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.     

Non-Linear Optical Microscopy Sheds Light on Cardiovascular Disease

Many cardiac diseases have been associated with increased fibrosis and changes in the organization of fibrillar collagen. The degree of fibrosis is routinely analyzed with invasive histological and immunohistochemical methods, giving a limited and qualitative understanding of the tissue''s morphological adaptation to disease. Our aim is to quantitatively evaluate the increase in fibrosis by three-dimensional imaging of the collagen network in the myocardium using the non-linear optical microscopy techniques Two-Photon Excitation microscopy (TPE) and Second Harmonic signal Generation (SHG). No sample staining is needed because numerous endogenous fluorophores are excited by a two-photon mechanism and highly non-centrosymmetric structures such as collagen generate strong second harmonic signals. We propose for the first time a 3D quantitative analysis to carefully evaluate the increased fibrosis in tissue from a rat model of heart failure post myocardial infarction. We show how to measure changes in fibrosis from the backward SHG (B_SHG) alone, as only backward-propagating SHG is accessible for true in vivo applications. A 5-fold increase in collagen I fibrosis is detected in the remote surviving myocardium measured 20 weeks after infarction. The spatial distribution is also shown to change markedly, providing insight into the morphology of disease progression.     

The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy

In this work, we report the implementation of interferometric second harmonic generation (SHG) microscopy with femtosecond pulses. As a proof of concept, we imaged the phase distribution of SHG signal from the complex collagen architecture of juvenile equine growth cartilage. The results are analyzed in respect to numerical simulations to extract the relative orientation of collagen fibrils within the tissue. Our results reveal large domains of constant phase together with regions of quasi-random phase, which are correlated to respectively high- and low-intensity regions in the standard SHG images. A comparison with polarization-resolved SHG highlights the crucial role of relative fibril polarity in determining the SHG signal intensity. Indeed, it appears that even a well-organized noncentrosymmetric structure emits low SHG signal intensity if it has no predominant local polarity. This work illustrates how the complex architecture of noncentrosymmetric scatterers at the nanoscale governs the coherent building of SHG signal within the focal volume and is a key advance toward a complete understanding of the structural origin of SHG signals from tissues.     

Characterization of lens capsule collagen: evidence for the presence of two unique chains in molecules derived from major basement membrane structures

A collagen fraction representing two-thirds of the collagenous sequences in bovine lens capsules has been isolated following limited pepsin digestion and purified by DEAE- and carboxymethyl-cellulose chromatography in native form. The denaturation products of this collagen contain two types of components. The more acidic components (C and 50K₁) are, respectively an α-chain-sized collagenous polypeptide and a mixture of smaller molecular weight proteolytic cleavage products of the C chain. The more basic components (80K and 50K₂) represent, respectively, a collagenous polypeptide with an apparent M_r = 80,000 and a mixture of smaller molecular weight components derived through proteolysis of the 80K component. The C chain and 80K components are unique with respect to chromatographic properties, amino acid composition, and cyanogen bromide cleavage products. These data are interpreted to indicate that lens capsule basement membrane collagen molecules collectively contain at least two genetically distinct collagen chains: the C chain representing the collagenous domain of one type of chain and the 80K component representing the major portion of the collagenous domain of a second type of chain, designated the D chain.     

Connective-tissue macromolecules in Golgi chicken tendon organs and at their interface with muscle fibers and adjoining tendinous structures.

Tendon organs from leg and forearm muscles of white leghorn chickens were examined with a library of monoclonal antibodies to determine the composition of their connective-tissue framework and the types of connective-tissue macromolecules that occur at the sites where muscle fibers attach to the receptors. The capsules of the tendon organs were positive for connective-tissue macromolecules typical of basal lamina (collagen type IV, laminin, and heparin sulfate proteoglycan) and for tenascin, collagen types III and VI, and fibronectin. Connective-tissue bundles in the lumen of a receptor reacted primarily with antibodies against collagen type I and 4-chondroitin sulfate. The narrow partitions that divide each lumen into compartments stained for collagen type III. Toward its tendinous end, a receptor made few contacts with muscle fibers. Instead, the capsule and the collagenous bundles blended gradually with the intermuscular portions of tendons. At the muscular end, the connections were more complex. Muscle fibers that attached in series to tendon organs split to produce basal lamina-covered, finger-like extensions, which were separated from each other by fissures. Tongues of connective tissue containing tenascin, collagen types I and VI, and fibronectin extended into the fissures. Distally the tongues were continuous with the tenascin in the capsule and just internal to the capsule, fibronectin and basal lamina macromolecules in the capsule, and collagen type I in the collagenous bundles. The uninterrupted presence of these macromolecules around terminating muscle fibers and in the capsule and/or the intraluminal collagen bundles suggests that muscle fibers that attach in series at the muscular end exert a force during muscular contraction on the intraluminal collagen bundles and on the receptor capsule.     

