Confocal raman microscope system alpha 300r

The WiTec Confocal Raman Microscope System Alpha 300R with UHTS300 spectrometer and DV401 CCD detector 600/mm grating were used to obtain confocal Raman spectroscopy samples. The WiTec spectrometer was calibrated with a mercury-argon lamp. A 532 nm wavelength Nd:YAG laser was used as the excitation source. A 100× air objective (NA 0.90; Nikon Instruments, Melville NY) was used for focusing the 532 nm excitation laser to the sample. Laser power at the objective was 10 mW as measured by an optical power meter (THORLABS, New Jersey). Resolution of the microscope was approximated as 0.3 μm with Abbe’s diffraction formula for lateral resolution. Cells were imaged at the mid-plane of each cell.

Confocal Raman spectroscopy measurements were conducted using a Confocal Raman Microscope System Alpha 300R (WITec, Ulm, Germany) with a UHTS300 spectrometer and DV401 CCD detector with 600/mm grating. The WITec spectrometer was calibrated with a Mercury-Argon lamp. A Nd:YAG laser (532 nm wavelength) was used as an excitation source. A 100× air objective (NA 0.90; Nikon Instrument, Melville, NY) was used for focusing the 532 nm excitation laser to the sample. The laser at the objective was 10 mW, as measured by an optical power meter (Thorlabs, Newton, NJ). The lateral resolution of the microscope was about 296 nm according to Abbe’s diffraction formula. Cell samples were frozen using a four-stage Peltier (Thermonamic Electronics Corp. Jiangxi, China) and a series 800 temperature controller (Alpha Omega Instruments Corp, Lincoln, RI). Cell samples were seeded at −6 °C with a liquid nitrogen cooled needle, cryopreserved at 1 °C/min to a holding temperature of −50 °C and held for 20 min before imaging. Condensation was minimized by creating a barrier around the imaging stage using plastic film (Bemis, Neenah, WI) and purging the space with dry nitrogen gas. About 1–3 µL of cell suspension was placed on the stage, covered with a piece of mica (TED PELLA, Redding, CA) and sealed with Kapton tape (Dupont, Wilmington, DE) to prevent sample evaporation/sublimation.

Raman spectra were obtained, for the intact archaeological bones, at the “Molecular Physical-Chemistry” R&D Unit of the University of Coimbra (QFM-UC, Portugal)⁴⁸ , in a WITec confocal Raman microscope system alpha300R, coupled to an ultra-high throughput spectrometer 300 VIS grating (f/4 300 mm focal length, 600 lines per millimetre blazed for 500 nm). The detection system was a 1650 × 200 pixels thermoelectrically cooled (– 55 °C at room temperature) charge-coupled device camera, front-illuminated with NIR/VIS antireflection coating, with a spectral resolution < 0.8 cm^–1/pixel. The excitation radiation used was a WITec 785 nm diode laser, ca. 20 mW at the sample position was applied. A 10 × objective (Zeiss Epiplan, NA 0.23, WD 16.1 mm) was used. 20 accumulations were collected per sample, with 30 s exposure time. Bone tissue often displays autofluorescence^{51 (link)} and fluorescent aromatic compounds may be formed during burning (mainly under anaerobic conditions)^{52 ,53} . This complicates Raman acquisition as fluorescence often masks the Raman signals, mainly for samples not subject to heat or burned at lower temperatures. The use of a near-infrared 785 nm laser enabled us to overcome this problem, providing good quality Raman data for most of the archaeological samples under study.

Raman spectroscopy analyses were conducted with powdered samples using a Confocal Raman Microscope System Alpha 300R (WITec, Ulm, Germany), with a UHTS300 spectrometer and with a DR316B_LD CCD detector employing a 300 g mm^–1 grating. A 785 nm laser set with a laser power of 90 mW in front of the objective was used. Spectral data were acquired using a 20× objective ((NA 0.4) EC Epiplan, Zeiss, Gina, Germany). Single spectra were recorded in a range of 100–3560 cm⁻¹, with an integration time of 4 s and with 10 accumulations. Each sample was measured in five different spots.
All acquired Raman spectra were further processed using the WITec ProjectSIX 6.1 software. All spectra were treated with a cosmic-ray-removal and a background subtraction procedure, and the “shape” function was applied, when necessary. After that, all spectra for each sample were averaged using the “average spectra” function.