3-dimensional imaging of collagen using second harmonic generation

Collagen is the most important structural protein of the animal body. Its unique triple-helix structure and extremely high level of crystallinity make it exceptionally efficient in generating the second harmonic of incident light, and we show here how this leads to a novel mode of microscopy of immediate practical significance in medicine and biology. In particular, it provides sensitive and high-resolution information on collagen distribution, discriminates between type I and type III collagen, and allows both a greater understanding of and a sensitive test for cirrhosis of the liver. Future research applications could include wound healing and hereditary collagen diseases such as osteogenesis imperfecta.     

Microscopic second harmonic generation (SHG) from tilt-placed collagen fibrils

We report a theoretical study of second-harmonic generation (SHG) from the collagen fibrils which have a tilt angle Φ between their axes with the plane of the polarization angle α of the linearly polarized focused light. The effects of Φ as well as α on SHG emission have been investigated. Our results show that the total strength of SHG electrical field (E(2ω)) and power (P(2ω)) reach their maximal values as Φ locates at a certain angle, here it is around 27° under all demonstrated α. When Φ < 27°, E(2ω) and P(2ω) gradually increase to their maximal value, while when Φ > 27°, they quickly drops. Specially, E(2ω) and P(2ω) are almost undetectable when Φ ≥ 45°. Also, Φ influences the distribution pattern of E(2ω), P(2ω) and their parallel (E(2ω,p), P(2ω,p)) and perpendicular (E(2ω,s), P(2ω,s)) components, such as the number of emission lobes and shape. α has unevenly impact on E(2ω) and P(2ω), the far deviation of α from x axis induce much great decrease of E(2ω) and P(2ω). α = 45° has special influence on the distribution pattern of E(2ω,s), E(2ω), P(2ω,s) as well as P(2ω) compared to those of α = 0° and 90°.     

Improved quantification of collagen anisotropy with polarization‐resolved second harmonic generation microscopy

Imaging tissue samples by polarization‐resolved second harmonic generation microscopy provides both qualitative and quantitative insights into collagen organization in a label‐free manner. Polarization‐resolved second harmonic generation microscopy goes beyond simple intensity‐based imaging by adding the laser beam polarization component and applying different quantitative metrics such as the anisotropy factor. It thus provides valuable information on collagen arrangement not available with intensity measurements alone. Current established approaches are limited to calculating the anisotropy factor for only a particular laser beam polarization and no general guidelines on how to select the best laser beam polarization have yet been defined. Here, we introduce a novel methodology for selecting the optimal laser beam polarization for characterizing tissues using the anisotropy in the purpose of identifying cancer signatures. We show that the anisotropy factor exhibits a similar laser beam polarization dependence to the second harmonic intensity and we combine it with the collagen orientation index computed by Fast Fourier Transform analysis of the recorded images to establish a framework for choosing the laser beam polarization that is optimal for an accurate interpretation of polarization‐resolved second harmonic generation microscopy images and anisotropy maps, and hence a better differentiation between healthy and dysplastic areas.

SHG image of skin tissue (a) and a selected area of interest for which we compute the SHG intensity (b) and anisotropy factor (c) dependence on the laser beam polarization and also the FFT spectrum (d) to evaluate the collagen orientation index.     

Quantitative second harmonic generation imaging of cartilage damage

Cartilage damage was studied using non-invasive multiphoton-excited autofluorescence and quantitative second harmonic generation (SHG) microscopy. Two cryopreservation techniques based upon freezing and vitrification methods, respectively, were employed to determine whether or not the collagen fiber structure of full thickness porcine articular cartilage was affected by cryopreservation and whether the level of collagen damage could be determined quantitatively in non-processed (non-fixed, non-sliced, non-stained) tissues. Multiphoton-induced autofluorescence imaging revealed the presence of chondrocytes, as well as collagenous structures in all fresh, vitrified and frozen cryopreserved cartilage samples. SHG imaging of the frozen cryopreserved specimens showed a dramatic loss of mean gray value intensities when compared to both fresh and vitrified tissues (P < 0.05), indicating structural changes of the extracellular matrix, in particular the deformation and destruction of the collagen fibers in the analyzed articular cartilage. A 0.9974 correlation coefficient was observed between the metabolic cell activity assessed by the alamarBlue technique, and retention of collagen structure between the three experimental groups. These studies suggest that multiphoton-induced autofluorescence imaging combined with quantitative SHG signal profiling may prove to be useful tools for the investigation of extracellular matrix changes in preserved cartilage, giving insights on the structural quality prior to implantation.     

