In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy

Yu, Che‐Hang; Tai, Shih-Peng; Kung, Chun-Ta; Wang, I‐Jong; Yu, Han-Chieh; Huang, Hsiang-Ju; Lee, Wei-Jei; Chan, Yi-Fan; Sun, Chi‐Kuang

doi:10.1364/oe.15.011167

Cited by 49 publications

(22 citation statements)

References 44 publications

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“…This core was filled by the SHG signal, arguing that the fiber was wide enough to produce an independent THG signal (induced by refraction index mismatch) on each side. A similar phenomenon was observed by others in the rat aortic wall, where autofluorescence from elastic fibers was surrounded by parallel THG signals [29].…”

Section: Resultssupporting

confidence: 86%

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

et al. 2011

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Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.

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Section: Resultssupporting

confidence: 86%

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

et al. 2011

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“…The term nonlinear microscopy now encompasses a broad class of techniques such as multiphoton-excited fluorescence imaging, multiple-harmonic generation imaging, and coherent anti-Stokes Raman imaging (Evans and others, 2007; Flusberg and others, 2005; Yu and others, 2007). While nonlinear microscopy has found applications in many biological studies, many in-vivo applications will require adapting this technology into an endoscopic format that requires delivering femtosecond optical pulses through optical fibers.…”

Section: Introductionmentioning

confidence: 99%

Ultrafast optical pulse delivery with fibers for nonlinear microscopy

Kim

Choi

Yazdanfar

et al. 2008

Microscopy Res & Technique

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Nonlinear microscopies including multiphoton excitation fluorescence microscopy and multipleharmonic generation microscopy have recently gained popularity for cellular and tissue imaging. The optimization of these imaging methods for minimally invasive use will require optical fibers to conduct light into tight space where free space delivery is difficult. The delivery of high peak power laser pulses with optical fibers is limited by dispersion resulting from nonlinear refractive index responses. In this paper, we characterize a variety of commonly used optical fibers in terms of how they affect pulse profile and imaging performance of nonlinear microscopy; the following parameters are quantified: spectral bandwidth and temporal pulse width, two-photon excitation efficiency, and optical resolution. A theoretical explanation for the measured performance of these is also provided.

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“…2(f)]. THG contrasts inside lamina propria revealed the moving erythrocytes in the capillary [18,19,33] due to the THG contrast on oxy-hemoglobin [34], as shown in Fig. 2(e) (arrowhead), which enables real-time observation of the capillary blood flow.…”

Section: Resultsmentioning

confidence: 99%

In vivo optical virtual biopsy of human oral mucosa with harmonic generation microscopy

Tsai

Chen

Shieh³

et al. 2011

Biomed. Opt. Express

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Recent clinical studies on human skin indicated that in vivo multi-harmonic generation microscopy (HGM) can achieve sub-micron resolution for histopathological analysis with a high penetration depth and leave no energy or photodamages in the interacted tissues. It is thus highly desired to apply HGM for in vivo mucosa histopathological diagnosis. In this paper, the first in vivo optical virtual biopsy of human oral mucosa by using epi-HGM is demonstrated. We modified an upright microscope to rotate the angle of objective for in vivo observation. Our clinical study reveals the capability of HGM to in vivo image cell distributions in human oral mucosa, including epithelium and lamina propria with a high penetration depth greater than 280 μm and a high spatial resolution better than 500 nm. We also found that the third-harmonic-generation (THG) contrast on nucleus depends strongly on its thicknesses, in agreement with a numerical simulation. Besides, 4% acetic acid was found to be able to enhance the THG contrast of nucleus in oral mucosa, while such enhancement was found to decay due to the metabolic clearance of the contrast enhancer by the oral mucosa. Our clinical study indicated that, the combined epi-THG and epi-second-harmonic-generation (SHG) microscopy is a promising imaging tool for in vivo noninvasive optical virtual biopsy and disease diagnosis in human mucosa.

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In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy

Cited by 49 publications

References 44 publications

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

Ultrafast optical pulse delivery with fibers for nonlinear microscopy

In vivo optical virtual biopsy of human oral mucosa with harmonic generation microscopy

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