Nah- und Fernfeld mid-infrarot Spektroskopie und Imaging für biomedizinische Anwendungen

Abweichender Titel nach Übersetzung der Verfasserin/des VerfassersMid-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.Die Spektroskopie im mittleren Infrarot (IR) ist eine markierungs- und zerstörungsfreie Technik um molekülspezifische Information zu erhalten. Diese Technik kann auch in der mikrobiologischen und biomedizinischen Forschung angewendet werden. Insbesondere im Bereich der bildgebenden Analyse mittels IR Mikroskopie können zur klassischen histologischen Untersuchung komplementäre sowie ergänzende Informationen gewonnen werden. Allerdings weist die IR Mikrospektroskopie eigene Limitierungen auf: erstens kann bei Verwendung von klassischen Fernfeldmikroskopie Systemen die räumliche Auflösung nicht besser als einige Mikrometer sein, zweitens kann die IR Spektroskopie keine Information bezüglich der Elementzusammensetzung der Probe liefern. Die erste Beschränkung kann durch die Anwendung einer neuen, im Nahfeld arbeitenden, bildgebenden Methode basierend auf der photothermisch induzierten Resonanz (PTIR) gelöst werden. Die zweite Beschränkung kann durch Anwendung einer zweiten, komplementären, Messtechnik und anschließender gemeinsamer Analyse der vereinten Datensätze aufgehoben werden. Der erste experimentelle Teil dieser Dissertation beschreibt die Charakterisierung eines selbstgebauten PTIR Systems und dessen Anwendung zur Analyse von verschiedenen biologischen Proben. Die Arbeit beinhaltet die weltweit erste zeitaufgelöste PTIR Untersuchung am Beispiel einer induzierten Sekundärstrukturänderung einer 200 nm dünnen Schicht aus poly-L-lysine (PLL). PLL liegt in Abhängigkeit seines Wassergehaltes in verschiedenen Sekundärstrukturen vor. Der Wassergehalt des PLL Films und somit dessen angenommene Sekundärstruktur kann bei konstanter Luftfeuchtigkeit über die Temperatur eingestellt werden. Ein kontrollierter Temperaturanstieg der Probe führt somit zu Änderungen in der Amide I Bande von PLL. Diese Veränderung konnte mit zeitaufgelöster PTIR verfolgt werden. Die erreichte PTIR Aufnahmezeit war 15 s. Kontrollanalysen mit einem kommerziellen FTIR Mikroskope bestätigten die spektroskopischen Erkenntnisse. Des Weiteren wurde die Alterung der AFM Spitze des PTIR Systems während fortlaufender Messungen eines Polymeres (Polystyrol, PS) und biologischer Proben (Zellen und Gewebe) untersucht. Es wurde gezeigt, dass lang andauernde Messungen von PS keinen Einfluss auf die Leistungsfähigkeit des PTIR Systems haben. Allerdings ließ die PTIR Empfindlichkeit bereits nach nur einigen Messungen von biologischen Proben merkbar nach. Eine Methode zur in-situ Kontrolle der Alterung und damit der Einsatzfähigkeit der AFM-Spitze wurde vorgeschlagen. Die AFM-Spitzenalterung wurde über die Signalintensität der ɷs(Si-CH3) Bande beurteilt, welche einer stabile Verunreinigung der goldbeschichteten Spitze zugeordnet wird. Die vorgeschlagene Technik wurde erfolgreich während der Messung von biologischen Proben (Zellen und Gewebe) angewandt. PTIR spektroskopische und bildgebende Messungen von einzelnen E.coli Bakterien, welche das PƌoteiŶ MeeƌƌettiĐhpeƌodžidase iŶ Foƌŵ ǀoŶ „iŶĐlusioŶ ďodies͞ ;IBsͿ enthielten, wurden durchgeführt. Es wurde festgestellt, dass die durchschnittliche Sekundärstruktur der IBs sich von der des restlichen Bakterium unterscheidet. Eine Methode zur quantitativen Analyse der vorhandenen IB wurde gezeigt. 4 PTIR und FTIR Spektren wurden von abgestorbenen (apoptotischen) und lebensfähigen Zellen nach einem histologischer Schnitt aufgenomment und analysiert. Die Ergebnisse der Nahfeldanalyse stimmen mit den mikroskopischen Messungen im Fernfeld überein. Inbesondere Änderungen der Sekundärstruktur von Proteinen und nichtlineare Eigenschaften der IR Absorption durch kondensierte Nukleinsäuren wurden festgestellt. Des Weiteren wurden einzelne apoptotische sowie lebensfähige Zellen mittels der PTIR Technik charakterisiert. Zwei Arten von apoptotische Zellen konnten unterschieden werden. Einige Zellen zeigen Unterschiede in der Proteinsekundärstruktur zwischen Zellkern und Cytoplasma. Andere Zellen weisen keinen Unterschied in der Proteinsekundärdtruktur auf. Die beide Arten von apoptotische Zellen zeigen einen Rückgang der Absorptionsbande für Nukleinsäuren (C-N-C Streckschwingung von Ribose-Phosphat). Lebensfähige (Kontrol) Zellen zeigen keine signifikante Änderungen in der Proteinsekundärstruktur zwischen Zellkern und Cytoplasma. Es wurde festgestellt, dass die allgemeine Proteinkonformation im Zellkern der apoptotischen und Kontrollzellen unterschiedlich ist. Der zweite experimentelle Teil beschäftigt sich mit der kombinierten (Multisensor) Bildanalyse von histologischen Proben unter Zuhilfenahme der molekülspezifischen Fourier- Transformation Infrarot (FTIR) Spektroskopie und der elementspezifischen Laserablation induktiv gekoppelte Plasma Massenspektrometrie (LA-ICP-MS). Die kombinierte Analyse von FTIR und LA-ICP-MS Bildern von Tumorproben zeigte eine statistische Korrelation zwischen elementarer und molekularer Information. Zudem wurde durch die Kombination und gemeinsame Auswertung von Messdaten dieser beiden Methoden eine Verbesserung der Clusteranalyse ermöglicht. Dadurch gelang auch eine verbesserte Unterscheidung der verschiedenen Stufen der Apoptose. Während der Multisensor Analyse von Dünnschnitten einer ischämischer Hirnläsion eines Rattenhirns wurden die Klassifikationsverfahren Partial Least Squares Diskriminanzanalyse (PLS-DA) und Random Decision Forest (RDF) angewendet und deren Leistungsfähigkeit bzgl. der Klassifizierung von verschiedenen Bereichen der Dünnschnitte verglichen. Beide Verfahren erlaubten zwischen unterschiedliche Gewebetypen unterscheiden zu können. Die Leistungsfähigkeit der Klassifikationsmodelle für den kombinierten (FTIR und LA-ICP-MS Daten) Datensatz wurde mit den Klassifikationsergebnissen basierend auf Daten der einzelnen Techniken verglichen. Die Multisensor Analyse war auch hier vorteilhaft. Unterschiedliche Gewebetypen wie weiße und graue Substanz, sowie Läsion und deren Umgebung konnten dadurch noch effektiver unterschieden werden. Des Weiteren zeigte die RDF genauere Klassifizierungsergebnisse als die PLS-DA. Die dargestellten Ergebnisse dieser Arbeit zeigen die Fähigkeiten und Fortschritte der Nahund Fern-Feld IR Mikrospektroskopie und der spektroskopischen bildgebenden Analyse von biologischen Proben. Im Zuge dieser Arbeit konnten auch die Vorteile einer bildgebenden Multisensoranalyse zur verbesserten Analyse von biologischen Proben gezeigt werden.13

