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We present a new autofluorescence-imaging method for bone analysis. This method, based on the autofluorescence of bone, provides color images in microscopic scale. The color images are created from three monochrome images acquired with optimal excitation- and emission-wavelengths combinations. The choice of these combinations were determined from t...
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... The TumorID remains an exciting methodology for discerning tumor from healthy tissue, however, there are several limitations to this work. Bone displayed a high level of reflectivity to the incident 405 nm radiation which presented challenges to data collection that have been described previously 47 . This is not an unexpected result as the difference in color as well as the frequent lack of blood and hemoglobin products on the surface would allow for much greater reflectivity. ...
Ten patients undergoing surgical resection for spinal tumors were selected. Samples of tumor, muscle, and bone were resected, de-identified by the treating surgeon, and then scanned with the TumorID technology ex vivo. This study investigates whether TumorID technology is able to differentiate three different human clinical fresh tissue specimens: spine tumor, normal muscle, and normal bone. The TumorID technology utilizes a 405 nm excitation laser to target endogenous fluorophores, thereby allowing for the detection of tissue based on emission spectra. Metabolic profiles of tumor and healthy tissue vary, namely NADH (bound and free emission peak, respectively: 487 nm, 501 nm) and FAD (emission peak: 544) are endogenous fluorophores with distinct concentrations in tumor and healthy tissue. Emission spectra analyzed consisted of 74 scans of spine tumor, 150 scans of healthy normal bone, and 111 scans of healthy normal muscle. An excitation wavelength of 405 nm was used to obtain emission spectra from tissue as previously described. Emission spectra consisted of approximately 1400 wavelength intensity pairs between 450 and 750 nm. Kruskal–Wallis tests were conducted comparing AUC distributions for each treatment group, α = 0.05. Spectral signatures varied amongst the three different tissue types. All pairwise comparisons among tissues for Free NADH were statistically significant (Tumor vs. Muscle: p = 0.0006, Tumor vs. Bone: p < 0.0001, Bone vs. Muscle: p = 0.0357). The overall comparison of tissues for FAD (506.5–581.5 nm) was also statistically significant (p < 0.0001), with two pairwise comparisons being statistically significant (Tumor vs. Muscle: p < 0.0001, Tumor vs. Bone: p = 0.0045, Bone vs. Muscle: p = 0.249). These statistically significant differences were maintained when stratifying tumor into metastatic carcinoma (N = 57) and meningioma (N = 17). TumorID differentiates tumor tissue from normal bone and normal muscle providing further clinical evidence of its efficacy as a tissue identification tool. Future studies should evaluate TumorID’s ability to serve as an adjunctive tool for intraoperative assessment of surgical margins and surgical decision-making.
... In this study, the molecular variations in human-mandibular osteoid and new bone inside cortical bone's osteon are analyzed by using Fourier-transform infrared (FTIR) imaging spectroscopy combined with autofluorescence imaging developed previously by Fauch et al. [13]. The FTIR method is commonly used for analyzing the chemical composition of bone, but has not been previously combined with autofluorescence imaging. ...
... The exposure time for each acquired image was set at 10 s and the gain at 255. After the acquisition, a triband image of 0.3-μm-spatial resolution and 420 × 320-μm field of view was created from the three acquired images according to the method described by Fauch et al. [13]. The spatial resolution of the triband images was reduced to provide color images of 1.1-μm-spatial resolution corresponding to the spatial resolution of the FTIR-hyperspectral images. ...
Osteoid is a layer of new-formed bone that is deposited on the bone border during the process of new bone formation. This deposition process is crucial for bone tissue, and flaws in it can lead to bone diseases. Certain bone diseases, i.e. medication related osteonecrosis, are overexpressed in mandibular bone. Because mandibular bone presents different properties than other bone types, the data concerning osteoid formation in other bones are inapplicable for human-mandibular bone. Previously, the molecular distribution of other bone types has been presented using Fourier-transform infrared (FTIR) spectroscopy. However, the spatial distribution of molecular components of healthy-human-mandibular-bone osteoid in relation to histologic landmarks has not been previously presented and needs to be studied in order to understand diseases that occur human-mandibular bone. This study presents for the first time the variation in molecular distribution inside healthy-human-mandibular-bone osteoid by juxtaposing FTIR data with its corresponding histologic image obtained by autofluorescence imaging of its same bone section. During new bone formation, bone-forming cells produce an osteoid constituted primarily of type I collagen. It was observed that in mandibular bone, the collagen type I increases from the osteoblast line with the distance from the osteoblasts, indicating progressive accumulation of collagen during osteoid formation. Only later inside the collagen matrix, the osteoid starts to mineralize. When the mineralization starts, the collagen accumulation diminishes whereas the collagen maturation still continues. This chemical-apposition process in healthy mandibular bone will be used in future as a reference to understand different pathologic conditions that occur in human-mandibular bone.
