2.2 Optical coherence tomography and image processing
Optical Coherence Tomography (OCT) is a 3D imaging technique that allows for the non-invasive, real-time imaging of the mesoscopic biofilm structure (Martin, Bolster, Derlon, Morgenroth, & Nerenberg, 2014; Wagner & Horn, 2017). A brief summary regarding the working principle of OCT has already been provided in (Blauert et al., 2015; Gierl et al., 2020).
A spectral domain tomograph (GANYMEDE I, Thorlabs GmbH, Dachau, Germany) with an optical resolution of 8 × 8 × 2.1 µm3 (x × y × z, LSM03 objective lens) in water (\(n\) = 1.33) was used to monitor biofilm development. OCT images (A-scan averaging = 3) with a size of 7 × 6 × 0.5 mm3were acquired on a daily basis. Image post-processing included the calculation of structural biofilm parameters. OCT datasets were cropped to a volume of 7 × 5 × 0.25 mm3 (due to autocorrelation artifacts). A mean filter with a radius of 2 px was applied and binary datasets were generated using Fiji (Schindelin et al., 2012). Substratum coverage (SC), mean biofilm thickness\({(\overset{\overline{}}{L}}_{F})\), textural entropy (TE), kurtosis \((R_{\text{KU}})\), skewness \({(R}_{\text{SK}})\), fractal dimension (FD), angular second moment \((ASM)\), inverse difference moment \((IDM)\) as well as average horizontal \((AHRL)\) and average vertical run lengths \((AVRL)\) were calculated from binary datasets according to (Wagner & Horn, 2017), (Blauert et al., 2015) and by use of the MiToBo plugin (Fiji) for biofilms (Möller, Glaß, Misiak, & Posch, 2016). In-house macros were used to render topographic representations of OCT C-scans (e.g., height maps representing the bulk-biofilm interface) (Wagner & Horn, 2017).
An overview of all structural parameters and their calculation is given in (Beyenal, Donovan, Lewandowski, & Harkin, 2004; Wagner & Horn, 2017; X. Yang, Beyenal, Harkin, & Lewandowski, 2000). ParametersSC, \(R_{\text{KU}}\) and \(R_{\text{SK}}\) were analyzed using Fiji’s plugin function “analyze”. An overview regarding the interpretation of the structural biofilm parameters is presented below (see Table 2).