Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy

International audienceSoft connective tissues such as skin, tendon or cornea are made of about 90% of extracellular matrix proteins, fibrillar collagens being the major components. Decreased or aberrant collagen synthesis generally results in defective tissue mechanical properties as the classic form of Elhers-Danlos syndrome (cEDS). This connective tissue disorder is caused by mutations in collagen V genes and is mainly characterized by skin hyperextensibility. To investigate the relationship between the microstructure of normal and diseased skins and their macroscopic mechanical properties, we imaged and quantified the microstructure of dermis of ex vivo murine skin biopsies during uniaxial mechanical assay using multiphoton microscopy. We used two genetically-modified mouse lines for collagen V: a mouse model for cEDS harboring a Col5a2 deletion (a.k.a. pN allele) and the transgenic K14-COL5A1 mice which overexpress the human COL5A1 gene in skin. We showed that in normal skin, the collagen fibers continuously align with stretch, generating the observed increase in mechanical stress. Moreover, dermis from both transgenic lines exhibited altered collagen reorganization upon traction, which could be linked to microstructural modifications. These findings show that our multiscale approach provides new crucial information on the biomechanics of dermis that can be extended to all collagen-rich soft tissues

Methods and equipment for tube cooling during hardening

22.00; Translated from Polish (Wiad. Hutn. 1986 v. 42(9) p. 173-176)Available from British Library Document Supply Centre- DSC:9022.06(BISI-Trans--26169)T / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

Fast monitoring of in-vivo conformational changes in myosin using single scan polarization- SHG microscopy

Fast imaging of molecular changes under high-resolution and label-free conditions are essential for understanding in-vivo processes, however, current techniques are not able to monitor such changes in real time. Polarization sensitive second harmonic generation (PSHG) imaging is a minimally invasive optical microscopy technique capable of quantifying molecular conformational changes occurring below the diffraction limit. Up to now, such information is generally retrieved by exciting the sample with different linear polarizations. This procedure requires the sample to remain static during measurements (from a few second to minutes), preventing the use of PSHG microscopy from studying moving samples or molecular dynamics in living organisms. Here we demonstrate an imaging method that is one order of magnitude faster than conventional PSHG. Based on circular polarization excitation and instantaneous polarimetry analysis of the second harmonic signal generated in the tissue, the method is able to instantaneously obtain molecular information within a pixel dwell time. As a consequence, a single scan is only required to retrieve all the information. This allowed us to perform PSHG imaging in moving C. elegans, monitoring myosin’s dynamics during the muscular contraction and relaxation. Since the method provides images of the molecular state, an unprecedented global understanding of the muscles dynamics is possible by correlating changes in different regions of the sample.Peer Reviewe

Quantitative discrimination between endogenous SHG sources in mammalian tissue, based on their polarization response

In this study, the second harmonic generation (SHG) response to polarization and subsequent data analysis is used to discriminate, in the same image, different SHG source architectures with pixel resolution. This is demonstrated in a mammalian tissue containing both skeletal muscle and fibrilar collagen. The SHG intensity variation with the input polarization (PSHG) is fitted pixel by pixel in the image using an algorithm based on a generalized biophysical model. The analysis provides the effective orientation, θe, of the different SHG active structures (harmonophores) at every pixel. This results in a new image in which collagen and muscle are clearly differentiated. In order to quantify the SHG response, the distribution of θe for every harmonophore is obtained. We found that for collagen, the distribution was centered at θe = 42.7° with a full width at half maximum of ∆θ = 5.9° while for muscle θe = 65.3°, with ∆θ = 7.7°. By comparing these distributions, a quantitative measurement of the discrimination procedure is provided.This work is supported by the Generalitat de Catalunya and by the Spanish government grant TEC2006-12654 SICO. Authors also acknowledge The Centre for Innovacio i Desenvolupament Empresarial-CIDEM (RDITSCON07-1-0006), Grupo Ferrer and the European Regional Development Fund. This research has been partially supported by Fundació Cellex Barcelona.Peer reviewe

Estimation of the effective orientation of the SHG source in primary cortical neurons

