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: 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 01/2014; 20(2):6800311-6800311. · 4.08 Impact Factor
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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. · 3.18 Impact Factor
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ABSTRACT: Injuries and damage to tendons plagues both human and equine athletes. At the site of injuries, various cells congregate to repair and re-structure the collagen. Treatments for collagen injury range from simple procedures such as icing and pharmaceutical treatments to more complex surgeries and the implantation of stem cells. Regardless of the treatment, the level of mechanical stimulation incurred by the recovering tendon is crucial. However, for a given tendon injury, it is not known precisely how much of a load should be applied for an effective recovery. Both too much and too little loading of the tendon could be detrimental during recovery. A mapping of the complex local environment imparted to any cell present at the site of a tendon injury may however, convey fundamental insights related to their decision making as a function of applied load. Therefore, fundamentally knowing how cells translate mechanical cues from their external environment into signals regulating their functions during repair is crucial to more effectively treat these types of injuries. In this paper, we examined systems of tendons with a variety of 2-photon-based imaging techniques to examine the local mechanical environment of cells in both normal and injured tendons. These tendons were chemically treated to instigate various extents of injury and in some cases, were injected with stem cells. The results related by each imaging technique distinguish with high contrast and resolution multiple morphologies of the cells' nuclei and the alignment of the collagen during injury. The incorporation of 2-photon FLIM into this study probed new features in the local environment of the nuclei that were not apparent with steady-state imaging. Overall, this paper focuses on horse tendon injury pattern and analysis at different 2photon confocal modalities useful for wide variety of application in damaged tissues.Methods 07/2013; · 3.64 Impact Factor