Biology can often be explained by structures and interactions between molecules. It is therefore of importance for medical sciences that the chemical reactions and molecular compositions of biological samples can be measured. The purpose of this thesis is twofold. Firstly, to develop methods for measuring the underlying mechanisms of the lung disorder pulmonary hypertension. Secondly, to investigating the possibility of classifying brain tissue for safer resection of brain tumors. A multimodal approach is often motivated, as there exist many different measurement techniques. In this thesis Raman spectroscopy has been applied both as the main measurement modality and in cooperation with other methods.

Raman spectroscopy is a label free optical measurement technique that measure inelastic scattering from materials that are illuminated by a monochromatic light source. Raman scattering results in a weak signal that is uniquely proportional to the chemical structure of the sample. When measuring biological samples, Raman scattering is accompanied by a strong intrinsic fluorescent signature that can overshadow the signal. To elucidate the underlying Raman spectrum, it is generally preprocessed to allow further analysis. There are many methods available for preprocessing; a selection of commonly used methodologies has been included here, as well as methods of my own design. The most novel approach being a neural network that was trained on synthetic spectra to perform preprocessing without relying on user defined variables, which is common for other methods (Paper A). The neural network resulted in improved preprocessing when compared to a control predictor, with test data from paraffin, ethanol, and polyethylene, as well as spectra based on simulations.

Hypoxic pulmonary hypertension (PH) is a condition where the arteries in the lung-walls are blocked due to prolonged oxygen deprivation or lung disease. People who suffer from PH, often experience shortness of breath and fatigue. If the condition persists the added strain upon the heart from the increased resistance in the arteries will result in right-heart failure. Hypoxic PH is the result of permanently constricted pulmonary arterial smooth muscle cells (PASMCs). PASMCs reside in the arterial walls and react locally to reduced oxygen content by constricting. This effect is called pulmonary vasoconstriction (HPV) and results in the regulation of deoxygenated blood to areas of the lung that have more oxygen available. Full understanding of the mechanisms of oxygen sensing in PASMCs has importance for the development of new treatments against PH. To this end a sealed microfluidic system was designed with the purpose to enable multimodal measurements of the response of cultivated PASMCs to acute hypoxia including Raman spectroscopy, patch clamp, and imaging (Paper B).  The microfluidic system was initially tested using Raman spectroscopy and oxygen sensing to investigate the reaction of PASMCs to hypoxia. The results were compared to an open flow system that showed a higher variation of the desired oxygen content (21% or 4%) compared to the designed closed microfluidic system. The system was later reworked and tested with simultaneous measurements using Raman spectroscopy, oxygen sensing, imaging, and patch-clamp (Paper C). With this setup it was possible to track the molecular response in the mitochondria in correlation with the activity of the calcium-ion channels and the mechanical response of the PASMCs.

The main modality used in clinics for brain tumor imaging is magnetic resonance imaging (MRI). Structural MRI gives neurosurgeons information regarding the size and mass effects of tumors. However, during surgery it can be difficult to assess the marginal zone of tumors. Improvements have been made by incorporating fluorescence guided resection (FGR) in the standard practice of many operating rooms in Europe. FGR relies on measuring the emission from metabolized precursor molecules. However, the drawback of FGR is that it is not tumor specific and has reduced sensitivity in low-grade tumors and children. In this thesis the option of incorporating a Raman probe setup, to fill in the gaps of other methods has been discussed. During this preliminary discussion it was noted that there is no standard approach for preprocessing and many different methodologies have been employed by various researcher. Therefore, measurement on fresh brain tumor tissue from a Raman microscopy setup was preprocessed using commonly applied methods, in addition to a pretrained neural network, to investigate the variations of the outcome (Paper D). It became apparent that different methods and variable choices can alter the distinctive spectral features. The conclusion being that it is important to be both transparent and specific when explaining how data has been prepared prior to analysis to enable reproducible results.