Monitoring dynamic collagen reorganization during skin stretching with fast polarization‐resolved second harmonic generation imaging

The mechanical properties of biological tissues are strongly correlated to the specific distribution of their collagen fibers. Monitoring the dynamic reorganization of the collagen network during mechanical stretching is however a technical challenge, because it requires mapping orientation of collagen fibers in a thick and deforming sample. In this work, a fast polarization‐resolved second harmonic generation microscope is implemented to map collagen orientation during mechanical assays. This system is based on line‐to‐line switching of polarization using an electro‐optical modulator and works in epi‐detection geometry. After proper calibration, it successfully highlights the collagen dynamic alignment along the traction direction in ex vivo murine skin dermis. This microstructure reorganization is quantified by the entropy of the collagen orientation distribution as a function of the stretch ratio. It exhibits a linear behavior, whose slope is measured with a good accuracy. This approach can be generalized to probe a variety of dynamic processes in thick tissues.      

Bimetallic ferrocenyl derivatives: Molecular second order hyperpolarizabilities and second harmonic generation

The ground and excited states dipole moments and the second order hyperpolarizabilities of a series of oxazolinyl-ferrocenyl derivatives have been measured. After complexation of the oxazoline group with a second metal ion, the new bimetallic complexes show an increased hyperpolarizability, as determined by the EFISHG technique. A copolymer of the same bimetallic complex with methyl methacrylate has been synthesized, and the second order susceptibility has been measured by second harmonic generation at 1.06µm.     

Analyzing the feasibility of discriminating between collagen types I and II using polarization‐resolved second harmonic generation

According to previous studies, the nonlinear susceptibility tensor ratio χ₃₃/χ₃₁ obtained from polarization‐resolved second harmonic generation (P‐SHG) under the assumption of cylindrical symmetry can be used to distinguish between fibrillar collagen types. Discriminating between collagen fibrils of types I and II is important in tissue engineering of cartilage. However, cartilage has a random organization of collagen fibrils, and the assumption of cylindrical symmetry may be incorrect. In this study, we simulated the P‐SHG response from different collagen organizations and demonstrated a possible method to exclude areas where cylindrical symmetry is not fulfilled and where fibrils are located in the imaging plane. The χ₃₃/χ₃₁‐ratio for collagen type I in tendon and collagen type II in cartilage was estimated to be 1.33 and 1.36, respectively, using this method. These ratios are now much closer than what has been reported previously in the literature, and the larger reported differences between collagen types can be explained by variation in the structural organization.      

Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure

Second-harmonic generation (SHG) microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. Because of its underlying physical origin, it is highly sensitive to the collagen fibril/fiber structure, and, importantly, to changes that occur in diseases such as cancer, fibrosis and connective tissue disorders. We discuss how SHG can be used to obtain more structural information on the assembly of collagen in tissues than is possible by other microscopy techniques. We first provide an overview of the state of the art and the physical background of SHG microscopy, and then describe the optical modifications that need to be made to a laser-scanning microscope to enable the measurements. Crucial aspects for biomedical applications are the capabilities and limitations of the different experimental configurations. We estimate that the setup and calibration of the SHG instrument from its component parts will require 2-4 weeks, depending on the level of the user's experience.     

Sensitivity of second harmonic generation from styryl dyes to transmembrane potential

In this article we present results from the simultaneous nonlinear (second harmonic generation and two-photon excitation fluorescence) imaging and voltage clamping of living cells. Specifically, we determine the sensitivity to transmembrane potential of second harmonic generation by ANEP-chromophore styryl dyes as a function of excitation wavelength and dye structure. We have measured second harmonic sensitivities of up to 43% per 100 mV, more than a factor of four better than the nominal voltage sensitivity of the dyes under "one-photon" fluorescence. We find a dependence of voltage sensitivity on excitation wavelength that is consistent with a two-photon resonance, and there is a significant dependence of voltage sensitivity on the structure of the nonchromophore portion of the dyes.     

High-resolution nonlinear optical imaging of live cells by second harmonic generation

By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.     

Monitoring micrometer-scale collagen organization in rat-tail tendon upon mechanical strain using second harmonic microscopy

We continuously monitored the microstructure of a rat-tail tendon during stretch/relaxation cycles. To that purpose, we implemented a new biomechanical device that combined SHG imaging and mechanical testing modalities. This multi-scale experimental device enabled simultaneous visualization of the collagen crimp morphology at the micrometer scale and measurement of macroscopic strain-stress response. We gradually increased the ultimate strain of the cycles and showed that preconditioning mostly occurs in the first stretching. This is accompanied by an increase of the crimp period in the SHG image. Our results indicate that preconditioning is due to a sliding of microstructures at the scale of a few fibrils and smaller, that changes the resting length of the fascicle. This sliding can reverse on long time scales. These results provide a proof of concept that continuous SHG imaging performed simultaneously with mechanical assay allows analysis of the relationship between macroscopic response and microscopic structure of tissues.     

