The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy

Couture, Charles-André; Bancelin, Stéphane; Kolk, Jarno van der; Popov, Konstantin; Rivard, Maxime; Légaré, Katherine; Martel, Gabrielle; Richard, Hélène; Brown, Cameron; Ramunno, Lora; Légaré, François

doi:10.1016/j.bpj.2015.10.040

Cited by 45 publications

(35 citation statements)

References 59 publications

(78 reference statements)

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“…On the contrary, in biological tissues, the contrast is usually lower which imposes a sharper limit (typically 100x100µm 2 ). Interestingly, the interferometric contrast obtained in the center of the image is similar to the one reported previously [38] using the sample scanning system. Therefore, this approach will be applicable in disorganized collagen structures such as skin or cartilage, but with a limited field of view.…”

Section: Fast I-shg In Periodically Poled Lithium Niobatesupporting

confidence: 86%

“…Finally, the peak widths measured in Fig. 6(a) allow to extract the ratio of fibrils pointing in opposite direction [34,38].…”

Section: Optimization Of the Phase Stepmentioning

confidence: 99%

“…The use of a sample scanning system decreases imaging speed considerably, which prevents the investigation of dynamical processes. We recently solved the first issue by implementing a new microscope based on a femtosecond excitation and optimizing the temporal overlap between the two beams by compensating the group velocity delay in the microscope [38].…”

Section: Introductionmentioning

confidence: 99%

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Fast interferometric second harmonic generation microscopy

Bancelin¹,

Couture²,

Légaré³

et al. 2016

Biomed. Opt. Express

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We report the implementation of fast Interferometric Second Harmonic Generation (I-SHG) microscopy to study the polarity of non-centrosymmetric structures in biological tissues. Using a sample quartz plate, we calibrate the spatially varying phase shift introduced by the laser scanning system. Compensating this phase shift allows us to retrieve the correct phase distribution in periodically poled lithium niobate, used as a model sample. Finally, we used fast interferometric second harmonic generation microscopy to acquire phase images in tendon. Our results show that the method exposed here, using a laser scanning system, allows to recover the polarity of collagen fibrils, similarly to standard I-SHG (using a sample scanning system), but with an imaging time about 40 times shorter.

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Section: Fast I-shg In Periodically Poled Lithium Niobatesupporting

confidence: 86%

“…Finally, the peak widths measured in Fig. 6(a) allow to extract the ratio of fibrils pointing in opposite direction [34,38].…”

Section: Optimization Of the Phase Stepmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Fast interferometric second harmonic generation microscopy

Bancelin¹,

Couture²,

Légaré³

et al. 2016

Biomed. Opt. Express

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“…Bulk strains were calculated based on the mean distance between the fibril end nodes. Organisation was classified using an anisotropy parameter r mean based on polarised optical parameters used for cartilage measurements [29,30,31], providing a means for comparison with experiments. In particular r mean is defined by…”

Section: Collagen Structure Simulationmentioning

confidence: 99%

Effect of crosslinking in cartilage-like collagen microstructures

Chen

Gaffney

et al. 2017

Journal of the Mechanical Behavior of Biomedical Materials

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The mechanical performance of biological tissues is underpinned by a complex and finely balanced structure. Central to this is collagen, the most abundant protein in our bodies, which plays a dominant role in the functioning of tissues, and also in disease. Based on the collagen meshwork of articular cartilage, we have developed a bottom-up spring-node model of collagen and examined the effect of fibril connectivity, implemented by crosslinking, on mechanical behaviour. Although changing individual crosslink stiffness within an order of magnitude had no significant effect on modelling predictions, the density of crosslinks in a meshwork had a substantial impact on its behaviour. Highly crosslinked meshworks maintained a 'normal' configuration under loading, with stronger resistance to deformation and improved recovery relative to sparsely crosslinked meshwork. Stress on individual fibrils, however, was higher in highly crosslinked meshworks. Meshworks with low numbers of crosslinks reconfigured to disease-like states upon deformation and recovery. The importance of collagen interconnectivity may provide insight into the role of ultrastructure and its mechanics in the initiation, and early stages, of diseases such as osteoarthritis.

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“…Although the mean value and the variance of noise intensity decreased significantly when the borosilicate substrate material was replaced with high-purity SiO 2 , the noise level persisted. This unexpected noise has not been considered in previous SHG studies of bulk samples 3,[5][6][7][8][9][10][11][12][13]30 . The problem might be negligible when the laser focus is significantly far from the substrate, or when the signal intensity generated by the specimen is sufficiently high.…”

mentioning

confidence: 99%

Second harmonic generation polarization microscopy as a tool for protein structure analysis

Kaneshiro

Okada

Shima

et al. 2018

Preprint

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Second-harmonic generation (SHG) is a nonlinear coherent scattering process that is sensitiveto molecular structures in illuminated materials. We report SHG polarization measurement for the detection of protein conformational changes in solutions of macromolecular protein assemblies such as microtubules and protein crystals. The results illustrate the potential of this method for protein structural analysis in physiological solutions at room temperature without labelling.Second-harmonic generation (SHG) microscopy is a powerful modality that enables the visualization of fibre structures without labelling. Such structures include the fibrillar collagen in tendons, myosin thick filaments in myosarcoma, and the densely bundled microtubules in living specimens 1-3 . SHG is a coherent process in which two photons that have the same energy are effectively 'combined' to generate a new photon with twice the energy; it was first discovered in 1961 4 . Twenty-five years later, SHG was used to determine the orientation of collagen fibres in a rat tail tendon 5 . Because SHG is sensitive to static electric field, SHG microscopy was also used to measure the action potential of a nerve cell 6,7 . The microscope design is based on that used for laser-scanning confocal fluorescence microscopes, so that the simultaneous observation of fluorescence and SHG is possible 8 .SHG is now also used for whole-animal imaging in vivo 9,10 .The nonlinear susceptibility of a material to SHG is expressed as the 3 rd -rank tensor (SHG tensor) originating from molecules with broken centrosymmetry and their alignments in macromolecular assemblies 1,2 . This unique feature may enable the acquisition of data about the spatial distributions of filament orientations, and can provide information on protein secondary structures, such as the axial pitch of alpha-helices 11 . Furthermore, SHG microscopy can be used to visualize the spatial distribution of fibre polarity directions in tissues with interferometric configurations 12,13 . Therefore, the SHG signal potentially carries structural information about proteins, which can be recorded optically without labelling.

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The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy

Cited by 45 publications

References 59 publications

Fast interferometric second harmonic generation microscopy

Fast interferometric second harmonic generation microscopy

Effect of crosslinking in cartilage-like collagen microstructures

Second harmonic generation polarization microscopy as a tool for protein structure analysis

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