The role of backscattering in SHG tissue imaging.

We investigate the properties of second-harmonic generation (SHG) tissue imaging for the functional biological unit fascia, skeletal muscle, and tendon. Fascia and Achilles tendon primarily consist of similar collagen type I arrays that can be imaged using SHG microscopy. For muscle, it is the myosin molecules represented within the A bands. For fascia and tendon tissue samples, we observe, in addition to a stronger signal in forward images, vastly different features for the backward versus the forward images. In vivo as well as intact ex vivo thick tissue imaging requires backward detection. The obtained image is a result of the direct backward components plus a certain fraction of the forward components that are redirected (backscattered) toward the objective as they propagate within the tissue block. As the forward and the backward images are significantly different from each other for the imaged collagen type I tissue, it is crucial to determine the fraction of the forward signal that contributes to the overall backward signal. For intact ex vivo SHG imaging of Achilles tendon, we observe a significant contribution of forward features in the resulting image. For fascia, the connective tissue immediately surrounding muscle, we only observe backward features, due to low backscattering in muscle.

[1]  Watt W Webb,et al.  Interpreting second-harmonic generation images of collagen I fibrils. , 2005, Biophysical journal.

[2]  B. Olsen,et al.  The contribution of collagenous proteins to tissue-specific matrix assemblies. , 1990, Current opinion in cell biology.

[3]  P. T. Nguyen,et al.  Dendritic Spine Abnormalities in Amyloid Precursor Protein Transgenic Mice Demonstrated by Gene Transfer and Intravital Multiphoton Microscopy , 2005, The Journal of Neuroscience.

[4]  Brian J Bacskai,et al.  Four-dimensional multiphoton imaging of brain entry, amyloid binding, and clearance of an amyloid-β ligand in transgenic mice , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[5]  E. Sevick-Muraca,et al.  Quantitative optical spectroscopy for tissue diagnosis. , 1996, Annual review of physical chemistry.

[6]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[7]  Riyi Shi,et al.  Coherent anti-stokes Raman scattering imaging of axonal myelin in live spinal tissues. , 2005, Biophysical journal.

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

[9]  T. Pufe,et al.  Structure of the human tibialis posterior tendon , 2004, Archives of Orthopaedic and Trauma Surgery.

[10]  C. Stosiek,et al.  In vivo two-photon calcium imaging of neuronal networks , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Hedi Mattoussi,et al.  Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy , 2004, Nature Medicine.

[12]  Josef Bille,et al.  Second harmonic generation imaging of collagen fibrils in cornea and sclera. , 2005, Optics express.

[13]  B. Tromberg,et al.  Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence , 2002, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[16]  L V Wang,et al.  Anisotropy in the absorption and scattering spectra of chicken breast tissue. , 1998, Applied optics.

[17]  Paul J Campagnola,et al.  Macromolecular structure of cellulose studied by second-harmonic generation imaging microscopy. , 2003, Optics letters.

[18]  Kenneth W. Dunn,et al.  Principles of Multiphoton Microscopy , 2006, Nephron Experimental Nephrology.

[19]  B. Olsen,et al.  New insights into the function of collagens from genetic analysis. , 1995, Current opinion in cell biology.

[20]  Karsten König,et al.  Imaging of cardiovascular structures using near-infrared femtosecond multiphoton laser scanning microscopy. , 2005, Journal of biomedical optics.

[21]  W. Webb,et al.  Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo , 2003, Science.

[22]  V. Centonze,et al.  Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. , 1998, Biophysical journal.

[23]  Sergey Plotnikov,et al.  Optical clearing for improved contrast in second harmonic generation imaging of skeletal muscle. , 2006, Biophysical journal.

[24]  I. S. Saidi,et al.  Mie and Rayleigh modeling of visible-light scattering in neonatal skin. , 1995, Applied optics.

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

[26]  Raymond P. Molloy,et al.  In vivo multiphoton microscopy of deep brain tissue. , 2004, Journal of neurophysiology.

[27]  T K Borg,et al.  Morphology of connective tissue in skeletal muscle. , 1980, Tissue & cell.

[28]  P. Maxwell,et al.  Organ-Specific Collagen Expression: Implications for Renal Disease , 2005, Nephron Experimental Nephrology.

[29]  Brian Seed,et al.  Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation , 2003, Nature Medicine.

[30]  Leslie M Loew,et al.  Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms , 2003, Nature Biotechnology.

[31]  Iris Riemann,et al.  High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. , 2003, Journal of biomedical optics.

[32]  Watt W Webb,et al.  Use of multiphoton imaging for studying cell migration in the mouse. , 2005, Methods in molecular biology.

[33]  L. Soslowsky,et al.  Development of tendon structure and function: regulation of collagen fibrillogenesis. , 2005, Journal of musculoskeletal & neuronal interactions.

[34]  D. Kleinfeld,et al.  In vivo dendritic calcium dynamics in neocortical pyramidal neurons , 1997, Nature.

[35]  W. Webb,et al.  Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Huafeng Ding,et al.  A primary method for determination of optical parameters of turbid samples and application to intralipid between 550 and 1630 nm. , 2006, Optics express.

[37]  Conor L Evans,et al.  Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

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

[39]  H. J. van Staveren,et al.  Light scattering in Intralipid-10% in the wavelength range of 400-1100 nm. , 1991, Applied optics.

[40]  A. Fabre,et al.  Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy , 2005, Nature Methods.

[41]  Arantxa González,et al.  Mechanisms of Disease: pathologic structural remodeling is more than adaptive hypertrophy in hypertensive heart disease , 2005, Nature Clinical Practice Cardiovascular Medicine.

[42]  Yaron Silberberg,et al.  Depth-resolved structural imaging by third-harmonic generation microscopy. , 2004, Journal of structural biology.

[43]  Zhongping Chen,et al.  Second harmonic optical coherence tomography , 2004, The 26th Annual International Conference of the IEEE Engineering in Medicine and Biology Society.

[44]  Leslie M Loew,et al.  Second harmonic imaging microscopy. , 2003, Methods in enzymology.

[45]  Mark J. Miller,et al.  Two-Photon Imaging of Lymphocyte Motility and Antigen Response in Intact Lymph Node , 2002, Science.

[46]  W. Webb,et al.  Measuring Serotonin Distribution in Live Cells with Three-Photon Excitation , 1997, Science.

[47]  K. Cutroneo.  How is Type I procollagen synthesis regulated at the gene level during tissue fibrosis , 2003, Journal of cellular biochemistry.

[48]  Ivan V. Tomov,et al.  High-resolution second-harmonic optical coherence tomography of collagen in rat-tail tendon , 2005 .