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A lens-based Raman spectrometer is characterized by studying the optical elements in the optical path and we study the measure of aberration–diffraction effects. This is achieved by measuring the spectral resolution (SR) thus encompassing almost all optical elements of a spectrometer that are mostly responsible for such effects. An equation for SR is used to determine the quality factor Q which measures aberration/diffraction effects occurring in a spectrometer. We show how the quality factor changes with different spectrometer parameters such as grating groove density, the wavelength of excitation, pinhole width, charge-coupled device (CCD) pixel density, etc. This work provides an insight into the quality of a spectrometer and helps to monitor the performance of the spectrometer over a certain period. Commercially available spectrometers or home-built spectrometers are prone to misalignment in optical elements and can benefit from this work that allows maintaining the overall quality of the setup. Performing such experiments over a period helps to minimize the aberration/ diffraction effects occurring as a result of time and maintaining the quality of measurements.
Characterization of brain tumours requires neuropathological expertise and is generally performed by histological evaluation and molecular analysis. One emerging technique to assist pathologists in future tumour diagnostics is multimodal optical spectroscopy. In the current clinical routine, tissue preprocessing with formalin is widely established and suitable for spectroscopic investigations since degradation processes impede the measurement of native tissue. However, formalin fixation results in alterations of the tissue chemistry and morphology for example by protein cross-linking. As optical spectroscopy is sensitive to these variations, we evaluate the effects of formalin fixation on multimodal brain tumour data in this proof-of-concept study. Nonfixed and formalin-fixed cross sections of different common human brain tumours were subjected to analysis of chemical variations using ultraviolet and Fourier-transform infrared microspectroscopy. Morphological changes were assessed by elastic light scattering microspectroscopy in the visible wavelength range. Data were analysed with multivariate data analysis and compared with histopathology. Tissue type classifications deduced by optical spectroscopy are highly comparable and independent from the preparation and the fixation protocol. However, formalin fixation leads to slightly better classification models due to improved stability of the tissue. As a consequence, spectroscopic methods represent an appropriate additional contrast for chemical and morphological information in neuropathological diagnosis and should be investigated to a greater extent. Furthermore, they can be included in the clinical workflow even after formalin fixation.
Scanning Near-Field Optical Microscopy (SNOM) has developed during recent decades into a valuable tool to optically image the surface topology of materials with super-resolution. With aperture-based SNOM systems, the resolution scales with the size of the aperture, but also limits the sensitivity of the detection and thus the application for spectroscopic techniques like Raman SNOM. In this paper we report the extension of solid immersion lens (SIL) technology to Raman SNOM. The hemispherical SIL with a tip on the bottom acts as an apertureless dielectric nanoprobe for simultaneously acquiring topographic and spectroscopic information. The SIL is placed between the sample and the microscope objective of a confocal Raman microscope. The lateral resolution in the Raman mode is validated with a cross section of a semiconductor layer system and, at approximately 180 nm, is beyond the classical diffraction limit of Abbe.