ABSTRACT
For this study, we have considered a new large field of view imaging dedicated to matrix collagen (no stained samples). To integrate a multidimensional scale (non-sliced samples), a femtosecond oscillator (two photon excitation laser) has been coupled with a large field optical setup to collect SHG signal. We introduced an index (F-SHG) based on decay time response measured by TCSPC for, respectively, Fluorescence (F) and Second Harmonic Generation (SHG) values. For samples where protein collagen is the major component of extracellular matrix (skin) or not (nacre), we compared the index distribution (from 2 to 12) obtained with large field optical setup. In this work, we showed for the first time that multiscale large field imaging combined to multimodality approaches (SHG-TCSPC) could be an innovative and non invasive technique to detect and identify some biological interest molecules (collagen) in biomedical topics.
Subject(s)
Collagen/ultrastructure , Extracellular Matrix/ultrastructure , Microscopy, Fluorescence, Multiphoton/methods , Nacre/analysis , Pinctada/ultrastructure , Skin/ultrastructure , Animals , Collagen/analysis , Extracellular Matrix/chemistry , Male , Pinctada/chemistry , Rats , Skin/chemistryABSTRACT
We propose an innovative invasiveless technique in the field of nonlinear optical imaging to facilitate monitoring of cell/scaffold combinations for tissue repair. By using a near infrared (NIR) femtosecond excitation, we were able to introduce a new index based on decay time response for fluorescence (F) and Second Harmonic Generation (SHG) obtained with Time Correlated Single Photon Counting (TCSPC) microscopy to monitor structural information on the state of the matrix collagen. Some human Mesenchymal Stem Cells (hMSCs) seeded in 3D scaffolds were tested with different culture times (from D7 to D56) to analyze the effect of Tumor Growth Factor beta 1 (TGF-ß1) on type-2 collagen expression in the matrix. After 14 days in the presence of TGF-ß1, our results showed an increase in the expression of type-2 collagen synthesized by hMSCs, and a change in collagen conformation, as an indication of its ability to be detected as a harmonophore by TCSPC-SHG without the need for an exogenous probe.
Subject(s)
Collagen/metabolism , Extracellular Matrix/metabolism , Lighting/methods , Mesenchymal Stem Cells/metabolism , Microscopy, Fluorescence/methods , Spectrometry, Fluorescence/methods , Spectrophotometry, Infrared/methods , Tissue Scaffolds , Cells, Cultured , HumansABSTRACT
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.
Subject(s)
Cartilage/pathology , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Microscopy, Fluorescence, Multiphoton/methods , Microscopy, Interference/instrumentation , Cartilage/metabolism , Cartilage, Articular/metabolism , Cartilage, Articular/pathology , Collagen/metabolism , Compressive Strength , Humans , Proteoglycans/metabolism , Stress, Mechanical , Stress, PhysiologicalABSTRACT
Imaging thick and opaque tissue, like blood vessel, in a noninvasive mode with high resolution, is nowadays possible with multiphoton technology. A near-infrared excitation presents the advantage to be compatible with living specimens and allows a deep penetration into tissues. The nonlinear excitation process is followed by several deactivation ways, among which fluorescence emission can be represented with Spectral or Lifetime imaging. Applied to ex vivo blood vessel imaging, these techniques enabled us to discriminate cell structures (nucleus, cytoskeleton) by fluorescent labelling (Hoechst, QDots). Another method, based on 2-photon excitation and which doesn't need any exogenous dye has also been experimented on arteries: SHG (Second Harmonic Generation) is a diffusion process generated from organized structures. Collagen molecules give rise to a strong SHG signal, enabling us to image the arterial wall (3-dimensional extracellular matrix).
