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. 2023:53:309-324.
doi: 10.1016/bs.mim.2023.05.005. Epub 2023 Jun 26.

Use of epifluorescence widefield deconvolution microscopy for imaging and three-dimensional rendering of Pseudomonas aeruginosa biofilms and extracellular matrix materials

Heidi J Smith  1   2 , Michael J Franklin  1   2
Affiliations

Use of epifluorescence widefield deconvolution microscopy for imaging and three-dimensional rendering of Pseudomonas aeruginosa biofilms and extracellular matrix materials

Heidi J Smith et al. Methods Microbiol. 2023.
No abstract available

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Figures

FIG. 1
FIG. 1
Maximum projection epifluorescence widefield image of a P. aeruginosa PAO1 (pMF230) biofilm cultured on the glass-bottom surface of a microtiter plate in biofilm minimal medium (BMM). Each image shows the same field of view. (A, E) showing the GFP-labelled cells, (B, F) showing the extracellular material that stains with cell mask orange false coloured red, and (C, G) showing the extracellular material that stains with Bodipy X-SE 630/650 false coloured cyan. Panels (D, H) show an overlay of the three fluorescent images. Panels (A–D) show the early biofilm prior to deconvolution. Panels (E–H) show the biofilms following computational deconvolution using the Leica Instant Computational Clearing method.
FIG. 2
FIG. 2
Maximum projection epifluorescence widefield image of a P. aeruginosa PAO1 (pMF230) biofilm cultured on the glass-bottom surface of a microtiter plate in BMM with 2 mM CaCl2. Each image shows the same field of view. (A, E) showing the GFP-labelled cells, (B, F) showing the extracellular material that stains with cell mask orange false coloured red, and (C, G) showing the extracellular material that stains with Bodipy X-SE 630/650 false coloured cyan. Panels (D, H) show an overlay of the three fluorescent images. Panels (A–D) show the early biofilm prior to deconvolution. Panels (E–H) show the biofilms following computational deconvolution using the Leica Instant Computational Clearing method.
FIG. 3
FIG. 3
Maximum projection epifluorescence widefield image of a P. aeruginosa PAO1 ΔpslA (pMF230) biofilm cultured on the glass-bottom surface of a microtiter plate in BMM. Each image shows the same field of view. (A, E) showing the GFP-labelled cells, (B, F) showing the extracellular material that stains with cell mask orange false coloured red, and (C, G) showing the extracellular material that stains with Bodipy X-SE 630/650 false coloured cyan. Panels (D, H) show an overlay of the three fluorescent images. Panels (A–D) show the early biofilm prior to deconvolution. Panels (E–H) show the biofilms following computational deconvolution using the Leica Instant Computational Clearing method.
FIG. 4
FIG. 4
Maximum projection epifluorescence widefield image of a P. aeruginosa PAO1 ΔpslA (pMF230) biofilm cultured on the glass-bottom surface of a microtiter plate in BMM with 2 mM CaCl2. Each image shows the same field of view. (A, E) showing the GFP-labelled cells, (B, F) showing the extracellular material that stains with cell mask orange false coloured red, and (C, G) showing the extracellular material that stains with Bodipy X-SE 630/650 false coloured cyan. Panels (D, H) show an overlay of the three fluorescent images. Panels (A–D) show the early biofilm prior to deconvolution. Panels (E–H) show the biofilms following computational deconvolution using the Leica Instant Computational Clearing method.
FIG. 5
FIG. 5
Expanded image of a microcolony of P. aeruginosa PAO1 (pMF230) from Fig. 1, showing the workflow used to enumerating biofilm cells and extracellular matrix components. All panels show the same field of view. (A) Microcolony showing the GFP-labelled cells following deconvolution using the Leica Instant Computational Clearing method, and (B) microcolony prior to deconvolution. (C) Surface map of biofilm cells generated from the deconvoluted image, using Imaris software to enumerate individual cells within the microcolony. (D) Surface map of deconvoluted microcolony image, generated using Imaris software. The surface map was used to calculate biovolumes for the extracellular matrix material within a microcolony with the cells shown in green, the cell mask orange-stained material shown in red, and the Bodipy X-SE 630/650-stained material shown in blue.
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