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.     

用二次谐波成像技术研究经飞秒激光切削后角膜变化

本文用二次谐波成像技术（second harmonic generation SHG）来研究飞秒激光切削后角膜结构的变化.在生物学研究,材料科学等方面都有很广泛应用的SHG成像技术能在不破坏的角膜情况下获得高对比度的角膜层析图像,分辨率为500 nm,实验装置是利用现有的双光子显微镜.本文还根据成像结果评价了飞秒激光在角膜切削中的质量,为飞秒激光微米级的精确切削和临床应用提供了实验支持.     

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.     

二次谐波显微成像技术

二次谐波非线性显微成像技术是近年发展起来的一种新型光学成像方法,已广泛应用于生物医学的各个领域。介绍了光学二次谐波产生的原理、成像装置及其技术发展,描述了二次谐波的成像特点和它与双光子荧光成像的异同,并对其在生物医学上的应用及发展前景做出展望。     

生物活组织的背向二次谐波成像

光学二次谐波成像技术由于具有三维高分辨率、不需要荧光标记、对生物样品的杀伤效应小等特点,在生物医学研究上具有广阔的应用前景.在双光子荧光成像基础上,实现了适合对厚组织样品观测的背向光学二次谐波成像,探讨了背向二次谐波成像的特点和影响因素.通过对多种生物组织样品进行大量实验,发现胶原纤维和肌肉纤维均可以产生很强的背向二次谐波,并成功地将背向二次谐波成像技术应用于糖尿病患者皮肤的观测.背向二次谐波成像技术可望推广到病理检查等临床应用中.     

基于非线性热扩散效应的亚衍射极限光声二次谐波显微成像技术

本文提出了一种基于非线性热扩散效应的光声二次谐波显微SH-PAM成像技术,用于实现亚衍射极限光声成像。生物组织受到强度调制的高斯激光束辐射时,组织吸收光子形成高斯分布的温度场,由于热扩散系数非线性热效应引起的非线性光声PA效应,从而产生光声二次谐波信号。模拟和试验结果均表明,重建后的光声二次谐波成像的横向分辨率超过了传统光学成像分辨率。本文通过仿体样品验证了该方法的可行性,并且对人表层皮肤细胞进行了成像,以证明其对生物样品的成像能力。该方法扩展了传统光声成像的范围,为超分辨成像开辟了新的可能性,为生物医学成像和材料检测提供了新的方法。     

Second Harmonic Generation from Ultrathin Gold Nanotubes

Plasmonics - We numerically study second harmonic generation from plasmonic nanotubes of air-core and ultrathin gold shell using classical nonlocal hydrodynamic model of free electron. We...     

Enhancement of Second Harmonic Generation in Metal-Insulator-Metal Plasmonic Waveguides

Nonlinear effects such as second harmonic generation (SHG) are important for applications such as switching and wavelength conversion. In this study, the generation of second harmonic in metal-insulator-metal (MIM) plasmonic waveguides was investigated for both symmetric and asymmetric structures. This study considered two different structures as plasmonic waveguides for the generation of second harmonic, and analysis was performed using the finite-difference time-domain method. Besides, the structure has grating on both sides for more coupling between photons and plasmons. The wavelength duration of grating per unit length (number of grooves) was optimized to reach the highest second harmonic generation. To perform this optimization, the wavelength of operation (λ = 458 nm) was considered. It was shown that field enhancement in symmetric MIM waveguides can result in the enhancement of SHG magnitude when compared to literature values. Also, asymmetric devices result in more than two orders of magnitude enhancement in SHG, as compared to the symmetric structure. It has been shown that the electric field of the second harmonic depends on the thickness of the crystal (insulator). Hence, its thickness was optimized to achieve the highest electric field.     

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.     

Investigation of Second Harmonic Generation in Asymmetric Metal-Insulator-Metal Plasmonic Waveguides

Plasmonics - In this study, the second harmonic generation in metal-insulator-metal (MIM) plasmonic waveguides was investigated for both symmetric and asymmetric structures. Nonlinear processes...     

