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Assessment of shifted excitation Raman difference spectroscopy in highly fluorescent biological samples
Shifted excitation Raman difference spectroscopy (SERDS) can be used as an instrumental baseline correction technique to retrieve Raman bands in highly fluorescent samples. Genipin (GE) cross-linked equine pericardium (EP) was used as a model system since a blue pigment is formed upon cross-linking, which results in a strong fluorescent background in the Raman spectra. EP was cross-linked with 0.25% GE solution for 0.5 h, 2 h, 4 h, 6 h, 12 h, and 24 h, and compared with corresponding untreated EP. Raman spectra were collected with three different excitation wavelengths. For the assessment of the SERDS technique, the preprocessed SERDS spectra of two excitation wavelengths (784 nm-786 nm) were compared with the mathematical baseline-corrected Raman spectra at 785 nm excitation using extended multiplicative signal correction, rubberband, the sensitive nonlinear iterative peak and polynomial fitting algorithms. Whereas each baseline correction gave poor quality spectra beyond 6 h GE crosslinking with wave-like artefacts, the SERDS technique resulted in difference spectra, that gave superior reconstructed spectra with clear collagen and resonance enhanced GE pigment bands with lower standard deviation. Key for this progress was an advanced difference optimization approach that is described here. Furthermore, the results of the SERDS technique were independent of the intensity calibration because the system transfer response was compensated by calculating the difference spectrum. We conclude that this SERDS strategy can be transferred to Raman studies on biological and non-biological samples with a strong fluorescence background at 785 nm and also shorter excitation wavelengths which benefit from more intense scattering intensities and higher quantum efficiencies of CCD detectors. This journal is
Label-free multimodal imaging of infected Galleria mellonella larvae
Non-linear imaging modalities have enabled us to obtain unique morpho-chemical insights into the tissue architecture of various biological model organisms in a label-free manner. However, these imaging techniques have so far not been applied to analyze the Galleria mellonella infection model. This study utilizes for the first time the strength of multimodal imaging techniques to explore infection-related changes in the Galleria mellonella larvae due to massive E. faecalis bacterial infection. Multimodal imaging techniques such as fluorescent lifetime imaging (FLIM), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) were implemented in conjunction with histological HE images to analyze infection-associated tissue damage. The changes in the larvae in response to the infection, such as melanization, vacuolization, nodule formation, and hemocyte infiltration as a defense mechanism of insects against microbial pathogens, were visualized after Enterococcus faecalis was administered. Furthermore, multimodal imaging served for the analysis of implant-associated biofilm infections by visualizing biofilm adherence on medical stainless steel and ePTFE implants within the larvae. Our results suggest that infection-related changes as well as the integrity of the tissue of G. mellonella larvae can be studied with high morphological and chemical contrast in a label-free manner.