(12), 120501 (November 22, 2011). doi:10.1117/1.3659715
History: Received September 12, 2011; Revised October 18, 2011; Accepted October 21, 2011; Published November 22, 2011; Online November 22, 2011
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* Address all correspondence to: Jianxin Chen, Fujian Normal University, School of Physics and OptoElectronics Technology, Fuzhou, Fujian 350007 China. Tel: 0086-591-22686078 Fax: 0086-591-83465373; E-mail: chenjianxin@fjnu.edu.cn.

Most colorectal cancers arise from dysplastic lesions, such as adenomatous polyps, and these lesions are difficult to be detected by the current endoscopic screening approaches. Here, we present the use of an intrinsic second-harmonic generation (SHG) signal as a novel means to differentiate between normal and dysplastic human colonic tissues. We find that the SHG signal can quantitatively identify collagen change associated with colonic dysplasia that is indiscernible by conventional pathologic techniques. By comparing normal with dysplastic mucosa, there were significant differences in collagen density and collagen fiber direction, providing substantial potential to become quantitative intrinsic biomarkers for in vivo clinical diagnosis of colonic dysplasia.

Figures in this Article

Colorectal cancer (CRC) is a major cause of cancer-related deaths in the world.1 It is commonly believed that adenomatous polyps, or adenomas, appear to be major precursors to CRC, and the morbidity rate from CRC could be significantly reduced if these dysplastic lesions were detected.23 However, it is challenging that the current endoscopic screening methods with white light detect these dysplastic lesions.45 Thus, the development of new approaches to identify such lesions is of great medical significance.

Recently, the remodeling of the stroma has been implicated in colonic dysplasia,67 and these alterations may provide a better biomarker of these lesions than currently available screening methods. The stroma is composed primarily of type-I collagen, which is known to induce a second harmonic generation (SHG) signal.89 SHG microscopy, in recent years, has emerged to be a promising technique for the qualitative and quantitative characterization of stromal biology with advantages of being label-free, inherent three-dimension resolution, near-IR excitation for superior optical penetration, lower photodamage, and capable of providing quantitative information.811 Therefore, it has been recognized as promising approaches for cancer diagnostics.9,1214 To date, we have found no previous studies that quantitatively investigated the remodeling of the stroma in dysplastic colonic mucosa, and this is what motivated us to do this work. In this study, we used SHG microscopy to analyze the collagen change of normal and dysplastic colonic mucosa.

SHG microscopy was achieved using a nonlinear optical system which has been described previously.14 In brief, SHG images were acquired using a commercial laser scanning microscopic imaging system (Zeiss LSM 510 META, Jena, Germany) coupled to a femtosecond Ti:sapphire laser (Coherent Mira 900-F) operating at 800 nm. The polarization direction of the laser light is the horizontal polarization. An oil immersion objective (×63 and NA = 1.4) was employed for focusing the excitation beam into tissue samples (average power less than 15 mW) and was also used to collect the backscattered intrinsic SHG signals. The images were obtained at 2.56 μs per pixel. A fine focusing stage (HRZ 200 stage, Carl Zeiss) was used to change the focus position for recording various optical sections.

A total of nine fresh ex vivo colon tissue samples were obtained immediately after resection from patients undergoing a colectomy for familial adenomatous polyposis. Prior to study participation, all patients signed an informed consent, and this study was approved by the Institutional Review Board of Fujian Medical University. Colonic adenomas are dysplastic lesions. Typical adenomas are polypoid structures and the adenomas are surrounded by normal tissue. In this study, the surrounding normal regions and the polyp regions were imaged. After SHG imaging, the tissue specimens were fixed in 10% formalin and prepared for pathologic examination using standard protocols. In this work, all data were presented as a mean value plus-or-minus its standard deviation, and analyzed using Student's t-test. Differences were considered to be statistically significant when the P-values were less than 0.05.

Shown in Fig. 1 are the representative en face SHG images from normal and dysplastic colonic mucosa. As can be seen from Fig. 1, the stroma can be identified because the epithelium consists mainly of columnar epithelial cells and goblet cells that are not effective in generating SHG signals and the stroma is composed primarily of type-I collagen that is capable of emitting strong SHG signals. Morphologically, normal and dysplastic colonic mucosa can be easily discriminated. In a normal case, the collagen displays a denser matrix, and the collagen fibers are almost parallel to the interface of epithelium and stroma. In dysplasia, a looser collagen matrix is observed, and the collagen fibers are located at an angle to the interface of epithelium and stroma. It is worth pointing out that these features are not visible in standard hematoxylin and eosin-stained sections, as presented in Fig. 2.