... A differential autofluorescence emission signal between a young and an older bone has been demonstrated after an excitation-light wavelength of around 350 nm (Prentice, 1967). This phenomenon may be attributed to the cellular components of the different tissue compartments, as well as to the collagen content (Fauch et al., 2019). ...
Traditional thin sectioning microscopy of large bone and dental tissue samples using demineralization may disrupt structure morphologies and even damage soft tissues, thus compromising the histopathological investigation. Here, we developed a synergistic and original framework on thick sections based on wide-field multi-fluorescence imaging and spectral Principal Component Analysis (sPCA) as an alternative, fast, versatile, and reliable solution, suitable for highly mineralized tissue structure sustain and visualization. Periodontal 2-mm thick sections were stained with a solution containing five fluorescent dyes chosen for their ability to discriminate close tissues, and acquisitions were performed with a multi-zoom macroscope for blue, green, red, and NIR (near-infrared) emissions. Eigen-images derived from both standard scaler (Std) and Contrast Limited Adaptive Histogram Equalization (Clahe) pre-preprocessing significantly enhanced tissue contrasts, highly suitable for histopathological investigation with an in-depth detail for sub-tissue structure discrimination. Using this method, it is possible to preserve and delineate accurately the different anatomical/morphological features of the periodontium, a complex tooth-supporting multi-tissue. Indeed, we achieve characterization of gingiva, alveolar bone, cementum, and periodontal ligament tissues. The ease and adaptability of this approach make it an effective method for providing high-contrast features that are not usually available in standard staining histology. Beyond periodontal investigations, this first proof of concept of an sPCA solution for optical microscopy of complex structures, especially including mineralized tissues opens new perspectives to deal with other chronic diseases involving complex tissue and organ defects. Overall, such an imaging framework appears to be a novel and convenient strategy for optical microscopy investigation.
... It is well known that bone tissue has strong autofluorescence [62,63]. This makes it rather difficult to analyze the operated tissue with immunolabeling (using fluorophorelabeled antibody) because there was an abundant autofluorescence in the regenerating region ( Figure 6). ...
Biomaterial use is a promising approach to facilitate wound healing of the bone tissue. Biomaterials induce the formation of membrane capsules and the recruitment of different types of macrophages. Macrophages are immune cells that produce diverse combinations of cytokines playing an important role in bone healing and regeneration, but the exact mechanism remains to be studied. Our work aimed to identify in vivo macrophages in the Masquelet induced membrane in a rat model. Most of the macrophages in the damaged area were M2-like, with smaller numbers of M1-like macrophages. In addition, high expression of IL-1β and IL-6 cytokines were detected in the membrane region by RT-qPCR. Using an innovative combination of two hybridization techniques (in situ hybridization and in situ hybridization chain reaction (in situ HCR)), M2b-like macrophages were identified for the first time in cryosections of non-decalcified bone. Our work has also demonstrated that microspectroscopical analysis is essential for macrophage characterization, as it allows the discrimination of fluorescence and autofluorescence. Finally, this work has revealed the limitations of immunolabelling and the potential of in situ HCR to provide valuable information for in vivo characterization of macrophages.
... Macrophages were identified based on the expression of CD68 mRNA ( Figure 5). CD68 mRNA expression was strong in the bone marrow ( Figure 5A Autofluorescence of bone tissue was previously described [50][51][52]. To confirm that the detected fluorescence resulted from an in situ-HCR signal and did not derive from nonspecific binding of an Alexa Fluor 488 molecule or from autofluorescence, hybridization was evaluated by microspectrofluorimetry ( Figure 6). ...
... The emission spectra of the fluorescence peaked at 520 nm, corresponding to Alexa Fluor 488 fluorophore spectra. Biomedicines 2022, 10, x FOR PEER REVIEW 11 of 15 Autofluorescence of bone tissue was previously described [50][51][52]. To confirm that the detected fluorescence resulted from an in situ-HCR signal and did not derive from nonspecific binding of an Alexa Fluor 488 molecule or from autofluorescence, hybridization was evaluated by microspectrofluorimetry ( Figure 6). ...
