A novel signal processing algorithm for quantifying structural disorder in biological tissue using second harmonic generation (SHG) imaging is described. Both the magnitude and the pattern of disorder in collagenous tissues can be determined with this method. Mathematical models are used to determine the range of disordered states over which the algorithm can be used, because highly disordered biological samples do not generate second harmonic signals. The method is validated by measuring disorder in heated fascicles using SHG and showing that results are significantly correlated with morphometric determination. Applicability of the method to tissue pathology is demonstrated by analysis of a mouse model of intervertebral disk injury. Disks were subjected to tensile or compressive forces in vivo for one week. Structural disorder in the annulus fibrosus was measured by SHG scanning and by standard morphometric analysis. Values for disorder obtained by SHG scanning were significantly correlated with values obtained by morphometry (p<0.001). Quantitation of disorder using SHG offers significant advantages over morphometric determination. Data obtained in this study suggest that this method can be used to discriminate between reversible and irreversible tissue damage.
Collagen possesses a strong second order nonlinear susceptibility; when it is irradiated with intense laser light, some of the reflected and transmitted light will have twice the frequency of the incident beam, a phenomenon known as second harmonic generation (SHG). Polarization modulation of an ultra-short pulse laser beam can be used to simultaneously measure collagen fiber orientation, SHG intensity, and a parameter related to the second order non-linear susceptibility. This technique has made it possible to discriminate among patterns of fibrillar orientation in many tissues. In the present study the role that organizational complexity plays in the relationship between nonlinear optical properties and collagen structure is investigated. As a component of tissues and organs, collagen’s structure and function is inextricably intertwined with that of the many other matrix components; to what extent do these noncollagenous components affect its nonlinear properties? To answer this, we investigated SHG in two different collagenous tissues, liver and cartilage; in addition we looked at the effect of progressive pathological changes in these tissues on SHG. At the other end of the spectrum, we studied collagen organized at the minimal level of complexity necessary for SHG detection: fibrils generated from solutions containing only a single type of collagen. Data obtained from these studies suggest that collagen’s strong nonlinear susceptibility, a property no other biologically significant macromolecule shares to the same degree, may serve as more than the basis of a novel imaging device for soft tissue. Collagen’s nonlinear optical properties in conjunction with its vast capacity for self-initiated conformational change--through self-assembly, site recognition, post-translational modification, and the like -make it an attractive candidate molecule for any of several demanding engineering applications, such as nanopatterning.
The effects of structural perturbation on second harmonic generation in collagen were investigated. Type I collagen fascicles obtained from rat tails were structurally modified by increasing nonenzymatic cross-linking, by thermal denaturation, by collagenase digestion, or by dehydration. Changes in polarization dependence were observed in the dehydrated samples. Surprisingly, no changes in polarization dependence were observed in highly crosslinked samples, despite significant alterations in packing structure. Complete thermal denaturation and collagenase digestion produced samples with no detectable second harmonic signal. Prior to loss of signal, no change in polarization dependence was observed in partially heated or digested collagen.
We use polarization-modulated second harmonic generation to image fiber orientation in collagen tissues, with an axial resolution of about 10 micrometers and a transverse resolution of up to 1 micrometers . A linearly polarized ultra-short pulse (200 fs) Ti:Sapphire laser beam is modulated using an electro-optic modulator and quarter-wave plate combination and focused onto a translation stage mounted sample using a microscope objective. The generated second harmonic light is collected using a photomultiplier tube and demodulated using phase sensitive detection to obtain signal intensity and fiber orientation information. In order to obtain second harmonic generation images of different types of collagen organization, we analyze several different tissues, including rat-tail tendon, mouse aorta, mouse fibrotic liver, and porcine skin. We can use our technique to image fibrotic tissue in histological sections of damaged liver and to identify burned tissue in porcine skin to a depth of a few hundred microns. Polarization-modulated second harmonic generation potentially could be a useful clinical technique for diagnosing collagen related disease or damage, especially in the skin.
The measurement of the second order nonlinear susceptibility of collagen in various biological tissues has potential applications in the detection of structural changes which are related to different pathological conditions. We investigate second harmonic generation in a rat-tail tendon, a highly organized collagen structure consisting of parallel fibers. Using an electro-optic modulator and a quarter-wave plate, we modulate the linear polarization of an ultra-short pulse laser beam that is used to measure second harmonic generation in a confocal microscopy setup. Phase-sensitive detection of the generated signal, coupled with a simple model of the collagen protein structures, allows us to measure a parameter (gamma) related to nonlinear susceptibility and to determine the relative orientation of the structures. Our preliminary results indicate that it may be possible to use this parameter to characterize the structure.
