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.     

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.     

Polarization-resolved second-harmonic generation in tendon upon mechanical stretching

Collagen is a triple-helical protein that forms various macromolecular organizations in tissues and is responsible for the biomechanical and physical properties of most organs. Second-harmonic generation (SHG) microscopy is a valuable imaging technique to probe collagen fibrillar organization. In this article, we use a multiscale nonlinear optical formalism to bring theoretical evidence that anisotropy of polarization-resolved SHG mostly reflects the micrometer-scale disorder in the collagen fibril distribution. Our theoretical expectations are confirmed by experimental results in rat-tail tendon. To that end, we report what to our knowledge is the first experimental implementation of polarization-resolved SHG microscopy combined with mechanical assays, to simultaneously monitor the biomechanical response of rat-tail tendon at macroscopic scale and the rearrangement of collagen fibrils in this tissue at microscopic scale. These experiments bring direct evidence that tendon stretching corresponds to straightening and aligning of collagen fibrils within the fascicle. We observe a decrease in the SHG anisotropy parameter when the tendon is stretched in a physiological range, in agreement with our numerical simulations. Moreover, these experiments provide a unique measurement of the nonlinear optical response of aligned fibrils. Our data show an excellent agreement with recently published theoretical calculations of the collagen triple helix hyperpolarizability.     

Investigating mechanisms of collagen thermal denaturation by high resolution second-harmonic generation imaging

We apply the technique of second-harmonic generation (SHG) microscopy to obtain large area submicron resolution image of Type I collagen from rat tail tendon as it is heated from 40 degrees C to 70 degrees C for 0-180 min. The change in the collagen structure as reflected in its SHG image is observed at length scales from submicron to hundreds of microns. We observed that heating the tendon below the temperature of 54 degrees C does not produce any change in the averaged SHG intensity. At the heating temperature of 54 degrees C and above, we find that increasing the heating temperature and time leads to decreasing SHG intensity. As the tendon is heated above 54 degrees C, the regions where the SHG signal vanish and form a tiger-tail like pattern. In addition, a decrease in the SHG signal occurs uniformly throughout the tendon. By comparing the relative SHG intensities in small and large areas, we found that the denaturation process responsible for forming the tiger-tail like pattern occurs at a higher rate than the global denaturation process occurring throughout the tendon. We also measured the fibril spacing and found that it remains constant at 1.61 +/- 0.04 micron for all heating temperature and times. The constant fibril density shows that the global denaturation process occurs at a length scale smaller than the size of the fibril. Our results show that second-harmonic generation microscopy is effective in monitoring the thermal damage to collagen and has potential applications in biomedicine.     

High resolution imaging of collagen organisation and synthesis using a versatile collagen specific probe

Collagen is the protein primarily responsible for the load-bearing properties of tissues and collagen architecture is one of the main determinants of the mechanical properties of tissues. Visualisation of changes in collagen three-dimensional structure is essential in order to improve our understanding of collagen fibril formation and remodelling, e.g. in tissue engineering experiments. A recently developed collagen probe, based on a natural collagen binding protein (CNA35) conjugated to a fluorescent dye, showed to be much more specific to collagen than existing fluorescent techniques currently used for collagen visualisation in live tissues. In this paper, imaging with this fluorescent CNA35 probe was compared to imaging with second harmonic generation (SHG) and the imaging of two- and three-dimensional collagen organisation was further developed. A range of samples (cell culture, blood vessels and engineered tissues) was imaged to illustrate the potential of this collagen probe. This images of collagen organisation showed improved detail compared to images generated with SHG, which is currently the most effective method for viewing three-dimensional collagen organisation in tissues. In conclusion, the fluorescent CNA35 probe allows easy access to high resolution imaging of collagen, ranging from very young fibrils to more mature collagen fibres. Furthermore, this probe enabled real-time visualisation of collagen synthesis in cell culture, which provides new opportunities to study collagen synthesis and remodelling.     

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.     

Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues

Sum frequency generation (SFG) and second harmonic generation (SHG) were observed from helical fibrils in spinal cord white matter isolated from guinea pigs. By combining SFG with coherent anti-Stokes Raman scattering microscopy, which allows visualization of myelinated axons, these fibers were found to be distributed near the surface of the spinal cord, between adjacent axons, and along the blood vessels. Using 20-microm-thick tissue slices, the ratio of forward to backward SHG signal from large bundles was found to be much larger than that from small single fibrils, indicating a phase-matching effect in coherent microscopy. Based on the intensity profiles across fibrils and the size dependence of forward and backward signal from the same fibril, we concluded that the main SHG signal directly originates from the fibrils, but not from surface SHG effects. Further polarization analysis of the SHG signal showed that the symmetry property of the fibril could be well described with a cylindrical model. Colocalization of the SHG signal with two-photon excitation fluorescence (TPEF) from the immunostaining of glial fibrillary acidic protein demonstrated that SHG arises from astroglial filaments. This assignment was further supported by colocalization of the SHG contrast with TPEF signals from astrocyte processes labeled by a Ca(2+) indicator and sulforhodamine 101. This work shows that a combination of three nonlinear optical imaging techniques--coherent anti-Stokes Raman scattering, TPEF, and SHG (SFG) microscopy--allows simultaneous visualization of different structures in a complex biological system.     

Second- and third-harmonic generation microscopies for the structural imaging of intact tissues

One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system ; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy.     

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.     

The role of backscattering in SHG tissue imaging

We investigate the properties of second-harmonic generation (SHG) tissue imaging for the functional biological unit fascia, skeletal muscle, and tendon. Fascia and Achilles tendon primarily consist of similar collagen type I arrays that can be imaged using SHG microscopy. For muscle, it is the myosin molecules represented within the A bands. For fascia and tendon tissue samples, we observe, in addition to a stronger signal in forward images, vastly different features for the backward versus the forward images. In vivo as well as intact ex vivo thick tissue imaging requires backward detection. The obtained image is a result of the direct backward components plus a certain fraction of the forward components that are redirected (backscattered) toward the objective as they propagate within the tissue block. As the forward and the backward images are significantly different from each other for the imaged collagen type I tissue, it is crucial to determine the fraction of the forward signal that contributes to the overall backward signal. For intact ex vivo SHG imaging of Achilles tendon, we observe a significant contribution of forward features in the resulting image. For fascia, the connective tissue immediately surrounding muscle, we only observe backward features, due to low backscattering in muscle.     

用光学二次谐波成像技术观察皮肤内不同种类的胶原

目的：用光学二次谐波成像的方法比较成熟皮肤与新生皮肤内不同种类胶原的含量,以及正常皮肤与创伤皮肤内胶原种类的变化。方法：用前向及背向二次谐波观察正常及创伤皮肤内的胶原,并与传统的天狼猩红染色法相对照。结果：与传统方法相比,二次谐波可以更快速,更灵敏地检测组织中的胶原。背向二次谐波信号强度随着切片厚度的增加而增强。结论：光学二次谐波成像技术是一种灵敏、简单、快速检测皮肤组织内胶原的新方法,具有很好的应用前景,可应用于活体检测。     

Hyperglycemia-Induced Abnormalities in Rat and Human Corneas: The Potential of Second Harmonic Generation Microscopy

Background

Second Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet’s membrane, in the posterior cornea.

Methodology/Principal Findings

We studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet’s membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats.

Conclusions/Significance

Our study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.     

Second Harmonic Generation Microscopy: A Tool for Spatially and Temporally Resolved Studies of Heat Induced Structural Changes in Meat

Myofibers and collagen show non-linear optical properties enabling imaging using second harmonic generation (SHG) microscopy. The technique is evaluated for use as a tool for real-time studies of thermally induced changes in thin samples of unfixed and unstained pork. The forward and the backward scattered SHG light reveal complementary features of the structures of myofibers and collagen fibers. Upon heating the myofibers show no structural changes before reaching a temperature of 53 °C. At this temperature the SHG signal becomes extinct. The extinction of the SHG at 53 °C coincides with a low-temperature endotherm peak observable in the differential scanning calorimetry (DSC) thermograms. DSC analysis of epimysium, the connective tissue layer that enfold skeletal muscles, produces one large endotherm starting at 57 °C and peaking at 59.5 °C. SHG microscopy of collagen fibers reveals a variability of thermal stability. Some fibers show severe shrinkage at 57 °C, before the signal for most of them vanishes between 59 °C and 61 °C and thus coinciding with the endotherm of the thermograms. However, in some areas, strong SHG signals from collagen can be visualized even after prolonged heating to 67 °C and thus indicating regions of much higher thermal stability. It is seen that the benefits of the structural and temporal information available from SHG microscopy reveals complementary information to a traditional DSC measurement and enables a more complete understanding of the thermal denaturation process.     

