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).