Three-dimensional structure of type IV collagen in the mammalian lens capsule.

The anterior lens capsule provides a thick, easily handled model system for the study of the organization of type IV collagen, the main component of basement membranes. We have used the technique of rapid freezing, deep-etch, and rotary replication to study the three-dimensional organization of the collagen skeleton in mammalian lens capsule after a variety of extraction procedures. In all cases the collagen appeared as a densely packed three-dimensional branching network of fine microfibrils. The organization of the microfibrils appears to show some regularity, with branch points approximately 40 nm apart. Most junctions are three-way and the network forms predominantly five-sided figures. This closely resembles the collagenous network described by Yurchenco and Ruben (1987, 1988) in human amniotic basement membrane and EHS tumor matrix, but extends their findings to another system for which X-ray diffraction data are available. The three-dimensional network is discussed in terms of molecular packing of type IV collagen in light of the information available from the diffraction data.     

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.     

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.     

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.     

Collagens serve as an extracellular store of bioactive interleukin 2

The binding of certain growth factors and cytokines to components of the extracellular matrix can regulate their local availability and modulate their biological activities. We show that interleukin 2 (IL-2), an important stimulator of T cell growth, preferentially binds to collagen types I, III, and VI and to a lesser degree to collagen types II, IV, and V, immobilized on polystyrene or nitrocellulose. These interactions are inhibited by denatured, single collagen chains or a subset of their cyanogen bromide peptides in a dose-dependent manner. Cross-inhibition experiments and ligand blotting of collagen-derived peptides point to a limited set of collagenous consensus sequences mediating the binding of IL-2. This interaction is saturable, with dissociation constants of approximately 10(-)(8) m, and estimated molar ratios of 4-6 molecules of IL-2 bound to one molecule of triple helical collagen. Furthermore, collagen-bound IL-2 stimulates proliferation of mouse lymphocytes. We conclude that its specific binding to the abundant interstitial collagens leads to a spatial pattern of bioavailable IL-2 which is dictated by the local organization of the collagenous extracellular matrix. This interaction may contribute to the particular phenotype of stromal lymphocytes and could be exploited for devising collagenous peptide analogues that modulate IL-2 bioactivity.     

Macromolecular organization of bovine lens capsule

Rabbit antisera to type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin were used to localize these proteins in cross-sections of bovine anterior lens capsule. The antisera were exposed to (a) 10-micron frozen-thawed sections of formaldehyde-fixed tissue for examination in the light microscope by the indirect immunofluorescence method and (b) formaldehyde-fixed and L. R. White plastic-embedded thin sections for electron microscopic examination by the protein A-gold technique. The intensity of immunofluorescence was both uniform and strong throughout for type IV collagen, laminin and entactin, but patchy and weak for fibronectin. Electron microscopic immunolabeling with protein A-gold showed that all five components were distributed throughout the full thickness of the membrane, albeit the density of gold particles was not identical for all basement membrane proteins. In general, the number of particles per micron2 was greatest for type IV collagen and entactin, moderate for laminin and heparan sulfate proteoglycan and low for fibronectin. The ultrastructure of the lens capsule as examined by the electron microscope revealed a relatively uniform parallel alignment of filaments, thought to be collagenous. Since the distribution of the filaments corresponds well with the observed immunocytochemical pattern it is concluded that type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin co-localize throughout the cross-section of the anterior lens capsule.     

The use of small angle light scattering in assessing strain induced collagen degradation in arterial tissue ex vivo

Collagen is the predominant load bearing component in many soft tissues including arterial tissue and is therefore critical in determining the mechanical integrity of such tissues. Degradation of collagen fibres is hypothesized to be a strain dependent process whereby the rate of degradation is affected by the magnitude of strain applied to the collagen fibres. The aim of this study is to investigate the ability of small angle light scattering (SALS) imaging to identify strain dependent degradation of collagen fibres in arterial tissue ex vivo, and determine whether a strain induced protection mechanism exists in arterial tissue as observed in pure collagen and other collagenous tissues. SALS was used in combination with histological and second harmonic generation (SHG) analysis to determine the collagen fibre architecture in arterial tissue subjected to strain directed degradation. SALS alignment analysis identified statistically significant differences in fibre alignment depending on the strain magnitude applied to the tissue. These results were also observed using histology and SHG. Our findings suggest a strain protection mechanism may exist for arterial collagen at intermediate strain magnitudes between 0% and 25%. These findings may have implications for the onset and progression of arterial disease where changes in the mechanical environment of arterial tissue may lead to changes in the collagen degradation rate.     

