W plan apochromat 63 1 0 m27

For in-situ measuring the incubation cell was placed onto the scan table of an alpha 500 R confocal Raman microscope (WITec GmbH, Ulm, Germany), fixed by 4 pins on the bottom of the cell. The Raman microscope is equipped with a 532-nm excitation laser, a UHTS 300 spectrometer, a DV401-BV CCD detector and a 63× water immersion objective with numerical aperture of 1.0 (W “Plan-Apochromat” 63/1,0 M27, Carl Zeiss, Jena, Germany). To obtain a strong signal without damaging the skin, the laser intensity was set to 25 mW, using a pinhole size of 50 µm. The DV401-BV CCD detector was cooled to −60 °C and a spectral range from 501 cm⁻¹ to 1635 cm⁻¹ with the spectral centre of 1100 cm⁻¹, obtained by an optical grating (1800 g/mm, spectral centre: 1100 cm⁻¹). Two-dimensional image scans of 5 µm width and 25 µm in depth were performed, acquiring 10 spectra per line and 50 lines per vertical dimension, with an integration time of 1.5 s per spectra.
To ensure the suitability of the setup for depth profiling, the depth resolution was measured, by scanning into a silica plate and determining the full width at half maximum of the depth profile corresponding the 521 cm⁻¹ band intensity [25 (link)]. Also, the thickness of a PET film was measured, in order to test whether valid depth profiles can be obtained.

Raman microspectroscopy (WITec alpha 300 ​R, Ulm, Germany) was performed at the interface of the fibrotic capsule as described previously [23 ]. A 63× objective (W Plan-Apochromat 63×/1.0 M27, Carl Zeiss AG) was used to image deparaffinized and hydrated tissue sections, sequential to sections subjected to IF staining. Spectral preprocessing and analysis were conducted in Project Five 5.2 (WITec GmbH). True component analysis (TCA) was applied to generate false-color coded intensity distribution heatmaps based on reference spectra of αSMA, Col I, Col III and nuclei generated in a previous study [23 ]. To obtain reference spectra of immune cells, Raman imaging was correlated with CD68/CCR7 or CD68/CD204 positive immunofluorescence images and spectra from co-localized pixel were extracted. Triplicate images were selected for each sample. Similar to ECM signatures, the retrieved spectra were used as reference spectra to identify macrophage polarization states via TCA. MGV was also performed to quantify the signal intensities of the specific proteins. The cell numbers were counted manually by using ImageJ V 1.52p.