Alpha 300r microscope

Raman imaging measurements were performed in reflection mode using a WITec ALPHA300R microscope at room temperature. For the Raman excitation in the near-infrared a Toptica XTRA laser with a nominal wavelength of 785 nm and a power of 15 mW, measured at the back aperture of the objective (Zeiss EC EPIPLAN 50x/0.7), was used. Because of the large size of the sample and the structures within, it was possible to choose parameters that reduced the measurement time for a single scan, i.e. a fibre with a core diameter of 100 μm to deliver the collected light to a spectrometer and a distance between scan points of 1 μm, which does not yield the highest possible lateral resolution of the microscope. The signal was analysed in the spectral range between 0 cm^-1 and 1776 cm^-1 with a spectral resolution of about 6 cm^-1. The integration time for a single scan point was optimized near the scan region prior to each measurement to yield a good signal to noise ratio without causing damage to the sample.

In order to evaluate the thickness of API-containing layers on the product granules, confocal Raman microscopy was chosen, as it is capable of identifying chemical compounds in microscopic dimensions in three-dimensional volumes. It was conducted using an Alpha 300R microscope (WITec GmbH, Germany) with an excitation wavelength of 532 nm, operated at a laser power of approximately 5.4 mW and an integration time of 0.1 s. Depth scans of the granules with a size of 10 µm × 10 µm were performed with a step size of 0.2 µm using 100-fold magnification. The data were postprocessed using cosmic ray removal, noise filtering and background subtraction. Afterwards, datasets were compared with reference data of pure naproxen and carrier materials in order to visualize the naproxen distribution in a Raman chemical map.

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).