Article

Quantitative analysis of forward and backward second-harmonic images of collagen fibers using Fourier transform second-harmonic-generation microscopy.

Laboratory for Photonics Research of Bio/nano Environments (PROBE), Department of Electrical and Computer Engineering,University of Illinois Urbana-Champaign, 1406 W Green Street, Urbana, Illinois 61801, USA.
Optics Letters (Impact Factor: 3.18). 12/2009; 34(24):3779-81. DOI: 10.1364/OL.34.003779
Source: PubMed

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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    • "In order to examine SHG microscopy's potential for assessing bone structure, we choose an example study where we quantify the structural changes in the collagen fiber organization of bone due to age. For quantification, we employ an approach called Fourier transformsecond harmonic generation (FT-SHG) imaging— recently developed for quantifying collagen fiber organization in biological tissues [26] [32] [33]. Here, relative collagen fiber orientation is considered as a parameter for quantification. "
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    ABSTRACT: We propose the use of second-harmonic generation (SHG) microscopy for imaging collagen fibers in porcine femoral cortical bone. The technique is compared with scanning electron microscopy (SEM). SHG microscopy is shown to have excellent potential for bone imaging primarily due its intrinsic specificity to collagen fibers, which results in high contrast images without the need for specimen staining. Furthermore, this technique's ability to quantitatively assess collagen fiber organization is evaluated through an exploratory examination of bone structure as a function of age, from very young to mature bone. In particular, four different age groups: 1 month, 3.5 months, 6 months, and 30 months, were studied. Specifically, we employ the recently developed Fourier transform-second harmonic generation (FT-SHG) imaging technique for the quantification of the structural changes, and observe that as the bone develops, there is an overall reduction in porosity, the number of osteons increases, and the collagen fibers become comparatively more organized. It is also observed that the variations in structure across the whole cross-section of the bone increase with age. The results of this work show that quantitative SHG microscopy can serve as a valuable tool for evaluating the structural organization of collagen fibers in ex vivo bone studies.
    Bone 12/2011; 50(3):643-50. DOI:10.1016/j.bone.2011.11.013 · 4.46 Impact Factor
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    • "Second-harmonic generation (SHG) images of collagen fibers in keratoconic corneas have shown both significant thinning and a decrease in the number of collagen lamellae in the stroma as well as breaks in the lamellae of the Bowman's layer as shown in Fig. 3 (Morishige et al., 2007). SHG microscopy, in recent years has become a popular imaging technique to study the structural organization of collagen fibers (Ambekar Ramachandra Rao et al., 2009a,b; Campagnola and Loew, 2003). Thinning of the collagen lamellae has been attributed to the decrease in the number of cross-links (bonds between and within collagen fibrils) (Meek et al., 2005). "
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    04/2011; 4(3):223-36. DOI:10.1016/j.jmbbm.2010.09.014
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    • "collagen from the fluorescence signal and from the backward SHG signal [6]. The detected fluorescence was first obtained from the autofluorescence signal with respect to cellular properties or scaffold material (constitutive type-1 collagen) and second from fluorophores coupled to antibody specific type-2 collagen (cell synthesis). "
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    ABSTRACT: We propose an innovative invasiveless technique in the field of nonlinear optical imaging to facilitate monitoring of cell/scaffold combinations for tissue repair. By using a near infrared (NIR) femtosecond excitation, we were able to introduce a new index based on decay time response for fluorescence (F) and Second Harmonic Generation (SHG) obtained with Time Correlated Single Photon Counting (TCSPC) microscopy to monitor structural information on the state of the matrix collagen. Some human Mesenchymal Stem Cells (hMSCs) seeded in 3D scaffolds were tested with different culture times (from D7 to D56) to analyze the effect of Tumor Growth Factor beta 1 (TGF-β1) on type-2 collagen expression in the matrix. After 14 days in the presence of TGF-β1, our results showed an increase in the expression of type-2 collagen synthesized by hMSCs, and a change in collagen conformation, as an indication of its ability to be detected as a harmonophore by TCSPC-SHG without the need for an exogenous probe.
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