Dry Ice Fixation of Myofibrils for Scanning Electron Microscopy

A rapid method of fixation of myofibrils using dry ice is reported. A glass slide or coverslip containing a drop of glutaraldehyde-fixed suspension of myofibrils is placed on dry ice causing the myofibrils to adhere to the glass surface. The specimens are then dehydrated through the alcohols, air dried and metal coated. This technique gives the myofibrils a corrugated appearance under the scanning electron microscope corresponding to the sarcomere banding.     

Tip-Enhanced Second Harmonic Generation: an Approach for Hyper-Raman Spectroscopy

Plasmonics - The near-field optical response generated at the apex of a scanning probe in tip-enhanced Raman spectroscopy (TERS) enhances and confines the interaction of light and matter to a few...     

Broadband Wide-Angle Second Harmonic Generation in Magnetized Plasma: Double Resonant Effect

Conditions for the phase synchronism between high-frequency electromagnetic waves with frequencies ω and 2ω propagating in magnetized plasma are investigated. The variety of the values of the plasma density and magnetic field, as well as of wave polarizations, obeying the synchronism conditions are shown to provide resonant broadband wide-angle nonlinear generation of the second harmonic of the pumping wave. Special attention is given to oblique propagation of interacting waves. The coupling strengths for the resonant mode conversion in magnetized collisional plasma are obtained. The double resonance ensuring efficient nonlinear generation of extraordinary mode in the vicinity of the electron cyclotron resonance (ω(2k) = ω _ce ) is considered. Examples illustrating these nonlinear phenomena for some plasma and radiation parameters are presented.     

Plasmon-Enhanced Second Harmonic Generation: from Individual Antennas to Extended Arrays

We analyze the emission yield of the second harmonic generation (SHG) from dense ordered arrays of L-shaped Au nanoantennas within a well-defined collection angle and compare it to that of the isolated nanostructures designed with the same geometrical parameters. Thanks to the high antenna surface density, arrays display one order of magnitude higher SHG yield per unit surface with respect to isolated nanoantennas. The difference in the collected nonlinear signals becomes even more pronounced by reducing the collection angle, because of the efficient angular filtering that can be attained in dense arrays around the zero order. Albeit this key-enabling feature allows envisioning application of these platforms to nonlinear sensing, a normalization of the SHG yield to the number of excited antennas in the array reveals a reduced nonlinear emission from each individual antenna element. We explain this potential drawback in terms of resonance broadening, commonly observed in densely packed arrays, and angular filtering of the single antenna emission pattern provided by the array 0th order.

     

Detecting Subtle Plasma Membrane Perturbation in Living Cells Using Second Harmonic Generation Imaging

