Abstract
| Original language | English |
|---|---|
| Article number | 100209 |
| Pages (from-to) | 1-8 |
| Number of pages | 9 |
| Journal | Biofilm |
| Volume | 8 |
| Early online date | 3 Jul 2024 |
| DOIs | |
| Publication status | Published (in print/issue) - 30 Dec 2024 |
Bibliographical note
Publisher Copyright:© 2024 The Authors
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.Funding
Bright-field microscopy was performed on biofilms after the respective chemical treatment. The agar plates were inverted and placed on the stage of an inverted bright-field microscope (Nikon Ti\u2013S). Images were acquired with a 10x objective using a camera (GS3-U3-51S5M, Grasshopper). For confocal imaging, after treatments with the chemical compounds, the biofilms were stained in-situ using 10 \u03BCL of 10 \u03BCM Syto63 (Sigma, S11345) and incubated for 30 min. After staining, agar supporting the biofilm samples which had been stained were cut using a scalpel blade and carefully transferred to a microscope slide attached to a 25 \u03BCL geneframe (Sigma, UK). The geneframe was sealed with a #1.5 coverslip. Imaging was performed on a Leica SP8 confocal laser scanning microscope (CLSM) using a 100x oil immersion objective. Syto63 was excited at a wavelength of 660 nm and the emission filter was set between 670 and 750 nm. A total of 5 random fields of view (FOV) measuring 101 \u03BCm \u00D7 101 \u03BCm were imaged at 1024 pixels \u00D7 1024 pixels, yielding a pixel calibration of 98.6 nm/pixel. The pinhole was set at 1 Airy units (AU). Imaging was performed 10 \u03BCm above the coverslip. Images were pre-processed in ImageJ by applying a contrast-limited adaptive histogram equalization (CLAHE) filter with settings: box size = 127 pixels, block size = 50, histogram = 256, maximum = 2, for denoising and contrast enhancement [9]. Image smoothing was performed with a Gaussian filter of sigma of 2 pixels and then a Laplacian of Gaussian filter with a radius of 2 pixels before binarization using the Otsu thresholding method. The packing or area fraction (\u03C6) was then calculated as the ratio of cells to void space using relation \u03C6 = Acells/Avoid.J.C. acknowledges funding from Engineering and Physical Sciences Research Council (EPSRC), United Kingdom through award number EP/K039083/1 to Newcastle University. S.C. gratefully acknowledges EPSRC Doctoral Training Program studentship from Newcastle University. Authors are grateful for assistance from members of Bioimaging Unit at Newcastle University. J.C. acknowledges funding from Engineering and Physical Sciences Research Council (EPSRC), United Kingdom through award number EP/K039083/1 to Newcastle University. S.C. gratefully acknowledges EPSRC Doctoral Training Program studentship from Newcastle University. Authors are grateful for assistance from members of Bioimaging Unit at Newcastle University .
| Funders | Funder number |
|---|---|
| EP/K039083/1 | |
| Newcastle University | |
| S11345, 670 | |
| Engineering and Physical Sciences Research Council | EP/K039083/1 |
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