Effects of collagen density on cardiac fibroblast behavior and gene expression

Interactions between cells and the extracellular matrix (ECM) play essential roles in modulating cell behavior during development and disease. The myocardial ECM is composed predominantly of interstitial collagen type I and type III. The composition, organization, and accumulation of these collagens are altered concurrent with cardiovascular development and disease. Changes in these parameters are thought to play significant roles in myocardial function. While a number of studies have examined how changes in the ECM affect myocardial function as a whole, much less is known regarding the response at the cellular level to changes in the collagenous ECM. Experiments were carried out to determine the effects of alterations in collagen density and ECM stiffness on the behavior of isolated heart fibroblasts. In vitro bioassays were performed to measure the effects of changes in collagen concentration (0.75-1.25 mg/ml) on adhesion, migration, spreading, and gene expression by heart fibroblasts. Increased density of collagen in 3-dimensional gels resulted in more efficient adhesion, spreading, and migration by heart fibroblasts. These experiments indicated that the density of the collagen matrix has a significant impact on fibroblast function. These studies begin to elucidate the effects of ECM density at the cellular level in the myocardium.     

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.     

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.      

Characterization of the skin collagen of a surface water fish Scomberoides commersonianus.

The molecular organization of the skin collagen in the fish S. commersonianus has been investigated. The contents of imino acids proline and hydroxyproline are less in the skin collagen. The major collagenous component of fish skin is homologous to type I collagen. The number of CNBr peptides in fish skin collagen alpha 1 chains is two times that of rat skin alpha 1 CNBr peptides. The proline-hydroxyproline ratio in the six peptides studied was 1.66-3.5 as compared to that of rat skin collagen (0.67-1.94). This indicates that proline and hydroxyproline are not uniformly distributed in the collagen molecule in fish skin collagen.     

Quantification of liver fibrosis via second harmonic imaging of the Glisson's capsule from liver surface

Liver surface is covered by a collagenous layer called the Glisson's capsule. The structure of the Glisson's capsule is barely seen in the biopsy samples for histology assessment, thus the changes of the collagen network from the Glisson's capsule during the liver disease progression are not well studied. In this report, we investigated whether non‐linear optical imaging of the Glisson's capsule at liver surface would yield sufficient information to allow quantitative staging of liver fibrosis. In contrast to conventional tissue sections whereby tissues are cut perpendicular to the liver surface and interior information from the liver biopsy samples were used, we have established a capsule index based on significant parameters extracted from the second harmonic generation (SHG) microscopy images of capsule collagen from anterior surface of rat livers. Thioacetamide (TAA) induced liver fibrosis animal models was used in this study. The capsule index is capable of differentiating different fibrosis stages, with area under receiver operating characteristics curve (AUC) up to 0.91, making it possible to quantitatively stage liver fibrosis via liver surface imaging potentially with endomicroscopy.

     

Type XIII collagen and some other transmembrane collagens contain two separate coiled-coil motifs,which may function as independent oligomerization domains

Type XIII collagen is a homotrimeric transmembrane collagen composed of a short intracellular domain, a single membrane-spanning region, and an extracellular ectodomain with three collagenous domains (COL1-3) separated by short non-collagenous domains (NC1-4). Several collagenous transmembrane proteins have been found to harbor a conserved sequence next to their membrane-spanning regions, and in the case of type XIII collagen this sequence has been demonstrated to be important for chain association. We show here that this 21-residue sequence is necessary but not sufficient for NC1 association. Furthermore, the NC1 association region was predicted to form an alpha-helical coiled-coil structure, which may already begin at the membrane-spanning region, as is also predicted for the related collagen types XXIII and XXV. Interestingly, a second coiled-coil structure is predicted to be located in the NC3 domain of type XIII collagen and in the corresponding domains of types XXIII and XXV. It is found experimentally that the absence of the NC1 coiled-coil domain leads to a lack of disulfide-bonded trimers and misfolding of the membrane-proximal collagenous domain COL1, whereas the COL2 and COL3 domains are correctly folded. We suggest that the NC1 coiled-coil domain is important for association of the N-terminal part of the type XIII collagen alpha chains, whereas the NC3 coiled-coil domain is implicated in the association of the C-terminal part of the molecule. All in all, we propose that two widely separated coiled-coil domains of type XIII and related collagens function as independent oligomerization domains participating in the folding of distinct areas of the molecule.     

Isolation and partial characterization of a novel basement membrane collagen

A guanidine-HCl extraction of lens capsule basement membrane dissolves collagenous material. This material was fractionated on an Agarose A-5M column. Fractions 1, 2 and 3 were further purified and partially characterized immunochemically and by amino acid analysis. Fraction 3 has a molecular weight of 55,000 when compared with collagen type I standard. The CNBr peptide pattern and composition of fraction 3 are different from those of alpha 1 (IV) 95K and alpha 2 (IV) 95K chains. The results described suggest the presence of a new chain in lens capsule basement membrane.