Bone is a very complex tissue that is constantly changing throughout the lifespan. The precise mechanism of bone regeneration remains poorly understood. Large bone defects can be caused by gunshot injury, trauma, accidents, congenital anomalies and tissue resection due to cancer. Therefore, understanding bone homeostasis and regeneration has considerable clinical and scientific importance in the development of bone therapy. Macrophages are well known innate immune cells secreting different combinations of cytokines and their role in bone regeneration during bone healing is essential. Here, we present a method to identify mRNA transcripts in cryosections of non-decalcified rat bone using in situ hybridization and hybridization chain reaction to explore gene expression in situ for better understanding the gene expression of the bone tissues.
... In this regard, however, the multiple fluorophores shared between the center and outer border are more difficult to reconcile, as the center is believed to be a mineralization hub only earlier in skeletal development (see above). In the case of the center region, perhaps the AF signal is related to tissue age and degree of mineralization, as in bone [56]. The tissue properties and components at the root of the AF differences among the center, peripheral and outer border regions require characterization; however, their autofluorescent characteristics and the accretionary growth of tesserae argue these differences are linked to the regions having specific developmental roles and/or representing important turning points in the mineralization of the skeleton. ...
Tessellated cartilage is a distinctive composite tissue forming the bulk of the skeleton of cartilaginous fishes (e.g. sharks and rays), built from unmineralized cartilage covered at the surface by a thin layer of mineralized tiles called tesserae. The finescale structure and composition of elasmobranch tessellated cartilage has largely been investigated with electron microscopy, micro-computed tomography and histology, but many aspects of tissue structure and composition remain uncharacterized. In our study, we demonstrate that the tessellated cartilage of a stingray exhibits a strong and diverse autofluorescence, a native property of the tissue which can be harnessed as an effective label-free imaging technique. The autofluorescence signal was excited using a broad range of wavelengths in confocal and light sheet microscopy, comparing several sample preparations (fresh; demineralized and paraffin-embedded; non-demineralized and plastic-embedded) and imaging the tissue at different scales. Autofluorescence varied with sample preparation with the signal in both plastic- and paraffin-embedded samples strong enough to allow visualization of finescale (≥ 1 μm) cellular and matrix structures, such as cell nuclei and current and former mineralization fronts, identifiable by globular mineralized tissue. A defined pericellular matrix (PCM) surrounding chondrocytes was also discernible, described here for the first time in elasmobranchs. The presence of a PCM suggests similarities with mammalian cartilage regarding how chondrocytes interact with their environment, the PCM in mammals acting as a transducer for biomechanical and biochemical signals. A posterior analysis of hyperspectral images by an MCR-ALS unmixing algorithm allowed identification of several distinct fluorescence signatures associated to specific regions in the tissue. Some fluorescence signatures identified could be correlated with collagen type II, the most abundant structural molecule of cartilage. Other fluorescence signatures, however, remained unidentified, spotlighting tissue regions that deserve deeper characterization and suggesting the presence of molecules still unidentified in elasmobranch skeletal cartilage. Our results show that autofluorescence can be a powerful exploratory imaging tool for characterizing less-studied skeletal tissues, such as tessellated cartilage. The images obtained are largely comparable with more commonly used techniques, but without the need for complicated sample preparations or external staining reagents standard in histology and electron microscopy (TEM, SEM).
... 50 In the University of Eastern Finland there are currently research projects to develop digital imaging and digital histological staining techniques for oral tissues (teeth, oral mucosa, alveolar bone), using spectral imaging. 51,52 ...
Despite universal health coverage and a strong public health system, the oral health profile of Finland falls behind in an international age-matched prevalence of oral diseases. The oral healthcare system is organised and funded mainly by municipalities. Other stakeholders include the Finnish Student Health Service foundation (FSHS), government and private practices, where the Social Insurance Institution of Finland plays a major role in funding. Rise in the treatment need in recent years due to the increasing dentulous ageing population has challenged the healthcare system. Governmental response to the demand is an ongoing social and healthcare reform and increase of oral health professional education since 2004. However, the current and future treatment need is not met only by conventional prevention strategies and physical service provision. Finland has over the years supported a determined policy of building a digital healthcare architecture. This applies also to all fields of oral healthcare: virtual education, digital diagnostics, digital clinical workflow, national electronic patient records, patient-generated data registers, electronic prescriptions, remote consultation, digital service management, as well as research and big data mining. These tools could play an important role in improving national oral health and increasing equity. This is an overview of the above-mentioned fields of e-Oral health and teledentistry in Finland based on current scientific literature, national reports, strategies and legislation.