In this paper we provide, for the first time to our knowledge, the effective orientation of the SHG source in cultured cortical neuronal processes in vitro. This is done by the use of the polarization sensitive second harmonic generation (PSHG) imaging microscopy technique. By performing a pixel-level resolution analysis we found that the SHG dipole source has a distribution of angles centered at θe =33.96°, with a bandwidth of ∆θe = 12.85°. This orientation can be related with the molecular geometry of the tubulin heterodimmer contained in microtubules.This work is supported by the Generalitat de Catalunya and by the Spanish government grant TEC2006-12654. Authors also acknowledge The Centre for Innovacio i Desenvolupament Empresarial - CIDEM (RDITSCON07-1-0006), Grupo Ferrer and the European Regional Development Fund. This research has been partially supported by Fundació Cellex Barcelona.Peer reviewe

Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons

Neuronal death can be preceded by progressive dysfunction of axons. Several pathological conditions such as ischemia can disrupt the neuronal cytoskeleton. Microtubules are basic structural components of the neuronal cytoskeleton that regulate axonal transport and neuronal function. Up-to-date, high-resolution observation of microtubules in living neuronal cells is usually accomplished using fluorescent-based microscopy techniques. However, this needs exogenous fluorescence markers to produce the required contrast. This is an invasive procedure that may interfere with the microtubule dynamics. In this work, we show, for the first time to our knowledge, that by using the endogenous (label-free) contrast provided by second harmonic generation (SHG) microscopy, it is possible to identify early molecular changes occurring in the microtubules of living neurons under ischemic conditions. This is done by measuring the intensity modulation of the SHG signal as a function of the angular rotation of the incident linearly polarized excitation light (technique referred to as PSHG). Our experiments were performed in microtubules from healthy control cultured cortical neurons and were compared to those upon application of several periods of oxygen and glucose deprivation (up to 120 min) causing ischemia. After 120-min oxygen and glucose deprivation, a change in the SHG response to the polarization was measured. Then, by using a three-dimensional PSHG biophysical model, we correlated this finding with the structural changes occurring in the microtubules under oxygen and glucose deprivation. To our knowledge, this is the first study performed in living neuronal cells that is based on direct imaging of axons and that provides the means of identifying the early symptoms of ischemia. Live observation of this process might bring new insights into understanding the dynamics and the mechanisms underlying neuronal degeneration or mechanisms of protection or regeneration.This work is supported by the Spanish government through the Ministry of Economy and Competitiveness, grants No. TEC2009-09698 and No. FIS2009-09928; the Ministerio de Sanidad, grant No. FIS PI081932; the Laserlab-Europe Cont. grant No. JRA4:Optobio 212025; and the Photonics for Life Networks of Excellence. This research has also been partially supported by Fundació Cellex Barcelona and partially conducted at the Institute of Photonic Sciences’ Super-Resolution Light Nanoscopy Facility.Peer reviewe

Media 1: Fast monitoring of in-vivo conformational changes in myosin using single scan polarization-SHG microscopy

Originally published in Biomedical Optics Express on 01 December 2014 (boe-5-12-4362

Imaging amylopectin's order in starch using 3-dimensional polarization SHG

In minimally destructive SHG biomedical imaging (high resolution optical slicing) is greatly desirable to extract the maximum of information from the light matter interaction. Here we develop a 3-D biophysical model and a methodology, which extracts molecular information below the experimental resolution limit. Firstly, it provides the pitch angle (SHG effective orientation) of the SHG source helix of the sample. This information is used to characterize and categorize the SHG sources among them. And secondly, it provides the degree of organization of the SHG source molecules. This can be used as a quantitative imaging biomarker able to characterize the degree of organization (homeostasis) of the sample. Here we applied the model in dried and hydrated wheat starch granules. Our results show that the SHG source molecule in starch is amylopectin. We also conclude that under hydration, the amylopectin molecules are further organized but they do not change structure. This organization is reflected to the width of the pitch angles pixels’ histograms’ distributions. The shorter the width is, the more organized the amylopectin molecules in starch are.Peer Reviewe