Collagen type I and type V are present in the same fibril in the avian corneal stroma

The distribution, supramolecular form, and arrangement of collagen types I and V in the chicken embryo corneal stroma were studied using electron microscopy, collagen type-specific monoclonal antibodies, and a preembedding immunogold method. Double-label immunoelectron microscopy with colloidal gold-tagged monoclonal antibodies was used to simultaneously localize collagen type I and type V within the chick corneal stroma. The results definitively demonstrate, for the first time, that both collagens are codistributed within the same fibril. Type I collagen was localized to striated fibrils throughout the corneal stroma homogeneously. Type V collagen could be localized only after pretreatment of the tissue to partially disrupt collagen fibril structure. After such pretreatments the type V collagen was found in regions where fibrils were partially dissociated and not in regions where fibril structure was intact. When pretreated tissues were double labeled with antibodies against types I and V collagen coupled to different size gold particles, the two collagens colocalized in areas where fibril structure was partially disrupted. Antibodies against type IV collagen were used as a control and were nonreactive with fibrils. These results indicate that collagen types I and V are assembled together within single fibrils in the corneal stroma such that the interaction of these collagen types within heterotypic fibrils masks the epitopes on the type V collagen molecule. One consequence of the formation of such heterotypic fibrils may be the regulation of corneal fibril diameter, a condition essential for corneal transparency.     

Elastic Moduli of Collagen Gels Can Be Predicted from Two-Dimensional Confocal Microscopy

We quantitatively compare data obtained from imaging two-dimensional slices of three-dimensional unlabeled and fluorescently labeled collagen gels with confocal reflectance microscopy (CRM) and/or confocal fluorescence microscopy (CFM). Different network structures are obtained by assembling the gels over a range of concentrations at various temperatures. Comparison between CRM and CFM shows that the techniques are not equally sensitive to details of network structure, with CFM displaying higher fidelity in imaging fibers parallel to the optical axis. Comparison of CRM of plain and labeled collagen gels shows that labeling itself induces changes in gel structure, chiefly through inhibition of fibril bundling. Despite these differences, image analyses carried out on two-dimensional CFM and CRM slices of collagen gels reveal identical trends in structural parameters as a function of collagen concentration and gelation temperature. Fibril diameter approximated from either CRM or CFM is in good accord with that determined via electron microscopy. Two-dimensional CRM images are used to show that semiflexible polymer theory can relate network structural properties to elastic modulus successfully. For networks containing bundled fibrils, it is shown that average structural diameter, rather than fibril diameter, is the length scale that sets the magnitude of the gel elastic modulus.     

Live imaging of collagen remodeling during angiogenesis

To better understand interstitial matrix remodeling during angiogenesis, we probed endogenous optical signatures of collagen fibrils and cells with multiphoton microscopy to noninvasively visualize, in real-time, changes to fibril organization around angiogenic sprouts and growing neovessels. From analyses of the second-harmonic generation signal from fibrillar collagen and two-photon excited fluorescence, as well as coherent transmitted light from vascular cells, we found that microvessel fragments interacting with the collagen matrix exhibited two key features: a strong association of fibrillar collagen around the parent vessel fragment during vessel construct reconstitution and a substantial collagen fibril reorganization by sprout and neovessel tips. Results indicate that angiogenic sprouts and growing neovessels actively and differentially remodel existing collagen fibrils. This imaging approach to assess local changes in matrix organization may have a broader impact on tissue biology and mechanics during angiogenesis and allow for new insights in cardiovascular, diabetes, and cancer research.     

Collagen fibril formation in a wound healing model

Control of tissue composition and organization will be a key feature in the development of successful products through tissue engineering. However, the mechanism of collagen fibril formation, growth, and organization is not yet fully understood. In this study we have examined collagen fibril formation in a wound healing model in which the newly formed fibrils were kept distinct from preexisting tissue through use of a porous tubular biomaterial implant. Samples were examined after 4, 6, 14, and 28 days by light microscopy, in situ hybridization, and immunofluorescence microscopy. These showed a normal wound healing response, with significant collagen formation at 14 and 28 days. Individual collagen fibrils were isolated from these samples by gentle extraction in a gentamicin-containing buffer which allowed extraction of a large proportion of intact fibrils. Examination by transmission electron microscopy showed that approximately 80% of the intact fibrils showed a single polarity reversal, with both ends of each fibril comprising collagen amino-terminal domains; the remaining fibrils had no polarity reversal. All fibrils had similar diameters at both time points. Immunoelectron microscopy showed that all labeled fibrils contained both type I and III collagens. These data indicate that this wound healing model provides a system in which collagen fibril formation can be readily followed.     

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.     

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.