Method for Time-Resolved Monitoring of a Solid State Biological Film Using Photothermal Infrared Nanoscopy on the Example of Poly‑l‑lysine

We report time-resolved photothermal infrared nanoscopy measurements across a spectral range of more than 100 cm^–1 (1565 cm^–1 to 1729 cm^–1) at nanoscale spatial resolution. This is achieved through a custom-built system using broadly tunable external cavity quantum cascade lasers in combination with a commercially available atomic force microscope. The new system is applied to the analysis of conformational changes of a polypeptide (poly-l-lysine) film upon temperature-induced changes of the humidity in the film. Changes of the secondary structure from β-sheet to α-helix could be monitored at a time resolution of 15 s per spectrum. The time-resolved spectra are well comparable to reference measurements acquired with conventional Fourier transform infrared microscopy

Phosphonate coating of SiO2 nanoparticles abrogates inflammatory effects and local changes of the lipid composition in the rat lung: a complementary bioimaging study

Abstract Background The well-known inflammatory and fibrogenic changes of the lung upon crystalline silica are accompanied by early changes of the phospholipid composition (PLC) as detected in broncho-alveolar lavage fluid (BALF). Amorphous silica nanoparticles (NPs) evoke transient lung inflammation, but their effect on PLC is unknown. Here, we compared effects of unmodified and phosphonated amorphous silica NP and describe, for the first time, local changes of the PLC with innovative bioimaging tools. Methods Unmodified (SiO2-n), 3-(trihydroxysilyl) propyl methylphosphonate coated SiO2-n (SiO2-p) as well as a fluorescent surrogate of SiO2-n (SiO2-FITC) nanoparticles were used in this study. In vitro toxicity was tested with NR8383 alveolar macrophages. Rats were intratracheally instilled with SiO2-n, SiO2-p, or SiO2-FITC, and effects on lungs were analyzed after 3 days. BALF from the right lung was analyzed for inflammatory markers. Cryo-sections of the left lung were subjected to fluorescence microscopy and PLC analyses by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MS), Fourier transform infrared microspectroscopy (FT-IR), and tandem mass spectrometry (MS/MS) experiments. Results Compared to SiO2-p, SiO2-n NPs were more cytotoxic to macrophages in vitro and more inflammatory in the rat lung, as reflected by increased concentration of neutrophils and protein in BALF. Fluorescence microscopy revealed a typical patchy distribution of SiO2-FITC located within the lung parenchyma and alveolar macrophages. Superimposable to this particle distribution, SiO2-FITC elicited local increases of phosphatidylglycerol (PG) and phosphatidylinositol (PI), whereas phoshatidylserine (PS) and signals from triacylgyceride (TAG) were decreased in the same areas. No such changes were found in lungs treated with SiO2-p or particle-free instillation fluid. Conclusions Phosphonate coating mitigates effects of silica NP in the lung and abolishes their locally induced changes in PLC pattern. Bioimaging methods based on MALDI-MS may become a useful tool to investigate the mode of action of NPs in tissues

