Journal: International Journal of Molecular Sciences

Article Title: Insights into S. aureus -Induced Bone Deformation in a Mouse Model of Chronic Osteomyelitis Using Fluorescence and Raman Imaging

doi: 10.3390/ijms24119762

Figure Lengend Snippet: Confocal images of immunofluorescence-labelled pelvis cryo-sections LF1, LF2, LF3, and LF4 (as indicated in c). ( Top row ): Immunofluorescence images. Fluorescence staining highlights cell nuclei (DAPI, blue), actin-cytoskeleton (I555-Phalloidin, red), S. aureus (specific antibody AF488: green). The pink colour arises here from the overlay of nuclei and actin-cytoskeleton. Alphabetically labelled regions (a, c–g, i) refer to panel labels of enlarged views in . ( Bottom row ): Colour-coded slices according to structures. Trabecular bone (green) and lesions (blue) are visible in all slices. Muscle tissue (red) is still attached to the periosteum. Staphylococcal abscess communities (SACs) are visible in section LF3 (yellow).

Article Snippet: Next, the secondary antibody, AF488 donkey anti-rabbit Fab fragment (abnova, Taipei, Taiwan) in 1:200 dilution or DyLight405 donkey anti-rabbit IgG (dianova/BIOZOL, Hamburg, Germany) at 1:200 dilution plus Dylight650 donkey anti-goat IgG (Thermo Fisher Scientific) dilution 1:250 in permeabilization buffer were applied for 10 days at room temperature while shaking followed by washing thrice for 1 h and twice for 10 min in permeabilization buffer.

Techniques: Immunofluorescence, Fluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Insights into S. aureus -Induced Bone Deformation in a Mouse Model of Chronic Osteomyelitis Using Fluorescence and Raman Imaging

doi: 10.3390/ijms24119762

Figure Lengend Snippet: Detailed images of host tissue structures in infected pelvis. Colour-code of channels is given below. ( a ) Accumulation of immune cells around the SAC (green-stained S. aureus ), e.g., neutrophils (white circle), lymphocytes (orange circle), macrophages (in contour) assigned based on the nuclear shape (DAPI) and size (immunofluorescence, LF3) ( b ) Presence of NETs (white arrows) in close vicinity to S. aureus infiltrates (green arrows) in the lesion (immunofluorescence, RF2). ( c ) Orthogonal view of a foamy macrophage with bacteria phagocytosed (immunofluorescence, LF4). ( d ) Accumulation of fibroblast (immunofluorescence, LF1). ( e ) Deposition of collagen observed using SHG microscopy in inflamed tissue next to nuclei of fibroblasts (immunofluorescence, LF1). ( f ) Ortho-view showing osteocalcin deposits (pink) at the edges of mineralised, collagen-rich (orange) bone (immunofluorescence and SHG microscopy, LF2). ( g ) Immunofluorescence image of osteocalcin-rich (pink) area in soft tissue. Single bacteria were identified (green arrows) in addition to some cells with high osteocalcin production (white arrows) (LF1). ( h ) H&E staining reveals spindle-like cells in the trabecular spaces of infected bone tissue (RH1). ( i ) Broken trabecular structure infiltrated with the lesion (immunofluorescence LF3). Channel colours for the immunofluorescence images mark the following features (and used fluorophore): blue: nuclei (DAPI or SYTOX green), red: actin-cytoskeleton (I555-Phalloidin), green: S. aureus (DY405 or AF488), pink: osteocalcin (Dy650), orange: collagen (SHG). Note that for clarity not all channels were shown in all panels.

Article Snippet: Next, the secondary antibody, AF488 donkey anti-rabbit Fab fragment (abnova, Taipei, Taiwan) in 1:200 dilution or DyLight405 donkey anti-rabbit IgG (dianova/BIOZOL, Hamburg, Germany) at 1:200 dilution plus Dylight650 donkey anti-goat IgG (Thermo Fisher Scientific) dilution 1:250 in permeabilization buffer were applied for 10 days at room temperature while shaking followed by washing thrice for 1 h and twice for 10 min in permeabilization buffer.

Techniques: Infection, Staining, Immunofluorescence, Bacteria, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Insights into S. aureus -Induced Bone Deformation in a Mouse Model of Chronic Osteomyelitis Using Fluorescence and Raman Imaging

doi: 10.3390/ijms24119762

Figure Lengend Snippet: Detailed images of Staphylococci found in various host environments. ( a ) Gram-staining of an abscess in the right pelvis (RG1). ( b ) Enlarged view of the SACs in section LF3 shows a dense accumulation of bacteria surrounded by a capsule. The capsule is broken to one side allowing bacteria to spread into the surrounding lesion. The white square is shown in ( c ). ( c ) Enlarged view of bacteria spreading imaged using 2 photon excitation. ( d ) Clusters of bacteria between host cells, originating from RF1 ( e ) Individual S. aureus between host cells in inflamed tissue (LF1). ( f ) Occasionally, S. aureus were detected in trabecular bone regions (RF1). ( g ) Occasionally, bacteria were also detected in muscle tissue (LF3). ( h ) Intracellular location of S. aureus within an immune cell (maybe a neutrophil) as visible from the ortho-view (LF3). ( i ) Bacterial accumulation with actin in the background (RF1). ( b – i ) Channel colours for the immunofluorescence images mark the following features (and used fluorophore): green: S. aureus (Dy405 or AF488), blue: cell nuclei (DAPI or SYTOXgreen), red: actin-cytoskeleton (I555-Phalloidin), and orange: collagen (SHG).

Article Snippet: Next, the secondary antibody, AF488 donkey anti-rabbit Fab fragment (abnova, Taipei, Taiwan) in 1:200 dilution or DyLight405 donkey anti-rabbit IgG (dianova/BIOZOL, Hamburg, Germany) at 1:200 dilution plus Dylight650 donkey anti-goat IgG (Thermo Fisher Scientific) dilution 1:250 in permeabilization buffer were applied for 10 days at room temperature while shaking followed by washing thrice for 1 h and twice for 10 min in permeabilization buffer.

Techniques: Staining, Bacteria, Immunofluorescence