Optical clearing for improved contrast in second harmonic generation imaging of skeletal muscle

Using second harmonic generation (SHG) imaging microscopy, we have examined the effect of optical clearing with glycerol to achieve greater penetration into specimens of skeletal muscle tissue. We find that treatment with 50% glycerol results in a 2.5-fold increase in achievable SHG imaging depth. Signal processing analyses using fast Fourier transform and continuous wavelet transforms show quantitatively that the periodicity of the sarcomere structure is unaltered by the clearing process and that image quality deep in the tissue is improved with clearing. Comparison of the SHG angular polarization dependence also shows no change in the supramolecular organization of acto-myosin complexes. By contrast, identical treatment of mouse tendon (collagen based) resulted in a strong decrease in SHG response. We suggest that the primary mechanism of optical clearing in muscle with glycerol treatment results from the reduction of cytoplasmic protein concentration and concomitant decrease in the secondary inner filter effect on the SHG signal. The lack of glycerol concentration dependence on the imaging depth indicates that refractive index matching plays only a minor role in the optical clearing of muscle. SHG and optical clearing may provide an ideal mechanism to study physiology in highly scattering skeletal or cardiac muscle tissue with significantly improved depth of penetration and achievable imaging depth.     

Interest of second harmonic generation imaging for diagnosis in thick and opaque tissue

In articular hyaline cartilage, chondrocytes are surrounded by an extracellular matrix which is mainly composed by collagen and proteoglycanes. Pathological specimens show a partial or complete degradation of this matrix. Therefore, it could be interesting to know how mechanical or biochemical constraints applied to cartilage specimens induce modifications of the cartilage network. Multiphoton technology combined to Second Harmonic Generation (SHG) enables to image cartilage specimens in a non-invasive mode with high resolution at deep penetration. By placing a band pass filter in front of the transmitted light detector, SHG signal with frequency doubled can be isolated for a new contrast imaging. SHG (second harmonic generation) is a diffusion process generated from organized structures and does not need any fluorescent staining. Due to their non-centrosymetric structure, collagen fibrilles present a high second-order non-linear susceptibility and thus give rise to a strong SHG signal when exposed to high enough electric fields produced by a focal point of a femtosecond pulsed laser (multiphoton microscopy). As the extracellular matrix of cartilage is in part constituted by collagen fibers, it can be imaged with this contrast tool. The intensity of SHG signals strongly depends on the organization of collagen fibers. Thus a modification of the extracellular matrix in terms of 3D-organization of collagen induced by mechanical stress can be shown with this contrast tool.     

Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections

We performed second harmonic generation (SHG) imaging of collagen in rat-tendon cryosections, using femtosecond laser scanning confocal microscopy, both in backscattering and transmission geometries. SHG transmission images of collagen fibers were spatially resolved due to a coherent, directional SHG component. This effect was enhanced with the use of an index-matching fluid (n(i) = 1.52). The average SHG intensity oscillated with wavelength in the backscattered geometry (isotropic SHG component), whereas the spectral profile was consistent with quasi-phase-matching conditions in transmission geometry (forward propagating, coherent SHG component) around 440 nm (lambda(p) = 880 nm). Collagen type I from bovine Achilles tendon was imaged for SHG in the backscattered geometry and its first-order effective nonlinear coefficient was determined (|d(eff)| approximately 0.085(+/-0.025)x10(-12)mV(-1)) by comparison to samples of inorganic materials with known effective nonlinear coefficients (LiNbO3 and LiIO3). The SHG spectral response of collagen type I from bovine Achilles tendon matched that of the rat-tendon cryosections in backscattered geometry. Collagen types I, II, and VI powders (nonfibrous) did not show any detectable SHG, indicating a lack of noncentrosymmetric crystalline structure at the molecular level. The various stages of collagen thermal denaturation were investigated in rat-tendon cryosections using SHG and bright-field imaging. Thermal denaturation resulted in the gradual destruction of the SHG signal.     

