Narrowband-autofluorescence imaging for bone analysis

Fauch, Laure; Palander, Anni; Dekker, Hannah; Schulten, E.A.J.M.; Koistinen, Arto; Kullaa, Arja; Keinänen, Markku

doi:10.1364/boe.10.002367

Cited by 7 publications

(9 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…CD68 mRNA expression was strong in the bone marrow (Figure 5A,C) and in the interface between the periosteum and cortical bone (Figure 5B,D) with both in situ techniques. 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).…”

Section: Resultsmentioning

confidence: 94%

See 1 more Smart Citation

In Situ Gene Expression in Native Cryofixed Bone Tissue

Nikovics¹,

Castellarin²,

Holy³

et al. 2022

Biomedicines

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 94%

“…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 ).…”

Section: Resultsmentioning

confidence: 99%

In Situ Gene Expression in Native Cryofixed Bone Tissue

Nikovics¹,

Castellarin²,

Holy³

et al. 2022

Biomedicines

View full text Add to dashboard Cite

show abstract

“…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 fluorophore-labeled antibody) because there was an abundant autofluorescence in the regenerating region ( Figure 6 ).…”

Section: Resultsmentioning

confidence: 99%

Macrophages Characterization in an Injured Bone Tissue

Nikovics¹,

Durand

Castellarin³

et al. 2022

Biomedicines

View full text Add to dashboard Cite

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.

show abstract

“…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.…”

Section: Tesseraementioning

confidence: 99%

Autofluorescence of stingray skeletal cartilage: hyperspectral imaging as a tool for histological characterization

et al. 2021

View full text Add to dashboard Cite

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).

show abstract

Narrowband-autofluorescence imaging for bone analysis

Cited by 7 publications

References 43 publications

In Situ Gene Expression in Native Cryofixed Bone Tissue

In Situ Gene Expression in Native Cryofixed Bone Tissue

Macrophages Characterization in an Injured Bone Tissue

Autofluorescence of stingray skeletal cartilage: hyperspectral imaging as a tool for histological characterization

Contact Info

Product

Resources

About