Multifunctional Imaging of Endogenous Contrast by Simultaneous Nonlinear and Optical Coherence Microscopy of Thick Tissues

A variety of high resolution optical microscopy techniques have been developed in recent years for basic and clinical studies of biological systems. We demonstrate a trimodal microscope combining optical coherence microscopy (OCM) with two forms of nonlinear microscopy, namely two‐photon excited fluorescence (2PF) and second harmonic generation (SHG), for imaging turbid media. OCM combines the advantages of confocal detection and coherence gating for structural imaging in highly scattering tissues. Nonlinear microscopy enables the detection of biochemical species, such as elastin, NAD(P)H, and collagen. While 2PF arises from nonlinear excitation of fluorescent species, SHG is a form of nonlinear scattering observed in materials that lack a center of inversion symmetry, such as type I collagen. Characterization of the microscope showed nearly diffraction‐limited spatial resolution in all modalities. Images were obtained in fish scales and excised human skin samples. The primary endogenous sources of contrast in the dermis were due to elastin autofluorescence and collagen SHG. Multimodal microscopy allows the simultaneous visualization of structural and functional information of biological systems. Microsc. Res. Tech., 2007. © 2007 Wiley‐Liss, Inc.

[1]  Melissa C Skala,et al.  Multiphoton microscopy of endogenous fluorescence differentiates normal, precancerous, and cancerous squamous epithelial tissues. , 2005, Cancer research.

[2]  J. Fujimoto,et al.  Optical coherence microscopy in scattering media. , 1994, Optics letters.

[3]  P. So,et al.  Single-Photon Counting Multicolor Multiphoton Fluorescence Microscope , 2005, Journal of Fluorescence.

[4]  K R Wilson,et al.  Third harmonic generation microscopy. , 1998, Optics express.

[5]  J. Izatt,et al.  Optical coherence microscopy in gastrointestinal tissues , 1996, Summaries of papers presented at the Conference on Lasers and Electro-Optics.

[6]  B R Masters,et al.  Two-photon excitation fluorescence microscopy. , 2000, Annual review of biomedical engineering.

[7]  Siavash Yazdanfar,et al.  Molecular contrast in optical coherence tomography by use of a pump-probe technique. , 2003, Optics letters.

[8]  T Wilson,et al.  Size of the detector in confocal imaging systems. , 1987, Optics letters.

[9]  W. Webb,et al.  Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm , 1996 .

[10]  B. R. Masters,et al.  Optical Biopsy of In Vivo Human Skin: Multi-photon Excitation Microscopy , 1998, Lasers in Medical Science.

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

[12]  Siavash Yazdanfar,et al.  Interferometric second harmonic generation microscopy. , 2004, Optics express.

[13]  K. Seung,et al.  Visualization of coronary atherosclerotic plaques in patients using optical coherence tomography: comparison with intravascular ultrasound. , 2002, Journal of the American College of Cardiology.

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

[15]  L M Loew,et al.  High-resolution nonlinear optical imaging of live cells by second harmonic generation. , 1999, Biophysical journal.

[16]  Zhongping Chen,et al.  Imaging thermally damaged tissue by Polarization Sensitive Optical Coherence Tomography. , 1998, Optics express.

[17]  E. Cocker,et al.  Fiber-optic fluorescence imaging , 2005, Nature Methods.

[18]  S. A. Boppart,et al.  Integrated structural and functional optical imaging combining spectral-domain optical coherence and multiphoton microscopy , 2005, physics/0512161.

[19]  Scott E Fraser,et al.  Multiphoton excitation spectra in biological samples. , 2003, Journal of biomedical optics.

[20]  Bruce J Tromberg,et al.  Combined multiphoton microscopy and optical coherence tomography using a 12-fs broadband source. , 2006, Journal of biomedical optics.

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

[22]  P. So,et al.  High-speed, two-photon scanning microscope. , 1999, Applied optics.

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

[24]  J Mertz,et al.  Combined scanning optical coherence and two-photon-excited fluorescence microscopy. , 1999, Optics letters.

[25]  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.

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

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

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

[29]  M Deutsch,et al.  Second-harmonic microscopy of biological tissue. , 1986, Optics letters.

[30]  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.

[31]  K. Suslick,et al.  Engineered microsphere contrast agents for optical coherence tomography. , 2003, Optics letters.

[32]  S Pelet,et al.  A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation. , 2004, Biophysical journal.

[33]  P. So,et al.  In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy. , 2001, The Journal of investigative dermatology.

[34]  R. Webb.  Confocal optical microscopy , 1996 .

[35]  P. So,et al.  Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra. , 2005, Journal of biomedical optics.

[36]  P. So,et al.  Confocal microscopy and multi-photon excitation microscopy of human skin in vivo. , 2001, Optics express.

[37]  J. Izatt,et al.  Real-time in vivo imaging of human gastrointestinal ultrastructure by use of endoscopic optical coherence tomography with a novel efficient interferometer design. , 1999, Optics letters.

[38]  Chen-Yuan Dong,et al.  Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy. , 2005, Optics letters.

[39]  Jianan Y Qu,et al.  Two-photon autofluorescence spectroscopy and second-harmonic generation of epithelial tissue. , 2005, Optics letters.

[40]  J. Fujimoto,et al.  Optical biopsy and imaging using optical coherence tomography , 1995, Nature Medicine.

[41]  J. Fujimoto,et al.  In vivo endoscopic optical biopsy with optical coherence tomography. , 1997, Science.

[42]  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.

[43]  J G Fujimoto,et al.  High-resolution optical coherence microscopy for high-speed, in vivo cellular imaging. , 2003, Optics letters.

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

[45]  Tzu-Ming Liu,et al.  Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser. , 2001, Optics letters.

[46]  Y. Uesu,et al.  Multi-purpose nonlinear optical microscope. Its principle and applications to polar thin-film observation , 1999 .