Projectfour plus software

A CM3050 S cryomicrotome (Leica) was used to prepare 16 µm thick sections for imaging with an Alpha300RA Raman instrument (WITec GmbH, Ulm, Germany) as described previously (Gierlinger et al., 2012). The samples were excited using a linear polarized VIS laser (λ_ex = 532 nm, laser power 30 mW) via a 100× oil immersion objective lens (Zeiss, numerical aperture 1.4, coverslip correction 0.17 mm). Raman signals were backscattered through the same objective, directed through a 50 µm optical multifibre to a UHTS 300 spectrometer (WITec) fitted with a 600 g mm⁻¹ grating, and finally to a DU401 BV CCD camera (Andor Technology Ltd, Belfast, UK). control four acquisition software (WITec) was used for data collection with 0.3 µm steps at 0.07 s per spectrum, and the Projectfour plus software (WITec) was used for data analysis. After cosmic ray removal, Raman images were calculated based on band integration, and a hierarchical cluster analysis was performed as described previously (Gierlinger et al., 2012).

Confocal Raman microspectroscopy analyses were performed on digestive gland and gill tissues of mussels exposed for 21 days to TiO₂ NMs using an Alpha300 R microscope (WITec) equipped with a 532‐nm laser source, a 600‐g/mm grating, and a charge‐coupled device cooled down to −61 °C. All measurements were conducted using a 63× water immersion objective (W Plan‐Apochromat 63×/1.0; Zeiss). Paraffin‐embedded tissue samples of digestive glands were cut in 5–10‐µm‐thick sections and mounted onto glass slides. After deparaffinization, water‐mounted mussel tissue was scanned with a laser power of approximately 35 mW at 532 nm. Raman spectra were collected pixel‐wise with an integration time of approximately 0.07 s. Acquired spectra were processed using the Project FOUR PLUS software (WITec).