Abstract
In this work, we have considered a new multimodality imaging for macroscopy based on Second Harmonic Generation (SHG) method to monitor invasivelessly the matrix collagen. As the triple helicoidally structure of collagen molecules appearing as not centrosymetric, very organized and spatially oriented, collagen fibrils give rise to a very strong SHG signal and can be imaged without any exogenous dye. To integrate a multidimensional scale with a large field of view (non-sliced samples), we have adapted and validated an instrumental coupling between a two photon excitation laser and a macroscope to collect cartography of 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 various sample where protein collagen is the major component of extracellular matrix (vessel, skin, carotide vessel, rat femoral head cartilage, mouse tumor, human wharton’s jelly and rat tendon) or not (nacre), we compared the index distribution obtained with MacroSHG. In this work, we showed for the first time that multiscale large field imaging (Macroscopy) 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.
Highlights
To appreciate a high level of complexity in 3D organization of collagen network into biological matrix, optical microscopy, as non-destructive imaging technique, has a number of strong advantages over alternative imaging modalities (MRI, CT-scan, X-ray, arthro-scan, OCT, etc.)
This technique is well appreciated to estimate the topography of network of collagen in extracellular matrix (ECM) but it is not dedicated to quantify the amount of fibrillar collagen molecules
We proposed a study based on new Second Harmonic Generation (SHG) imaging contrast in macroscopy to detect “collagen index” from various tissues
Summary
To appreciate a high level of complexity in 3D organization of collagen network into biological matrix, optical microscopy, as non-destructive imaging technique, has a number of strong advantages over alternative imaging modalities (MRI, CT-scan, X-ray, arthro-scan, OCT, etc.). The red Sirius as staining agent is specific of fibrillar collagens in polarized light (Sweat, 1964) and is sensitive to the alignment of the molecules of collagen (red color for the extracellular matrix and yellow color for some other structures) This technique is well appreciated to estimate the topography of network of collagen in ECM but it is not dedicated to quantify the amount of fibrillar collagen molecules. In TCSPC-SHG microscopy, the limitations due to photobleaching and phototoxicity in biological samples are overcome This multimodality technique could be considered as a precise tool for biodiagnosis to detect in safety conditions collagen synthetized by cells seeded in scaffolds. This new approach in near-infrared SHG microscopy may not allow for sufficient tissue penetration to observe with a large field of viewing an organized network collagen. We showed that multiscale detection of SHG signal specific to collagen network is a non-invasive and potential technology in tomography to characterize the organized matrix
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