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ∼50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells.As the epicenter for many cellular functions, understanding the dynamics of the plasma membrane is important to monitoring biological phenomena. External forces acting upon the plasma membrane (e.g., electric, mechanical) have been shown to cause rapid disturbances, often resulting in dramatic changes in cell physiology (1–3). To understand this interaction, a minimally invasive, highly sensitive imaging technique that enables monitoring the structure of the plasma membrane is needed. Lipophilic dyes, which embed themselves into lipid membranes, are sensitive to the surrounding electric field and, therefore, report changes in membrane fluidity as well as voltage due to the capacitive nature of the membranes (4,5). This sensitivity is typically detected as a shift in the fluorescence emission spectrum. Localization of the fluorescence signal to only the plasma membrane is difficult because the probes also label internal membrane structures. Thus, to overcome this lack of spatial selectivity, second harmonic generation (SHG) has been used as an alternative to fluorescence for membrane imaging (6,7).In SHG, a second-order nonlinear polarization is induced by electronic disruption of a probe molecule from the electromagnetic field of the incident laser beam. This polarization generates oscillating dipole moments that reradiate light at twice the energy of the excitation beam. The induction of this dipole is sensitive to the static electric field surrounding the probe and the steady-state molecular polarization of the probe molecule. These properties make SHG probes useful for monitoring changes in biological membranes.First, as the voltage potential across the membrane changes, the static electric field around the probe also shifts, making the probe sensitive to these variations (7). Several SHG probes have, therefore, been employed to monitor plasma membrane potential (7,8).Second, because the dipole is affected by the steady-state molecular polarization of the probe itself, a SHG signal is only produced in materials that lack a center of inversion symmetry. In the centrosymmetric case, any emitted radiation is cancelled out by destructive interference. The properties of an interfacial environment, such as a cellular plasma membrane, not only provide the necessary asymmetry, but cause the polarized lipophilic dyes to be aligned in respect to the interface, instead of being randomly distributed as they would in a bulk environment. This alignment allows the generation of a coherent SHG signal from the plasma membrane while the rest of the cell remains nearly signal-free (6,7).We investigated whether the alignment sensitivity of the SHG response could be used to detect minute changes in the organization of the plasma membrane. Jurkat clone E6-1 human T-lymphocytes with a spherical morphology were selected for optimum signal clarity and cultured as directed by American Type Culture Collection (ATCC, Manassas, VA) with 1 I.U./mL penicillin and 0.1 μg/mL streptomycin. Cells were added to 35-mm poly-L-lysine-coated glass-bottomed dishes (MatTek, Ashland, MA) and incubated for 1 h in growth media to allow adherence. Before loading, the cells were rinsed with a buffer consisting of 135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM HEPES, 10 mM glucose, 2 mM CaCl2, pH 7.4, 290–310 mOsm. SHG probes, Di-4-ANEPPDHQ (Di-4) (5 μM final concentration), FM4-64 (15 μM) or ATR (100 μM, 1 mg/mL BSA) were added to the buffer solution and incubated for 1 h. Cellular imaging was performed in the labeling buffer to limit diffusion of the probe molecules out of the cell membranes.A Ti:sapphire oscillator at 980 nm (Coherent Chameleon, 130 fs, 80 MHz, ∼15 mW at the sample; Coherent Laser, Santa Clara, CA) was coupled through the scan head of a modified model No. TCS SP5 II (Leica Geosystems, Norcross, GA) for SHG and multiphoton-excited fluorescence imaging (40×, water, 1.1 NA) using resonant scanning. SHG signal was collected in transmission by a photomultiplier tube after 680-nm shortpass and 485/25-nm bandpass filters; simultaneous fluorescence signal was collected in the epi-direction by two non-descanned photomultiplier tubes with 540/60-nm and 650/60-nm bandpass filters.Three SHG probes previously used to monitor voltage or membrane order in living cells were tested (8–10). Although ATR is reported to be effective in monitoring membrane voltage, we obtained nearly no SHG signal, despite successful loading as indicated by the fluorescence signal (Fig. 1). When FM4-64 and Di-4 were loaded to similar fluorescence intensities, nearly equivalent SHG signal was collected. Di-4 did appear to have a greater internalization of the dye. However, after the first frame, the FM4-64 signal dropped considerably (Fig. 1 b), an observation reported as a membrane voltage-independent bleaching effect (8). This drop in signal recovered after excitation was blocked for several seconds, but quantification of the response was difficult. Di-4 did not suffer as dramatic a drop in signal upon excitation, and still had sufficient SHG signal/noise after several seconds, so it was used in all further experiments.Open in a separate windowFigure 1(a) Fluorescence (top) and SHG (bottom) images for the three probes. (b) Signal/noise for the fluorescence and SHG for the initial frame and shortly after beginning acquisition. Error bars represent the mean ± SE (n = 10). Scale bar is 10 μm.To test whether Di-4 would report a rapid change in membrane organization, we applied a single nanosecond-duration pulsed electric field (nsPEF) to the labeled cell. These ultrabrief, high-intensity (MV/m) pulses differ from longer (μs-ms), lower-intensity (kV/m) pulses traditionally associated with electroporation in induced cellular response (3,11,12). Through selective uptake of small ions (Ca²⁺, Ti⁺) with limited uptake of propidium iodide, nsPEF have been previously postulated to cause nanopores (<2 nm diameter) in the plasma membrane. In contrast with a previous study observing poration resulting from traditional electroporation (13), the brevity of this apparently novel cellular insult allows for the decoupling of the mechanical effects of the pulse on the membrane from the electrical effects of the pulse itself. A single pulse, generated by a custom pulse generator, was delivered to the cells using a pair of 125-μm diameter tungsten electrodes, separated edge-to-edge by 150 μm, as previously described in Ibey et al. (14). For maximum visualization of changes in the SHG signal, a half-wave plate was placed before the scan head to align the polarization of the laser such that the brightest signals from the plasma membrane were at the poles facing the electrodes.The Di-4 SHG signal in response to a single 16.7 kV/cm, 600-ns nsPEF is shown in Fig. 2. Before the pulse, the intensity of the SHG signal is high at each of these poles. Immediately after the pulse, the SHG intensity drops by ∼50% on the side of the cell facing the anodic electrode, whereas little intensity is lost at the other pole. This response is plotted in Fig. 2 (pulse applied at 2 s), where it is apparent that the response is near instantaneous with little recovery in signal in the 5 s postexposure. The SHG response matches the previously observed effect of this stimulus, where ion uptake displayed a polar dependence and persisted for a number of minutes (11,12). Images taken 5 min after an nsPEF exposure are also shown in Fig. 2. These images confirm the eventual recovery of the cell and the corresponding return of SHG signal to preexposure levels.Open in a separate windowFigure 2(a) SHG images showing drop in signal on the anodic (or A-pole) of the cell. (b) Time trace of SHG response with the electrical pulse applied at 2 s that shows a near-instantaneous drop in the SHG signal at the anodic pole of the cell. (c) SHG image preexposure, immediately postexposure, and then 5-min postexposure showing recovery of the SHG signal.To decouple membrane disturbance from environmental changes around the membrane, we compared the SHG response to the simultaneously acquired fluorescence signal. Because fluorescence is not subject to the strict orientation requirement of SHG, the plasma membrane fluorescence signal provides an indication of the membrane fluidity and/or potential. Despite the dramatic shift in SHG intensity on the anodic pole upon the electrical pulse exposure (Fig. 3 a), the fluorescence channels display little response from the equivalent membrane sections with the exception of photobleaching and a slight increase in signal in both emission bands on the anodic side (Fig. 3, b and c). The shading in these graphs represents the mean ± SE for six cells. Although this slight increase may indicate that a small amount of dye is simply diffusing in or out of the membrane upon exposure, the fluorescence response is not as rapid or as lasting as the SHG response. Change in membrane fluidity or voltage can also be quantized using these fluorescence signals and a value known as the generalized polarization (GP) (4),GP=I515−570−I620−680I515−570+I620−680.(1)As with the raw intensity of the individual signals, the GP value for the membrane (Fig. 3 d) shows no significant shift, indicating that the membrane is likely not transitioning between a more raft- and fluidlike state. Thus, it seems likely that the dye was initially aligned in the tightly-packed ordered membrane so that the probes were able to generate a SHG photon. As shown in Fig. 3 e, we postulate that upon electrical pulse exposure, the membrane was disrupted by the formation with nanopores giving the probe molecules the flexibility to disorient within the membrane. The resulting alignment of the probes is more isotropic in nature, thereby limiting the probes probability of producing a SHG photon. The fluorescence signal remained, however, indicating that the probes remained active in the membrane.Open in a separate windowFigure 3(a) Average SHG signal showing the dramatic drop in signal on the anodic pole at the pulse application (2 s). (b and c) Simultaneous TPF signals showing nearly no instantaneous change at the pulse application. (d) GP showing no observable changes in the membrane potential or fluidity after the pulse. (Shaded areas) Fit to the mean ± SE for each trace (n = 6). (e) Conceptualization of the hypothesized membrane disruption underlying the observed change in SHG response.Thus, by taking advantage of the selection criteria of SHG, we were able to successfully use the SHG probe, Di-4, to monitor rapid disruption of the plasma membrane. Because SHG can only be generated when the probes are aligned in the plasma membrane, the SHG signal diminishes significantly upon disruption. The simultaneous collection of the multiphoton-excited fluorescence signal was advantageous in that it demonstrated that the probes did not simply diffuse out of the membrane, did not appear to be energetically disrupted by the electric pulse, and showed that the membrane changes were not simply a change in lipid order. We believe that this technique holds tremendous potential for use in the study of how external stimuli interact with and change the orientation of biological membranes. Such knowledge may allow for further understanding of how manipulation of cells and biological systems can be achieved using external stimuli.     

