Alpha 500 r confocal raman microscope

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.

Confocal Raman microscopic images were obtained using an alpha 500R confocal Raman microscope (WiTec GmbH, D-Ulm) equipped with a 532 nm excitation laser, UHTS 300 spectrometer and DV401-BV CCD camera. The specific areas were mapped using a 100x objective (numerical aperture 1.25). Details can be seen in the corresponding figures. Colour-coded images were calculated using WiTecProject Suite 4.04 software (WiTec GmbH, D-Ulm).

Spectra of the drug carriers were acquired using an alpha 500R confocal Raman microscope (WiTec GmbH, Ulm, Germany) equipped with a 532 nm excitation laser, UHTS 300 spectrometer, a 600 gr/mm grating and DV401-BV CCD camera. A 40x/0.6 NA objective was used (EC Epiplan-neofluor; Carl Zeiss AG, Oberkochen, Germany) in combination with a 50 μm optical fibre. Spectra were recorded on the surface of the loaded drug carriers in an area of 150 × 150μm with a step size of 3 µm. Integration time was 0.5 s. Laser power was 25 mW. Spectra of ibuprofen and an unloaded drug carrier were used to calculate reference spectra. These spectra were used to determine the spatial distribution of ibuprofen on the surface of drug-loaded carriers. All spectra were processed by cosmic ray removal and baseline correction with the software Project Plus 4 (WiTec GmbH, Ulm, Germany)