A confocal microscopy imaging system was devised to selectively detect second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing.
A confocal microscopy imaging system was devised to selectively detect second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing.
KEYWORDS: Argon ion lasers, Feedback control, Collagen, Tissues, Optical fibers, In vivo imaging, Control systems, Natural surfaces, Veins, Infrared radiation
An in vivo study of vascular welding with a fiber-delivered argon laser was conducted using a canine model. Longitudinal arteriotomies and venotomies were treated on femoral vein and artery. Laser energy was delivered to the vessel wall via a 400 micrometer optical fiber. The surface temperature at the center of the laser spot was monitored in real time using a hollow glass optical fiber-based two-color infrared thermometer. The surface temperature was limited by either a room-temperature saline drip or direct feedback control of the laser using a mechanical shutter to alternately pass and block the laser. Acute patency was evaluated either visually (leak/no leak) or by in vivo burst pressure measurements. Biochemical assays were performed to investigate the possible laser-induced formation or destruction of enzymatically mediated covalent crosslinks between collagen molecules. Viable welds were created both with and without the use of feedback control. Tissues maintained at 50 degrees Celsius using feedback control had an elevated crosslink count compared to controls, while those irradiated without feedback control experienced a decrease. Differences between the volumetric heating associated with open and closed loop protocols may account for the different effects on collagen crosslinks. Covalent mechanisms may play a role in argon laser vascular fusion.
The strength and stability of laser-welded tissue may be influenced, in part, by the effects of laser exposure on collagen crosslinking. We therefore studied the effects of diode laser exposure (805 nm, 1 - 8 watts, 30 seconds) plus indocyanine green dye (ICG) on calf tail tendon collagen crosslinks. The effect of ICG dye alone on crosslink content prior to laser exposure was investigated; unexpectedly, we found that ICG-treated tissue had significantly increased DHLNL and OHP, but not HLNL. Laser exposure after ICG application reduced elevated DHLNL and OHP crosslink content down to their native levels. The monohydroxylated crosslink HLNL was inversely correlated with laser output (p less than 0.01 by linear regression analysis). DHLNL content was highly correlated with content of its maturational product, OHP, suggesting that precursor-product relationships are maintained. We conclude that: (1) ICG alone induces DHLNL and OHP crosslink formation; (2) subsequent laser exposure reduces the ICG-induced crosslinks down to native levels; (3) excessive diode laser exposure destroys normally occurring HLNL crosslinks.
Nonenzymatic glycation alters collagen throughout the body, resulting in the histopathology that underlies diabetic disease in several organs. In the eye such changes in vitreous collagen could contribute to the progression of proliferative diabetic retinopathy by inducing vitreous degeneration. In this study, near infrared Fourier-transform Raman spectroscopy was performed on vitreous obtained at surgery from diabetic patients and from non-diabetic control subjects. The findings were compared to measurements obtained in untreated and glycated (in vitro) rat-tail tendon collagen, as well as demineralized chick bone, rich in crosslinks. The results demonstrated substantial changes in diabetic vitreous collagen not resulting from enzyme-mediated crosslinking, but most likely advanced nonenzymatic glycation. This approach appears to be useful as a means of characterizing the molecular changes induced by diabetes. Furthermore, this technique could be developed as a way of quantifying these changes in vivo in several tissues, so as to gauge the severity of disease and monitor the response to therapy.
In diabetes nonenzymatic glycation alters collagen throughout the body resulting in the histopathology that underlies diabetic disease in several organs. In the eye such changes in vitreous collagen could contribute to vitreous degeneration and the progression of proliferative diabetic retinopathy. Previous studies have demonstrated early glycation and advanced endproducts in the vitreous of humans with proliferative diabetic retinopathy. Near-infrared Fourier-transform Raman spectroscopy was performed on vitreous obtained at surgery from diabetic patients and from non-diabetic control subjects. The findings were compared to measurements obtained in untreated and glycated (in vitro) rat-tail tendon collagen. The results demonstrated substantial changes in diabetic vitreous collagen resulting from glycation, most likely advanced glycation endproducts. This approach appears to be useful as a means of characterizing the molecular changes induced by diabetes. Furthermore, this technique could be developed as a way of quantifying these changes in vivo in several tissues, so as to gauge the severity of non-enzymatic glycation and monitor the response to therapy.
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