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A. Balbekova:
"Near- and Far-Field Mid-Infrared Spectroscopy and Spectroscopic Imaging for Biomedical Applications";
Betreuer/in(nen), Begutachter/in(nen): B. Lendl, G. Friedbacher, C. Huck; Chemische Technologien und Analytik, 2017; Rigorosum: 19.12.2017.

Mid-Infrared (IR) spectroscopy allows for non-destructive and label free analysis providing
molecular specific information. This technique can thus be successfully applied in
microbiological and biomedical research. IR spectroscopic imaging is an advantageous
complimentary tool to established histological analysis. However, IR spectroscopy has
certain limitations: the spatial resolution of IR microscopic imaging cannot be better than a
few micrometers due to the diffraction limit in far-field microscopy and IR spectroscopy
cannot provide information on an elemental level.
The first limitation can be solved through the application of recently developed near-field
imaging techniques such as phothermal induced resonance (PTIR). The second limitation can
be overcome when an additional technique complementary to IR spectroscopy is used too
and the combined data-sets are jointly analysed.
The first experimental part of this thesis is devoted to the characterization of a custom made
PTIR system and its application to the measurements of various biological samples. The
second experimental part is devoted to the combined image analysis of histological samples
using commercially available techniques such as Fourier transform Infrared (FTIR)
spectroscopy and laser ablation inductively coupled mass spectrometry (LA-ICP-MS).
In the first part of the work time-resolved PTIR spectroscopic measurements are reported
for the first time on the example of a biopolymer (poly-L-lysine, PLL) 200 nm thick film. PLL
films can adopt different secondary structures depending on its water content, which can be
adjusted through temperature in an atmosphere of controlled humidity. A controlled
temperature ramp of the sample caused changes in the amide I band of the PLL. Those
changes were detected using PTIR time resolved measurements. The achieved acquisition
time of one PTIR spectrum is 15 s. Control analysis using a commercial FTIR microscope
corroborated the spectroscopic results.
Further, the PTIR performance was characterized regarding AFM tip aging during prolonged
measurements of the polymer polystyrene (PS) and biological samples (cells and tissues). It
was shown, that after the prolonged measurements of PS have no notable affect on the PTIR
performance. However, a notable decrease in PTIR sensitivity was observed after several
measurements of biological samples. A method for in-situ controlling aging if the AFM tip
was proposed. The AFM tip aging was judged by following the signal intensity of a band δs(Si-
CH3) associated with stable impurities of the gold coated tip. The proposed method was
successfully employed during the measurements of biological samples (cells and tissues).
PTIR spectroscopic and imaging measurements of individual E. Coli bacteria consisting of
aggregated proteins (horseradish peroxidase) as inclusion bodies (IBs) were performed. The
average secondary structure of the IBs found to be different from the host bacteria. A
method for the quantitative analysis of the present IBs was proposed.
PTIR and FTIR spectra of dead (apoptotic) and viable regions in a histological tumor section
were acquired and analysed. The results of the near-field analysis agree with the far-field
micro-spectroscopic measurements. In particular, changes in protein secondary structure and nonlinear properties in the absorption of IR radiation by condensed nucleic acids were
observed.
Further, using the PTIR technique individual dead (apoptotic) and viable mammalian cells
were characterized. Two types of apoptotic cells were discriminated. For some cells protein
secondary structure at nuclei and cytoplasm regions appeared to be different. Other cells
had no notable differences in the overall protein conformation. In both kinds of apoptotic
cells the decay of the band related to nucleic acids (C-N-C stretch of ribose-phosphate
skeletal vibrations in nucleic acids) was observed. Viable (control) cells demonstrated no
significant alterations in the overall protein secondary structure at nuclei and cytoplasm
regions. Additionally, the overall protein conformation in nuclei region for apoptotic and
control cells were found to be different.
In the second part of the work combined image analysis for discrimination and
characterization of biological tissues (tumor and ischemic brain) was performed. Combined
analysis of the tumor demonstrated statistical correlations between elemental and
molecular chemical maps. Additionally, the combination of data from the two techniques
(FTIR and LA-ICP-MS) facilitated an improved cluster analysis and allowed discrimination of
different stages of apoptosis within a tumor tissue. Besides, the combined (multi-sensor)
analysis helped to characterize different degrees of cellular death within different tumor
samples.
During the multi-sensor analysis of thin cuts of ischemic rat brain partial least squares
discriminant analysis (PLS-DA) and random decision forest (RDF) classification algorithms
were applied and their performance was compared. As a result, different tissue types were
distinguished. The performance of classification models built on the combined dataset was
compared with the classification results based on the individual datasets. Multi-sensor
analysis again improved classification. Here different tissue types such as white and gray
matter, as well as stroke region and its surroundings could be differentiated more efficiently.
Furthermore RDF classification appeared to me more precise than PLS-DA.
The results, presented in this thesis demonstrate the capabilities and advances of near- and
far-field IR spectroscopy and spectroscopic imaging applied to analysis of biological samples.
Further, the results demonstrate that the multi-sensor combined analysis incorporating FTIR
and LA-ICP-MS imaging facilitates an improved multivariate analysis and thus deeper
understanding of biochemical processes.