Detecting Subtle Plasma Membrane Perturbation in Living Cells Using Second Harmonic Generation Imaging

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ∼50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells.     

Organization of Astaxanthin within Oil Bodies of Haematococcus pluvialis Studied with Polarization-Dependent Harmonic Generation Microscopy

Nonlinear optical microscopy was used to image the localization of astaxanthin accumulation in the green alga, Haematococcus pluvialis. Polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and third harmonic generation (THG) microscopy was applied to study the crystalline organization of astaxanthin molecules in light-stressed H. pluvialis in vivo. Since astaxanthin readily forms H- and J-aggregates in aqueous solutions, PIPO THG studies of astaxanthin aggregates contained in red aplanospores were compared to PIPO THG of in vitro self-assembled H- and J-aggregates of astaxanthin. The PIPO THG data clearly showed an isotropic organization of astaxanthin in red aplanospores of H. pluvialis. This is in contrast to the highly anisotropic organization of astaxanthin in synthetic H- and J-aggregates, which showed to be uniaxial. Since carotenoids in vitro preferentially form H- and J-aggregates, but in vivo form a randomly organized structure, this implies that astaxanthin undergoes a different way of packing in biological organisms, which is either due to the unique physical environment of the alga or is controlled enzymatically.     

Structural Characterization of Edematous Corneas by Forward and Backward Second Harmonic Generation Imaging

The purpose of this study was to image and quantify the structural changes of corneal edema by second harmonic generation (SHG) microscopy. Bovine cornea was used as an experimental model to characterize structural alterations in edematous corneas. Forward SHG and backward SHG signals were simultaneously collected from normal and edematous bovine corneas to reveal the morphological differences between them. In edematous cornea, both an uneven expansion in the lamellar interspacing and an increased lamellar thickness in the posterior stroma (depth > 200 μm) were identified, whereas the anterior stroma, composed of interwoven collagen architecture, remained unaffected. Our findings of heterogeneous structural alteration at the microscopic scale in edematous corneas suggest that the strength of collagen cross-linking is heterogeneous in the corneal stroma. In addition, we found that qualitative backward SHG collagen fiber imaging and depth-dependent signal decay can be used to detect and diagnose corneal edema. Our work demonstrates that SHG imaging can provide morphological information for the investigation of corneal edema biophysics, and may be applied in the evaluation of advancing corneal edema in vivo.     

Imaging the noncentrosymmetric structural organization of tendon with Interferometric Second Harmonic Generation microscopy

We report the imaging of tendon with Interferometric Second Harmonic Generation microscopy. We observe that the noncentrosymmetric structural organization can be maintained along the fibrillar axis over more than 150 μm, while in the transverse direction it is ～1–15 μm. Those results are explained by modeling tendon as a heterogeneous distribution of noncentrosymmetric nano‐cylinders (collagen fibrils) oriented along the fibrillar axis. The preservation of the noncentrosymmetric structural organization over multiple tens of microns reveals that tendon is made of domains in which the ratio between fibrils with positive and negative polarity is unbalanced. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Interferometric Backward Third Harmonic Generation Microscopy for Axial Imaging with Accuracy Beyond the Diffraction Limit

A new nonlinear microscopy technique based on interference of backward-reflected third harmonic generation (I-THG) from multiple interfaces is presented. The technique is used to measure height variations or changes of a layer thickness with an accuracy of up to 5 nm. Height variations of a patterned glass surface and thickness variations of fibroblasts are visualized with the interferometric epi-THG microscope with an accuracy at least two orders of magnitude better than diffraction limit. The microscopy technique can be broadly applied for measuring distance variations between membranes or multilayer structures inside biological tissue and for surface height variation imaging.