radiance 2100 mp confocal scan head  (Bio-Rad)

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protocol for Identifying Natural Agents That Selectively Affect Adhesion, Thickness, Architecture, Cellular Phenotypes, Extracellular Matrix, and Human White Blood Cell Impenetrability of Candida albicans Biofilms

doi: 10.1128/AAC.01319-17

Figure Lengend Snippet: Procedure for generating biofilm preparations for analysis and the parameters that were assessed in comparing the effects of extracts and compounds on individual biofilm development. (A) Flowchart of the procedure. See Materials and Methods and Results for details. (B) Model of a control biofilm and the assessed parameters. Note that pseudohyphae (parameter 7) are not formed in untreated control biofilms. SE disc, silicone elastomer disc; sLee's medium, supplemented Lee's medium; BYCP, basal yeast cell polylayer; CLSM, confocal laser scanning microscopy, GFP-expressing PMNs, green fluorescent protein-expressing polymorphonuclear leukocytes.

Article Snippet: To assess the effects of the extracts or compounds on the biofilm architecture and PMN impenetrability, biofilms were fixed with paraformaldehyde, stained with calcofluor white, and scanned using a Bio-Rad Radiance 2100 MP multiphoton confocal laser scanning microscope (CLSM) equipped with a 20× Plan Fluor water immersion objective and a Nikon TE 2000 inverted microscope.

Techniques: Confocal Laser Scanning Microscopy, Expressing

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protocol for Identifying Natural Agents That Selectively Affect Adhesion, Thickness, Architecture, Cellular Phenotypes, Extracellular Matrix, and Human White Blood Cell Impenetrability of Candida albicans Biofilms

doi: 10.1128/AAC.01319-17

Figure Lengend Snippet: Individual CLSM scans at different heights of a control biofilm (untreated) and an extract 84701-treated biofilm, which exhibits a loss of vertical hypha orientation and defects in other biofilm parameters. (A) Scan at the 4-cell level through the basal yeast cell polylayer (BYCP) of a 48-h-old untreated (control) biofilm. (B) Scan at the same height as the scan of the control biofilm in panel A of a preparation treated with extract 84701 after 48 h. Note that the biofilm treated with extract 84701 lacked a BYCP and vertical hypha orientation. (C) Scan at the midpoint (80 μm) through the hyphal region of a 48-h-old untreated control biofilm. Note the punctate pattern of the vertically oriented hyphae. (D) Scan at the same height as the scan of the control in panel C of a preparation treated with extract 84701. Note the randomly oriented hyphae, as was the case at the lower level in panel B. (E) The matted top of a 48-h-old untreated (control) biofilm after the most distal ends had bent. (F) Visualization of the ECM at the midpoint of a 48-h-old control biofilm in the interstitial regions between cells by increasing midgrayscale intensity.

Article Snippet: To assess the effects of the extracts or compounds on the biofilm architecture and PMN impenetrability, biofilms were fixed with paraformaldehyde, stained with calcofluor white, and scanned using a Bio-Rad Radiance 2100 MP multiphoton confocal laser scanning microscope (CLSM) equipped with a 20× Plan Fluor water immersion objective and a Nikon TE 2000 inverted microscope.

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Protocol for Identifying Natural Agents That Selectively Affect Adhesion, Thickness, Architecture, Cellular Phenotypes, Extracellular Matrix, and Human White Blood Cell Impenetrability of Candida albicans Biofilms

doi: 10.1128/AAC.01319-17

Figure Lengend Snippet: Orthogonal (side) views of biofilm staining with calcofluor white or imaging of green fluorescent protein (GFP)-expressing PMNs after treatment with five pairs of extracts and derivative compounds previously shown to inhibit the growth of C. albicans. The five pairs of extracts and derivative agents are described in Table 1, and biofilm characteristics are described in Table 2. (A to D) Untreated biofilm control in the absence of DMSO (A, B) and untreated biofilm control in the presence of 0.4% DMSO (C, D). Note that all test biofilms treated with extracts or derivative agents contained 0.4% DMSO. (E to H) Representative biofilms treated with the extract of Pentagonia gigantifolia (E, F) or the derivative 6-nonadecynoic acid (G, H). (I to L) Representative biofilms treated with the extract of Duguetia hadrantha (I and J) or the derivative sampangine (K and L). (M to P) Representative biofilms treated with the extract of Piper coruscans (M, N) or the derivative coruscanone A (O, P). (Q to T) Representative biofilms treated with the extract of Liriodendron tulipifera (Q, R) or the derivative liriodenine methiodide (S, T). (U to X) Representative biofilms treated with the extract of Pellina eusiphonia (U, V) or the derivative phloeodictine mixture (W, X). The matted hyphae in the Pellina eusiphonia extract-treated preparation unwound when inverted, causing the biofilm, which was devoid of ECM, to appear thicker than it was. The dashed lines in panels U and V reflect the actual upright thickness in this case. The orthogonal views represent vertical slices (sections) through the stack of CLSM scans (projected image) that had been rotated 90°. The view is of an orthogonal slice taken 40 pixels into the projected image.

Article Snippet: To assess the effects of the extracts or compounds on the biofilm architecture and PMN impenetrability, biofilms were fixed with paraformaldehyde, stained with calcofluor white, and scanned using a Bio-Rad Radiance 2100 MP multiphoton confocal laser scanning microscope (CLSM) equipped with a 20× Plan Fluor water immersion objective and a Nikon TE 2000 inverted microscope.

Techniques: Staining, Imaging, Expressing