Polarization second harmonic generation microscopy provides quantitative enhanced molecular specificity for tissue diagnostics

Due to specific structural organization at the molecular level, several biomolecules (e.g., collagen, myosin etc.) which are strong generators of second harmonic generation (SHG) signals, exhibit unique responses depending on the polarization of the excitation light. By using the polarization second harmonic generation (p‐SHG) technique, the values of the second order susceptibility components can be used to differentiate the types of molecule, which cannot be done by the use of a standard SHG intensity image. In this report we discuss how to implement p‐SHG on a commercial multiphoton microscope and overcome potential artifacts in susceptibility (χ) image. Furthermore we explore the potential of p‐SHG microscopy by applying the technique to different types of tissue in order to determine corresponding reference values of the ratio of second‐order χ tensor elements. These values may be used as a bio‐marker to detect any structural alterations in pathological tissue for diagnostic purposes.

The SHG intensity image (red) in ( a ) shows the distribution of collagen fibers in ovary tissue but cannot determine the type of collagen fiber. However, the histogram distribution ( b ) for the values of the χ tensor element ratio can be used to quantitatively identify the types of collagen fibers.     

Second harmonic generation microscopy reveals altered collagen microstructure in usual interstitial pneumonia versus healthy lung

Background

It is not understood why some pulmonary fibroses such as cryptogenic organizing pneumonia (COP) respond well to treatment, while others like usual interstitial pneumonia (UIP) do not. Increased understanding of the structure and function of the matrix in this area is critical to improving our understanding of the biology of these diseases and developing novel therapies. The objectives herein are to provide new insights into the underlying collagen- and matrix-related biological mechanisms driving COP versus UIP.

Methods

Two-photon second harmonic generation (SHG) and excitation fluorescence microscopies were used to interrogate and quantify differences between intrinsic fibrillar collagen and elastin matrix signals in healthy, COP, and UIP lung.

Results

Collagen microstructure was different in UIP versus healthy lung, but not in COP versus healthy, as indicated by the ratio of forward-to-backward propagating SHG signal (F_SHG/B_SHG). This collagen microstructure as assessed by F_SHG/B_SHG was also different in areas with preserved alveolar architecture adjacent to UIP fibroblastic foci or honeycomb areas versus healthy lung. Fibrosis was evidenced by increased col1 and col3 content in COP and UIP versus healthy, with highest col1:col3 ratio in UIP. Evidence of elastin breakdown (i.e. reduced mature elastin fiber content), and increased collagen:mature elastin ratios, were seen in COP and UIP versus healthy.

Conclusions

Fibrillar collagen’s subresolution structure (i.e. “microstructure”) is altered in UIP versus COP and healthy lung, which may provide novel insights into the biological reasons why unlike COP, UIP is resistant to therapies, and demonstrates the ability of SHG microscopy to potentially distinguish treatable versus intractable pulmonary fibroses.     

A comprehensive optical second harmonic generation study of the non-centrosymmetric character of biological structures

The application of optical second harmonic generation (SHG) to the study of non-centrosymmetry in 71α-amino acids, 22 dipeptides, 6 tripeptides, 16 proteins, and 5 viruses is reported. Allα-amino acids exceptα-glycine, all peptides containing at least one enantiomericα-amino acid residue, and all proteins and viruses are found to be non-centrosymmetric. Of the 22 racemicα-amino acids investigated, 19 form centrosymmetric crystalline racemates. DL-alanine and DL-norleucine form non-centrosymmetric racemates whereas DL-glutamic acid does not form racemic crystals except when hydrated. These results lead us to suggest that non-centrosymmetry and its consequences (i.e., allowable piezoelectricity and possible pyroelectricity) is an essential and universal property of all biological systems. Furthermore, the concept of polarization homeostasis (i.e., biomolecules in vivo have a macroscopic equilibrium polarization in which the polarization ≠0) is proposed as a basic biological phenomenon and shown to be consistent with the dominance of one enantiomeric configuration ofα-amino acids in nature, natural selection and mutationally induced molecular diseases.     

Spatial orientation mapping of fibers using polarization‐sensitive second harmonic generation microscopy

In this work, we present a non‐invasive approach to determine azimuth and elevation angles of collagen fibers capable of generating second harmonic signal. The azimuth angle was determined using the minimum of second harmonic generation (SHG) signal while rotating the plane of polarization of excitation light. The elevation angle was estimated from the ratio of the minimal SHG intensity to the intensity when laser polarization and fiber directions were parallel to each other using experimentally determined calibration curve. Pixel‐resolution images of collagen fiber spatial orientation in tendon from bovine leg, chicken leg, and chicken skin were acquired using our approach of SHG polarization‐resolved microscopy. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)