Graphic Jump LocationF1 :

Representative SHG images from (a) normal and (b) dysplastic colonic tissues. The size of the images is 146 × 146 μm2.

Graphic Jump LocationF2 :

Typical histological images (magnification, 40×), from (a) normal and (b) dysplastic colonic tissues.

In the following, to further quantify these features, two quantitative analyses were performed. First, the depth-dependent decay (DDD) of the SHG signal was analyzed to determine the collagen density. In short, a series of SHG images were obtained at intervals of 1 μm up to a final depth of 30 μm; the average SHG intensity of selected regions at each depth was then calculated and plotted as a function of depth. Decrease in SHG signal intensity with increasing depth can be approximated using a first-order model: I = Ae−kx + C, where I is the SHG intensity, x is the imaging depth, A is a pre-exponential scaling factor, C is an arbitrary constant that adjusts the lowest signal intensity to zero, and k is the DDD constant.15 Figure 3 shows a typical plot of the exponential fits from normal and dysplastic colonic tissues. Recently, Dunn et al. found that although scattering plays a role in DDD, absorption is the main factor responsible for DDD in turbid media at least to a depth of focus of 412 μm. Thus, although other factors, such as direction, distribution, and size of collagen fibers, may contribute to changes in the DDD value, it is a reasonable measure of collagen density.16 Moreover, the collagen fiber angle relative to the tangent of the interface of epithelium and stroma was measured every 5 μm using IMAGEJ software (National Institutes of Health), as reported previously.17 As shown in Table 1, the dysplastic colonic mucosa has a lower DDD constant and a bigger collagen fiber angle (p < 0.05), indicating loss of the collagen density and the collagen fibers located at an angle to the interface of epithelium and stroma in dysplasia, consistent with the above-mentioned observations. Therefore, these differences in the collagen density and the collagen fiber direction may be used to quantitatively discriminate between normal and dysplastic colonic mucosa, and serve as quantitative intrinsic biomarkers for quantifying the diagnosis of colonic dysplasia.

Graphic Jump LocationF3 :

Typical plot of the exponential fits from normal (circles), and dysplastic (triangles) colonic tissues; intensity at depth 0 μm has been normalized to unity in each case.

Table Grahic Jump Location
Quantitative variables with dysplasia.

In conclusion, this study demonstrates the potential of intrinsic SHG imaging to provide biochemical and morphological biomarkers, including the collagen density and the collagen fiber direction, which can be used to discriminate between normal and dysplastic colonic mucosa. With the advent of the SHG-based endoscopy,18 we expect that the analysis of collagen change using intrinsic SHG imaging may become an important noninvasive methodology that provides quantitative information about the collagen change for diagnosing colonic dysplasia.

Acknowledgments

The project was supported by the National Natural Science Foundation of China (Grant Nos. NNSFC 60908043 and NNSFC 30970783 ), and the Natural Science Foundation of Fujian Province (Grant No. NSF 2011J01341 ).

