Patrick Stoller

Polarization-Modulated Second Harmonic Generation in Collagen

Collagen possesses a strong second-order nonlinear susceptibility, a nonlinear optical property characterized by second harmonic generation in the presence of intense laser beams. We present a new technique involving polarization modulation of an ultra-short pulse laser beam that can simultaneously determine collagen fiber orientation and a parameter related to the second-order nonlinear susceptibility. We demonstrate the ability to discriminate among different patterns of fibrillar orientation, as exemplified by tendon, fascia, cornea, and successive lamellar rings in an intervertebral disc. Fiber orientation can be measured as a function of depth with an axial resolution of approximately 10 microm. The parameter related to the second-order nonlinear susceptibility is sensitive to fiber disorganization, oblique incidence of the beam on the sample, and birefringence of the tissue. This parameter represents an aggregate measure of tissue optical properties that could potentially be used for optical imaging in vivo.

Polarization-dependent optical second-harmonic imaging of a rat-tail tendon.

Using scanning confocal microscopy, we measure the backscattered second harmonic signal generated by a 100 fs laser in rat-tail tendon collagen. Damage to the sample is avoided by using a continuous scanning technique, rather than measuring the signal at discrete points. The second harmonic signal varies by about a factor of 2 across a single cross section of the rat-tail tendon fascicle. The signal intensity depends both on the collagen organization and the backscattering efficiency. This implies that we cannot use intensity measurements alone to characterize collagen structure. However, we can infer structural information from the polarization dependence of the second harmonic signal. Axial and transverse scans for different linear polarization angles of the input beam show that second harmonic generation (SHG) in the rat-tail tendon depends strongly on the polarization of the input laser beam. We develop an analytical model for the SHG as a function of the polarization angle in the rat-tail tendon. We apply this model in determining the orientation of collagen fibrils in the fascicle and the ratio gamma between the two independent elements of the second-order nonlinear susceptibility tensor. There is a good fit between our model and the measured data.

Quantitative second-harmonic generation microscopy in collagen

The second-harmonic signal in collagen, even in highly organized samples such as rat tail tendon fascicles, varies significantly with position. Previous studies suggest that this variability may be due to the parallel and antiparallel orientation of neighboring collagen fibrils. We applied high-resolution second-harmonic generation microscopy to confirm this hypothesis. Studies in which the focal spot diameter was varied from approximately 1 to approximately 6 microm strongly suggest that regions in which collagen fibrils have the same orientation in rat tail tendon are likely to be less than approximately 1 microm in diameter. These measurements required accurate determination of the focal spot size achieved by use of different microscope objectives; we developed a technique that uses second-harmonic generation in a quartz reference to measure the focal spot diameter directly. We also used the quartz reference to determine a lower limit (dXXX > 0.4 pm/V) for the magnitude of the second-order nonlinear susceptibility in collagen.

Effects of high repetition rate and beam size on hard tissue damage due to subpicosecond laser pulses

We report the effects of the repetition rate and the beam size on the threshold for ultrashort laser pulse induced damage in dentin. The observed results are explained as cumulative thermal effects. Our model is consistent with the experimental results and explains the dependence of the threshold on repetition rate, beam size, and exposure time.

Imaging collagen orientation using polarization-modulated second harmonic generation

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.

Confocal imaging of biological tissues using second harmonic generation

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.

Effect of structural modification on second harmonic generation in collagen

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.

Measurement of the second-order nonlinear susceptibility of collagen using polarization modulation and phase-sensitive detection

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.

Three-Dimensional Geometry of Collagenous Tissues by Second Harmonic Polarimetry

Collagen is a biological macromolecule capable of second harmonic generation, allowing label-free detection in tissues; in addition, molecular orientation can be determined from the polarization dependence of the second harmonic signal. Previously we reported that in-plane orientation of collagen fibrils could be determined by modulating the polarization angle of the laser during scanning. We have now extended this method so that out-of-plane orientation angles can be determined at the same time, allowing visualization of the 3-dimensional structure of collagenous tissues. This approach offers advantages compared with other methods for determining out-of-plane orientation. First, the orientation angles are directly calculated from the polarimetry data obtained in a single scan, while other reported methods require data from multiple scans, use of iterative optimization methods, application of fitting algorithms, or extensive post-optical processing. Second, our method does not require highly specialized instrumentation, and thus can be adapted for use in almost any nonlinear optical microscopy setup. It is suitable for both basic and clinical applications. We present three-dimensional images of structurally complex collagenous tissues that illustrate the power of such 3-dimensional analyses to reveal the architecture of biological structures.

Second harmonic generation in collagen

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.