Quantitative analysis of forward and backward second-harmonic images of collagen fibers using Fourier transform second-harmonic-generation microscopy.
ABSTRACT Fourier transform second-harmonic generation (SHG) microscopy has been applied to quantitatively compare the information content between SHG images obtained from the forward and backward direction for three tissue types: porcine tendon, sclera, and ear cartilage. Both signal types yield consistent information on the preferred orientation of collagen fibers. For all specimens, the Fourier transform of the forward and backward SHG images produces several overlapping peaks in the magnitude spectrum at various depths into the tissues, indicating that some information present in the forward SHG images can be extracted from the backward SHG images. This study highlights the potential of backward SHG microscopy for medical diagnostics.
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ABSTRACT: We present a quantitative second-harmonic generation (SHG) imaging technique that quantifies the 2D spatial organization of collagen fiber samples under dynamic conditions, as an image is acquired. The technique is demonstrated for both a well-aligned tendon sample and a randomly aligned, sparsely distributed collagen scaffold sample. For a fixed signal-to-noise ratio, we confirm the applicability of this method for various window sizes (pixel areas) as well as with using a gridded overlay map that allows for correlations of fiber orientations within a given image. This work has direct impact to in vivo biological studies by incorporating simultaneous SHG image acquisition and analysis.Biomedical Optics Express 01/2013; 4(11):2546-2554. · 3.50 Impact Factor
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ABSTRACT: We demonstrate the use of Fourier transform-second-harmonic generation (FT-SHG) imaging of collagen fibers as a means of performing quantitative analysis of obtained images of selected spatial regions in porcine trachea, ear, and cornea. Two quantitative markers, preferred orientation and maximum spatial frequency are proposed for differentiating structural information between various spatial regions of interest in the specimens. The ear shows consistent maximum spatial frequency and orientation as also observed in its real-space image. However, there are observable changes in the orientation and minimum feature size of fibers in the trachea indicating a more random organization. Finally, the analysis is applied to a 3D image stack of the cornea. It is shown that the standard deviation of the orientation is sensitive to the randomness in fiber orientation. Regions with variations in the maximum spatial frequency, but with relatively constant orientation, suggest that maximum spatial frequency is useful as an independent quantitative marker. We emphasize that FT-SHG is a simple, yet powerful, tool for extracting information from images that is not obvious in real space. This technique can be used as a quantitative biomarker to assess the structure of collagen fibers that may change due to damage from disease or physical injury.Proceedings of SPIE - The International Society for Optical Engineering 02/2010; · 0.20 Impact Factor
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ABSTRACT: Optical imaging plays a major role in both basic biological research and clinical diagnostics, providing noninvasive or minimally invasive microscopic imaging capability to investigate biological tissues. Optical image acquisition through significant depths of biological tissues, however, presents a major challenge since tissue is extremely heterogeneous and the strong scattering of the various tissue components has restricted high-resolution optical imaging to superficial layers. Multiphoton microscopy (MPM) has significantly extended the penetration depth of high-resolution optical imaging, particularly for in vivo applications. Multiphoton imaging critically depends on ultrafast technologies, particularly pulsed excitation sources. In this paper, the basics of deep tissue MPM and its improvements utilizing soliton self-frequency shift (SSFS) are reviewed. Wavelength tunable, high-energy soliton generation through SSFS in large-mode-area (LMA) fibers and photonic crystal rods is presented. The application of these solitons to MPM enables noninvasive imaging in biological tissues with unprecedented depth. The main characteristics of the excitation source for deep tissue MPM, such as wavelength, pulse energy, and repetition rate, are discussed.IEEE Journal of Selected Topics in Quantum Electronics 03/2014; 20(2):6800311-6800311. · 3.47 Impact Factor