Jemal  A., , Siegel  R., , Ward  E., , Hao  Y., , Xu  J., , and Thun  M. J., “ Cancer statistics, 2009. ,” Ca-Cancer J. Clin.. 59, , 225–249  ((2009)).
Srivastava  S., , Henson  D. E., , and Gazdar  A.,  Molecular Pathology of Early Cancer. ,  IOS Press ,  Amsterdam  ((1998)).
Miller  S. J., , Joshi  B. P., , Feng  Y., , Gaustad  A., , Fearon  E. R., , and Wang  T. D., “ In vivo fluorescence-based endoscopic detection of colon dysplasia in the mouse using a novel peptide probe. ,” PLoS ONE. 6, , e17384  ((2011)).
van Rijn  J. C., , Reitsma  J. B., , Stoker  J., , Bossuyt  P. M., , van Deventer  S. J., , and Dekker  E., “ Polyp miss rate determined by tandem colonoscopy: a systematic review. ,” Am. J. Gastroenterol.. 101, , 343–350  ((2006)).
Gualco  G., , Reissenweber  N., , Cliche  I., , and Bacchi  C. E., “ Flat elevated lesions of the colon and rectum: A spectrum of neoplastic and nonneoplastic entities. ,” Ann. Diagn. Pathol.. 10, , 333–338  ((2006)).
Kalluri  R., and Zeisberg  M., “ Fibroblasts in cancer. ,” Nat. Rev. Cancer. 6, , 392–401  ((2006)).
Romer  T. J., , Fitzmaurice  M., , Cothren  R. M., , Richards-Kortum  R., , Petras  R., , Sivak  M. V.  Jr., , and Kramer  J. R.  Jr, “ Laser-induced fluorescence microscopy of normal colon and dysplasia in colonic adenomas: implications for spectroscopic diagnosis. ,” Am. J. Gastroenterol.. 90, , 81–87  ((1995)).
Zipfel  W. R., , Williams  R. M., , Christie  R., , Nikitin  A. Y., , Hyman  B. T., , and Webb  W. W., “ Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. ,” Proc. Natl. Acad. Sci. U.S.A.. 100, , 7075–7080  ((2003)).
Campagnola1  P. J., and Dong  C. Y., “ Second harmonic generation microscopy: principles and applications to disease diagnosis. ,” Laser Photonics Rev.. 5, , 13–26  ((2011)).
Zhuo  S. M., , Chen  J. X., , Luo  T. S., , Zou  D. S., , and Zhao  J. J., “  Multimode nonlinear optical imaging of the dermis in ex vivo human skin based on the combination of multichannel mode and Lambda mode . ,” Opt. Express. 14, , 7810–7820  ((2006)).
Zipfel  W. R., , Williams  R. M., , and Webb  W. W., “ Nonlinear magic: multiphoton microscopy in the biosciences. ,” Nat. Biotechnol.. 21, , 1369–1377  ((2003)).
Zhuo  S. M., , Chen  J. X., , Xie  S. S., , Hong  Z. B., , and Jiang  X. S., “ Extracting diagnostic stromal organization features based on intrinsic two-photon excited fluorescence and second-harmonic generation signals. ,” J. Biomed. Opt.. 14, , 020503  ((2009)).
Nadiarnykh  O., , LaComb  R. B., , Brewer  M. A., , and Campagnola  P. J., “ Alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy. ,” BMC Cancer. 10, , 94  ((2010)).
Zhuo  S. M., , Chen  J. X., , Wu  G. Z., , Xie  S. S., , Zheng  L. Q., , Jiang  X. S., , and Zhu  X. Q., “ Quantitatively linking collagen alteration and epithelial tumor progression by second harmonic generation microscopy. ,” Appl. Phys. Lett.. 96, , 213704  ((2010)).
Torkian  B. A., , Yeh  A. T., , Engel  R., , Sun  C. H., , Tromberg  B. J., , and Wong  B. J., “ Modeling aberrant wound healing using tissue-engineered skin constructs and multiphoton microscopy. ,” Facial. Plast. Surg.. 6, , 180–187  ((2004)).
Dunn  A. K., , Wallace  V. P., , Coleno  M., , Berns  M. W., , and Tromberg  B. J., “ Influence of optical properties on two-photon fluorescence imaging in turbid samples. ,” Appl. Opt.. 39, , 1194–1201  ((2000)).
Provenzano  P. P., , Eliceiri  K. W., , Campbell  J. M., , Inman  D. R., , White  J. G., , and Keely  P. J., “ Collagen reorganization at the tumor-stromal interface facilitates local invasion. ,” BMC Medicine. 4, , 38  ((2006)).
Bao  H. C., , Boussioutas  A., , Jeremy  R., , Russell  S., , and Gu  M., “ Second harmonic generation imaging via nonlinear endomicroscopy. ,” Opt. Express. 18, , 1255–1260  ((2010)).
© 2011 Society of Photo-Optical Instrumentation Engineers (SPIE)

Citation

Shuangmu Zhuo ; Xiaoqin Zhu ; Guizhu Wu ; Jianxin Chen and Shusen Xie
"Quantitative biomarkers of colonic dysplasia based on intrinsic second-harmonic generation signal", J. Biomed. Opt. 16(12), 120501 (November 22, 2011). ; http://dx.doi.org/10.1117/1.3659715


Figures

Graphic Jump LocationF1 :

Representative SHG images from (a) normal and (b) dysplastic colonic tissues. The size of the images is 146 × 146 μm2.

Graphic Jump LocationF2 :

Typical histological images (magnification, 40×), from (a) normal and (b) dysplastic colonic tissues.

Graphic Jump LocationF3 :

Typical plot of the exponential fits from normal (circles), and dysplastic (triangles) colonic tissues; intensity at depth 0 μm has been normalized to unity in each case.

Tables

Table Grahic Jump Location
Quantitative variables with dysplasia.

