Raman spectrometer

The measurements were carried out by a Renishaw InVia Raman spectrometer, attached to a Leica DMLM microscope. The spectra were acquired with a Leica 50 × N Plan (0.75 aperture) objective. Additionally, to visualize and focus, another Leica 5 × N Plan (0.12 aperture) and a 20 × N Plan EPI (0.40 aperture) object were also used. The spatial resolution for the 50 × objectives is 2 microns. The microscope is equipped with a Prior scientific motorized stage (XYZ) with a joystick to facilitate focusing on or searching the points of interest. The excitation wavelength was 514 nm (ion-argon laser, Modu-Laser) with 50 mW of nominal power at the source, and 20 mW of maximum power directed to the sample. A holographic net of 1800 lines/mm was employed. The power of the laser was reduced by neutral density filter in all of the measurements to avoid photo-decomposition of samples (burning). Each spectrum took 20 s, and 5 scans were accumulated with 10% of the maximum power of the 514 nm laser in the 150–3500 cm⁻¹ spectral window.

The Raman spectrometer equipped with a Leica microscope (Renishaw, Gloucestershire, UK) was used to analyze Raman spectra in extracted starches. The Raman system was calibrated at 532 cm⁻¹ by using a silicon semiconductor [20 (link)]. Sample spectra were scanned in the range of 3500–0 cm⁻¹ at a resolution of 5 cm⁻¹. The starch molecular order was characterized by the full-width at half-maximum (FWHM) band at 480 cm⁻¹ using the WIRE software 2.0 version (Wire Swiss GmbH, Zug, Switzerland).