From small blocks of frozen walnut shells, 20–30 µm thin sections were cut in the cryostat microtome and transferred on a standard glass slide. Samples were washed several times with dH2O, followed by D2O, and sealed with nail polish for Raman microscopic measurements. Spectra were acquired from microsections using a confocal Raman microscope (alpha300RA, WITec, Ulm, Germany) equipped with a ×100 oil immersion objective (NA 1.4, Carl Zeiss, Jena, Germany) and a piezoelectric scan stage. A laser (λ=532 nm) was passed through a polarization-preserving single-mode optical fibre and focused through the objective with a spatial resolution of 0.3 μm on the sample. The Raman scattering signal was detected by a CCD camera (Andor DV401 BV, Belfast, UK) behind a spectrometer (600 g mm−1 grating, UHTS 300 WITec, Ulm, Germany). The laser power was 40 mW. For measurement set-up, the software Control Four (WITec) was used. Raman analysis was performed with Project FOUR (WITec) and Opus 7.5 software (Bruker Optik GmbH, Ettlingen, Germany). After applying cosmic ray spike removal, Raman chemical images were generated based on the integration of relevant wavenumber regions (e.g. CH stretching). The indent was selected, and a non-negative matrix factorization (NMF) was performed in Project FOUR with six basic spectra.
Project four
Manufactured by WITec
Sourced in Germany
Project FOUR is a high-performance scanning probe microscope system. It provides advanced imaging and analysis capabilities for a wide range of samples and applications.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using project four
1
Raman Microscopy Analysis of Walnut Shell Sections
2
Raman Spectral Imaging of Diclofenac Formulations
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Raman spectral imaging (Smith & Dent, 2005 ) was performed to assess the location and distribution of diclofenac in the three formulations; testing was performed by WITec (Wissenschaftliche Instrumente und Technologie GmbH, Ulm, Germany). Samples were prepared and placed between two coverslips and excited with a 532 nm diode laser. Single spectra were obtained separately from each component of each formulation. All Raman images and spectra were recorded using an alpha 300 RA Raman confocal imaging microscope (WITec) equipped with a CCD camera operating at −64 °C that captured an image 1600 pixels wide by 200 pixels high. The microscope was also equipped with an ultra‐high throughput spectrometer that had, in addition to the diode laser, 300 g/mm grating, BLZ = 500 nm, with a spectral centre of 2090 (rel 1/cm) and a Zeiss 100× oil immersion objective. Spectra were analysed with proprietary software (WITec ProjectFOUR and WITec ProjectFOUR Plus).
3
Raman Spectroscopy Data Analysis
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Raman data analysis was performed with Project FOUR (WITec GmbH, Ulm, Germany) and ImageLab (EPINA GmbH, Pressbaum, Austria) software. The extracted spectra were analyzed with Opus 7.5 softwareTM (Bruker, Rheinstetten, Germany). Before the Raman images were generated based on integration of specific bands, a cosmic ray removal filter (spike half-width 2) was applied. Based on the integrated images, average spectra of distinct areas of the samples (e.g., CC, cell wall and deposits) were obtained by drawing areas of interests or using an intensity threshold. The principal component analysis (PCA) in the spectral range 200–3170 cm−1 was carried out with the software OriginPro 9.1 (OriginLab Corporation, Northampton MA, US).
4
Raman Microscopy of Walnut Sections
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From small blocks of frozen walnut shells 20-30 µm thin sections were cut in the cryostat microtome and transferred on a standard glass slide. Samples were washed several times with dH2O, followed by D2O and sealed with nail polish for Raman microscopic measurements. Spectra were acquired from micro sections using a confocal Raman microscope (alpha300RA, WITec, Ulm, Germany) equipped with a 100× oil immersion objective (NA 1.4, Carl Zeiss, Jena, Germany) and a piezoelectric scan stage.
A laser (λ = 532 nm) was passed through a polarization-preserving single-mode optical fibre and focused through the objective with a spatial resolution of 0.3 μm on the sample. The Raman scattering signal was detected by a CCD camera (Andor DV401 BV, Belfast) behind a spectrometer (600 g mm -1 grating, UHTS 300 WITec, Ulm, Germany). The laser power was 40mW. For measurement setup the software Control Four (WITec, Ulm, Germany) was used. Raman analysis was performed with Project FOUR (WITec, Ulm, Germany) and Opus 7.5 software (Bruker Optik GmbH, Ettlingen, Germany). After applying cosmic ray spike removal, Raman chemical images were generated based on the integration of relevant wavenumber regions (e.g., CH stretching). The indent was selected, and a non-negative matrix factorization (NMF) was performed in Project FOUR with six basis spectra.
A laser (λ = 532 nm) was passed through a polarization-preserving single-mode optical fibre and focused through the objective with a spatial resolution of 0.3 μm on the sample. The Raman scattering signal was detected by a CCD camera (Andor DV401 BV, Belfast) behind a spectrometer (600 g mm -1 grating, UHTS 300 WITec, Ulm, Germany). The laser power was 40mW. For measurement setup the software Control Four (WITec, Ulm, Germany) was used. Raman analysis was performed with Project FOUR (WITec, Ulm, Germany) and Opus 7.5 software (Bruker Optik GmbH, Ettlingen, Germany). After applying cosmic ray spike removal, Raman chemical images were generated based on the integration of relevant wavenumber regions (e.g., CH stretching). The indent was selected, and a non-negative matrix factorization (NMF) was performed in Project FOUR with six basis spectra.
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