References

Jemal  A., , Siegel  R., , Ward  E., , Hao  Y., , Xu  J., , and Thun  M. J., “ Cancer statistics, 2009. ,” Ca-Cancer J. Clin.. 59, , 225–249  ((2009)).
Srivastava  S., , Henson  D. E., , and Gazdar  A.,  Molecular Pathology of Early Cancer. ,  IOS Press ,  Amsterdam  ((1998)).
Miller  S. J., , Joshi  B. P., , Feng  Y., , Gaustad  A., , Fearon  E. R., , and Wang  T. D., “ In vivo fluorescence-based endoscopic detection of colon dysplasia in the mouse using a novel peptide probe. ,” PLoS ONE. 6, , e17384  ((2011)).
van Rijn  J. C., , Reitsma  J. B., , Stoker  J., , Bossuyt  P. M., , van Deventer  S. J., , and Dekker  E., “ Polyp miss rate determined by tandem colonoscopy: a systematic review. ,” Am. J. Gastroenterol.. 101, , 343–350  ((2006)).
Gualco  G., , Reissenweber  N., , Cliche  I., , and Bacchi  C. E., “ Flat elevated lesions of the colon and rectum: A spectrum of neoplastic and nonneoplastic entities. ,” Ann. Diagn. Pathol.. 10, , 333–338  ((2006)).
Kalluri  R., and Zeisberg  M., “ Fibroblasts in cancer. ,” Nat. Rev. Cancer. 6, , 392–401  ((2006)).
Romer  T. J., , Fitzmaurice  M., , Cothren  R. M., , Richards-Kortum  R., , Petras  R., , Sivak  M. V.  Jr., , and Kramer  J. R.  Jr, “ Laser-induced fluorescence microscopy of normal colon and dysplasia in colonic adenomas: implications for spectroscopic diagnosis. ,” Am. J. Gastroenterol.. 90, , 81–87  ((1995)).
Zipfel  W. R., , Williams  R. M., , Christie  R., , Nikitin  A. Y., , Hyman  B. T., , and Webb  W. W., “ Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. ,” Proc. Natl. Acad. Sci. U.S.A.. 100, , 7075–7080  ((2003)).
Campagnola1  P. J., and Dong  C. Y., “ Second harmonic generation microscopy: principles and applications to disease diagnosis. ,” Laser Photonics Rev.. 5, , 13–26  ((2011)).
Zhuo  S. M., , Chen  J. X., , Luo  T. S., , Zou  D. S., , and Zhao  J. J., “  Multimode nonlinear optical imaging of the dermis in ex vivo human skin based on the combination of multichannel mode and Lambda mode . ,” Opt. Express. 14, , 7810–7820  ((2006)).
Zipfel  W. R., , Williams  R. M., , and Webb  W. W., “ Nonlinear magic: multiphoton microscopy in the biosciences. ,” Nat. Biotechnol.. 21, , 1369–1377  ((2003)).
Zhuo  S. M., , Chen  J. X., , Xie  S. S., , Hong  Z. B., , and Jiang  X. S., “ Extracting diagnostic stromal organization features based on intrinsic two-photon excited fluorescence and second-harmonic generation signals. ,” J. Biomed. Opt.. 14, , 020503  ((2009)).
Nadiarnykh  O., , LaComb  R. B., , Brewer  M. A., , and Campagnola  P. J., “ Alterations of the extracellular matrix in ovarian cancer studied by second harmonic generation imaging microscopy. ,” BMC Cancer. 10, , 94  ((2010)).
Zhuo  S. M., , Chen  J. X., , Wu  G. Z., , Xie  S. S., , Zheng  L. Q., , Jiang  X. S., , and Zhu  X. Q., “ Quantitatively linking collagen alteration and epithelial tumor progression by second harmonic generation microscopy. ,” Appl. Phys. Lett.. 96, , 213704  ((2010)).
Torkian  B. A., , Yeh  A. T., , Engel  R., , Sun  C. H., , Tromberg  B. J., , and Wong  B. J., “ Modeling aberrant wound healing using tissue-engineered skin constructs and multiphoton microscopy. ,” Facial. Plast. Surg.. 6, , 180–187  ((2004)).
Dunn  A. K., , Wallace  V. P., , Coleno  M., , Berns  M. W., , and Tromberg  B. J., “ Influence of optical properties on two-photon fluorescence imaging in turbid samples. ,” Appl. Opt.. 39, , 1194–1201  ((2000)).
Provenzano  P. P., , Eliceiri  K. W., , Campbell  J. M., , Inman  D. R., , White  J. G., , and Keely  P. J., “ Collagen reorganization at the tumor-stromal interface facilitates local invasion. ,” BMC Medicine. 4, , 38  ((2006)).
Bao  H. C., , Boussioutas  A., , Jeremy  R., , Russell  S., , and Gu  M., “ Second harmonic generation imaging via nonlinear endomicroscopy. ,” Opt. Express. 18, , 1255–1260  ((2010)).

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