Fourier Transform Infrared (FT-IR) and Laser Ablation Inductively Coupled Plasma–Mass Spectrometry (LA-ICP-MS) Imaging of Cerebral Ischemia: Combined Analysis of Rat Brain Thin Cuts Toward Improved Tissue Classification

Microspectroscopic techniques are widely used to complement histological studies. Due to recent developments in the field of chemical imaging, combined chemical analysis has become attractive. This technique facilitates a deepened analysis compared to single techniques or side-by-side analysis. In this study, rat brains harvested one week after induction of photothrombotic stroke were investigated. Adjacent thin cuts from rats’ brains were imaged using Fourier transform infrared (FT-IR) microspectroscopy and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The LA-ICP-MS data were normalized using an internal standard (a thin gold layer). The acquired hyperspectral data cubes were fused and subjected to multivariate analysis. Brain regions affected by stroke as well as unaffected gray and white matter were identified and classified using a model based on either partial least squares discriminant analysis (PLS-DA) or random decision forest (RDF) algorithms. The RDF algorithm demonstrated the best results for classification. Improved classification was observed in the case of fused data in comparison to individual data sets (either FT-IR or LA-ICP-MS). Variable importance analysis demonstrated that both molecular and elemental content contribute to the improved RDF classification. Univariate spectral analysis identified biochemical properties of the assigned tissue types. Classification of multisensor hyperspectral data sets using an RDF algorithm allows access to a novel and in-depth understanding of biochemical processes and solid chemical allocation of different brain regions.This work was supported by MEIBio doctoral project of TU Wien and by the funding from the Austrian FFG within project 84247. This work was co-funded by NPRP grant no. NPRP-5-381-3-101 from the Qatar National Research Fund (a member of The Qatar Foundation)

Fourier Transform Infrared (FT-IR) and Laser Ablation Inductively Coupled Plasma–Mass Spectrometry (LA-ICP-MS) Imaging of Cerebral Ischemia : Combined Analysis of Rat Brain Thin Cuts Toward Improved Tissue Classification

Microspectroscopic techniques are widely used to complement histological studies. Due to recent developments in the field of chemical imaging, combined chemical analysis has become attractive. This technique facilitates a deepened analysis compared to single techniques or side-by-side analysis. In this study, rat brains harvested one week after induction of photothrombotic stroke were investigated. Adjacent thin cuts from rats’ brains were imaged using Fourier transform infrared (FT-IR) microspectroscopy and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The LA-ICP-MS data were normalized using an internal standard (a thin gold layer). The acquired hyperspectral data cubes were fused and subjected to multivariate analysis. Brain regions affected by stroke as well as unaffected gray and white matter were identified and classified using a model based on either partial least squares discriminant analysis (PLS-DA) or random decision forest (RDF) algorithms. The RDF algorithm demonstrated the best results for classification. Improved classification was observed in the case of fused data in comparison to individual data sets (either FT-IR or LA-ICP-MS). Variable importance analysis demonstrated that both molecular and elemental content contribute to the improved RDF classification. Univariate spectral analysis identified biochemical properties of the assigned tissue types. Classification of multisensor hyperspectral data sets using an RDF algorithm allows access to a novel and in-depth understanding of biochemical processes and solid chemical allocation of different brain regions

Additional file 1: of Phosphonate coating of SiO2 nanoparticles abrogates inflammatory effects and local changes of the lipid composition in the rat lung: a complementary bioimaging study

Figure S1. Effect of different SiO2 NP on lung histology. Figure S2. MALDI-MS/MS spectrum resulting from the fragmentation of precursor m/z 721.4. Figure S3. MALDI-MS/MS spectrum resulting from the fragmentation of precursor m/z 861.5. Figure S4. Ion images from a vehicle-treated control lung. Figure S5. Ion images from a SiO2-p-treated control lung. (DOCX 1889 kb