Deep Imaging in Tissue and Biomedical Materials
eBook - ePub

Deep Imaging in Tissue and Biomedical Materials

Using Linear and Nonlinear Optical Methods

Lingyan Shi, Robert R. Alfano, Lingyan Shi, Robert R. Alfano

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  2. English
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eBook - ePub

Deep Imaging in Tissue and Biomedical Materials

Using Linear and Nonlinear Optical Methods

Lingyan Shi, Robert R. Alfano, Lingyan Shi, Robert R. Alfano

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About This Book

The use of light for probing and imaging biomedical media is promising for the development of safe, noninvasive, and inexpensive clinical imaging modalities with diagnostic ability. The advent of ultrafast lasers has enabled applications of nonlinear optical processes, which allow deeper imaging in biological tissues with higher spatial resolution. This book provides an overview of emerging novel optical imaging techniques, Gaussian beam optics, light scattering, nonlinear optics, and nonlinear optical tomography of tissues and cells. It consists of pioneering works that employ different linear and nonlinear optical imaging techniques for deep tissue imaging, including the new applications of single- and multiphoton excitation fluorescence, Raman scattering, resonance Raman spectroscopy, second harmonic generation, stimulated Raman scattering gain and loss, coherent anti-Stokes Raman spectroscopy, and near-infrared and mid-infrared supercontinuum spectroscopy. The book is a comprehensive reference of emerging deep tissue imaging techniques for researchers and students working in various disciplines.

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Chapter 1

Overview of Second- and Third-Order Nonlinear Optical Processes for Deep Imaging

Sangeeta Murugkara and Robert W. Boydb
aDepartment of Physics, Carleton University,
Ottawa, Ontario K1S 5B6, Canada
bDepartment of Physics, University of Ottawa,
Ottawa, Ontario K1N 6N5, Canada

1.1 Introduction: Nonlinear Optical Contrast in Biological Imaging

The field of optical microscopic imaging has rapidly evolved because of tremendous advances made in laser and detection technology. Various types of linear and nonlinear light–matter interactions have been harnessed for providing contrast in the microscopic images. Simple microscopy techniques such as bright-field and differential-interference-contrast reveal structural information at the cellular level owing to the refractive index contrast of the sample medium. Fluorescence microscopy offers higher chemical specificity and is the most popular contrast mechanism used in biological studies. The contrast is achieved by means of targeted labeling of molecules using exogenous or endogenous fluorophores. However, external fluorophores are often perturbative since they may disrupt the native state of the sample, especially for small molecules whose size may be smaller than the fluorescent label itself. Besides, many molecular species are intrinsically nonfluorescent or only weakly fluorescent. It is also better to avoid external contrast agents for in vivo imaging applications since such contrast agents need concurrent development of appropriate delivery strategies and are often limited by problems of label specificity and induced toxicity. Vibrational microscopy techniques, on the other hand, are inherently label-free. They involve the excitation of molecular vibrations and offer intrinsic chemical specificity. Two such techniques include infrared absorption and Raman microscopy. Out of these, infrared microscopy has low spatial resolution owing to the long infrared wavelengths employed. In addition, water absorption of the infrared light is a major limitation for investigating live biological samples. Raman scattering, on the other hand, is based on the inelastic scattering of light by vibrating molecules and provides a molecular fingerprint of the chemical composition of a living cell or tissue. It offers a powerful label-free contrast mechanism and has been applied in various biological investigations. Linear contrast mechanisms based on fluorescence and Raman scattering typically employ continuous-wave visible light for excitation and sample scanning or laser scanning to generate an image. A confocal pinhole inserted at the detector facilitates a three-dimensionally sectioned image but unfortunately limits the sensitivity of detection.
In comparison, nonlinear optical microscopy or multiphoton microscopy employs near-infrared (near-IR) femtosecond or picosecond pulsed light to excite nonlinear optical processes that can only be accessed by application of two or more (multi) photons [1]. The nonlinear optical signal is generated only in the focal plane of the objective where the beam intensity is maximized. This results in the inherent three-dimensional sectioning capability without the need for a confocal pinhole. This also means that significantly greater sensitivity in signal detection is achieved since the confocal collection geometry is not necessary. The near-IR light used for excitation of the nonlinear optical signal enables deeper penetration in thick scattering samples and in addition is less biologically harmful. In addition, multiphoton microscopic imaging can take advantage of nonlinear optical processes involving endogenous contrast. This ability permits dynamic studies of live cells and tissue specimens in a label-free manner.
Two-photon-excited fluorescence (TPEF) or two-photon microscopy has been extensively applied for biological imaging over the past couple of decades [2, 3]. In TPEF, a single femtosecond pulsed laser beam is tightly focused in the specimen such that two low energy, near-IR photons are simultaneously absorbed by a fluorophore and then emitted as one photon at a higher frequency than the incident ligh...

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