Contrast enhancement in second harmonic imaging: discriminating between muscle and collagen

In this study, polarization second harmonic generation (SHG) imaging is used and data analysis is developed to gain contrast and to discriminate with pixel resolution, in the same image, SHG source architectures. We use mammalian tissue in which both skeletal muscle and fibrilar collagen can be found. The images are fitted point by point using an algorithm based on a biophysical model, where the coefficient of determination is utilized as a filtering mechanism. For the whole image we retrieve for every pixel, the effective orientation, θe , of the SHG active structures. As a result a new image is formed which its contrast depends on the values of θe . Collagen presented in the forward direction for a predefined region of interest (ROI), peak distribution of angles θe centered in the region of ~45°, while muscle in the region of ~65°. Consequently, collagen and muscle are represented in different colors in the same image. Thus, here we show that it is possible to gain contrast and to discriminate between collagen and muscle without the use of any exogenous labeling or any co-localization with fluorescence imaging.

[1]  J Mertz,et al.  Coherent scattering in multi-harmonic light microscopy. , 2001, Biophysical journal.

[2]  Vincent Fleury,et al.  Collagen and myosin characterization by orientation field second harmonic microscopy. , 2008, Optics express.

[3]  Patrick Stoller,et al.  Polarization-modulated second harmonic generation in collagen. , 2002, Biophysical journal.

[4]  William A Mohler,et al.  Measurement of muscle disease by quantitative second-harmonic generation imaging. , 2008, Journal of biomedical optics.

[5]  Mingshi Wang,et al.  Spectral moment invariant analysis of disorder in polarization-modulated second-harmonic-generation images obtained from collagen assemblies. , 2007, Journal of the Optical Society of America. A, Optics, image science, and vision.

[6]  K. Beck,et al.  Supercoiled Protein Motifs: The Collagen Triple-Helix and the α-Helical Coiled Coil , 1998 .

[7]  H Szmacinski,et al.  Fluorescence lifetime imaging. , 1992, Analytical biochemistry.

[8]  Oleg Nadiarnykh,et al.  Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation. , 2008, Biophysical journal.

[9]  Y. Shen,et al.  Surface properties probed by second-harmonic and sum-frequency generation , 1989, Nature.

[10]  Jeffrey Lotz,et al.  Quantitative analysis of structural disorder in intervertebral disks using second harmonic generation imaging: comparison with morphometric analysis. , 2007, Journal of biomedical optics.

[11]  Yaron Silberberg,et al.  Nonlinear scanning laser microscopy by third harmonic generation , 1997 .

[12]  Isaac Freund,et al.  Second harmonic generation in collagen , 1979 .

[13]  Xiaoxing Han,et al.  Second harmonic properties of tumor collagen: determining the structural relationship between reactive stroma and healthy stroma. , 2008 .

[14]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[15]  Robert W. Boyd.  Nonlinear Optics Ed. 3 , 2008 .

[16]  P. Rez,et al.  Starch-based backwards SHG for in situ MEFISTO pulse characterization in multiphoton microscopy , 2008 .

[17]  D. Dombeck,et al.  Polarized microtubule arrays in apical dendrites and axons , 2008, Proceedings of the National Academy of Sciences.

[18]  Tsung-Han Tsai,et al.  Studies of χ(2)/χ(3) Tensors in Submicron-Scaled Bio-Tissues by Polarization Harmonics Optical Microscopy , 2004 .

[19]  Julian Moger,et al.  Collagen fiber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy. , 2008, Journal of biomedical optics.

[20]  William A Mohler,et al.  Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. , 2002, Biophysical journal.

[21]  W. R. Wiley,et al.  Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering , 1999 .

[22]  Watt W. Webb,et al.  Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[23]  David Artigas,et al.  In vivo, pixel-resolution mapping of thick filaments' orientation in nonfibrilar muscle using polarization-sensitive second harmonic generation microscopy. , 2009, Journal of biomedical optics.

[24]  Thierry Boulesteix,et al.  Chiroptical effects in the second harmonic signal of collagens I and IV. , 2005, Journal of the American Chemical Society.

[25]  H. S. de Bruijn,et al.  In vivo nonlinear spectral imaging in mouse skin. , 2006, Optics express.

[26]  Chi-Hung Lin,et al.  Selective imaging in second-harmonic-generation microscopy by polarization manipulation , 2007 .

[27]  François Tiaho,et al.  Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy. , 2007, Optics express.

[28]  Israel Rocha-Mendoza,et al.  Sum frequency vibrational spectroscopy: the molecular origins of the optical second-order nonlinearity of collagen. , 2007, Biophysical journal.

[29]  William A Mohler,et al.  Characterization of the myosin-based source for second-harmonic generation from muscle sarcomeres. , 2006, Biophysical journal.

[30]  Martin Vogel,et al.  Scanning multi photon microscopy of SHG signals from single myofibrils of mammalian skeletal muscle , 2006, SPIE BiOS.