Dynamic and spatially resolved mid-infrared characterization of biofilms

In this contribution we present results on non-destructive chemical imaging in the mid-infrared (MIR) region of well-defined biofilms formed by Pseudomonas simiae. Biofilms were grown on stainless steel slides using a static biofilm model (incubation lasted for seven days at 10 °C, with repetitive medium changes). The MIR spectra correlate with fundamental molecular vibrations and are therefore characteristic for chemical composition and structure [1, 2]. Besides a brief insight into the systematic of how the investigated biofilms were grown the main focus will be on MIR spectroscopic measurements including dynamic observation of drying processes of bacteria, as well as spatially resolved scans of the steel plates with an MIR microscope. The obtained hyperspectral chemical images of biofilms were analyzed by various spectroscopic data analysis techniques.

Furthermore, the dynamic spectroscopic observation of the drying process of planktonic Pseudomonas simiae cultures in nutrient solution gave insight in dynamic variances in certain functional chemical groups of the bacteria.  These variances have also been observed in biofilm samples and may correlate with vitality. The MIR chemical images, where each pixel is composed of an entire MIR spectrum (4000-400 cm^-1) provide detailed information of the investigated biofilms such as their composition and spatial structure. The overlay with conventional microscope images relates spectroscopic to visual data, both laterally resolved in the µm-range, over a scan area of up to 10 x 40 mm².

The variation of the vibrational bands was screened, revealing high and low variance bands, to identify certain spectral regions suitable for classification of the investigated biofilm samples. Characteristic spectral bands were found and related to data from literature. Furthermore, differences in the spatial distribution of proteins and carbohydrates as part of the bacteria and extracellular polymeric substances were clearly identified.

Acknowledgment: 
This work was created within a research project of the des Austrian Competence Centre for Feed and Food Quality, Safety and Innovation (FFoQSI). The COMET-K1 competence centre FFoQSI is funded by the Austrian ministries BMVIT, BMDW and the Austrian provinces Niederoesterreich, Upper Austria and Vienna within the scope of COMET -Competence Centers for Excellent Technologies. The programme COMET is handled by the Austrian Research Promotion Agency FFG.

References:
[1] Andreas Schwaighofer, Markus Brandstetter and  Bernhard Lendl , “Quantum cascade lasers (QCLs) in biomedical spectroscopy”, Chem. Soc. Rev. 46, 5903-5924 (2017)
[2] Jakob Kilgus, Gregor Langer, Kristina Duswald, Robert Zimmerleiter, Ivan Zorin, Thomas Berer, and Markus Brandstetter, "Diffraction limited mid-infrared reflectance microspectroscopy with a supercontinuum laser," Opt. Express 26, 30644-30654 (2018)