Dmlm microscope

In order to enhance the detection of Raman signals emitted by high fluorescent paints such as Cadmium Yellow (CY), Cadmium Red (CR), and Primary Red Magenta (PRM), Raman analysis was carried out with two devices using different excitation wavelengths.
The first device is a Renishaw InVia 0310–02 System based on a continuous Nd:YAG laser excitation source at 532 nm. The diameter of the laser spot on the sample was diffraction limited to 1 μm by the objective lens (50×). The system is equipped with a Leica microscope (DMI 3000 M) and an electrically cooled CCD camera. The laser power was set between 0.15 and 0.30 mW.
The second device is a Renishaw Raman microscope RM1000 system coupled with an optical Leica DM LM microscope. The system is equipped with a refrigerated CCD camera and a CW He-Ne laser emitting at 632 nm, operating at a power of 3–30 mW, with a probing depth of 2 μm.
For all measurements, the spectral resolution was set at 4 cm⁻¹ with an integration time in the range 10–60 s, the final spectra resulting from the accumulation of three individual ones.

A Leica DM LM microscope, Leica Microsystems Ltd, Milton Keynes, UK was also used for this study. The images were then exported using OmniMet software attached to the microscope.

A Raman spectrometer (InVia Reflex Raman spectrometer; Renishaw, New Mills Wotton-under-Edge Gloucestershire, UK) equipped with an optical microscope (research-grade Leica DMLM microscope, Leica Microsystems, Wetzlar, Germany) and a laser with a 785 nm excitation line was used. The laser line was focused through a 20x objective lens on the sample. The system was equipped with a CCD detector (Peltier cooled and near-infrared enhanced). The power of the incident laser was 250 mW. The laser exposure time was 10 s; the laser power was set at 80%; there were 10 accumulations for all measurements.
Raman spectra were collected from different spots on femur and tibia periosteum in the entire diaphysis region. Next, the femurs were cut in the mid-diaphysis region by means of a scalpel. Raman spectra were also recorded from the endosteum.
Four regions of interest were isolated for further analysis in the Raman spectra: 800–900 cm⁻¹ (prolines), 900–990 cm⁻¹ (apatite), 990–1140 cm⁻¹ (carbonate) and the amide I envelope at 1590–1730 cm⁻¹. All regions in the Raman spectra were isolated and baselined. Further analysis included deconvolution and curve fitting of the sub-bands using Peakfit software (Peakfit© v4.0; Jandel Scientific, San Rafael, CA, USA). The typical spectral resolution was 2 cm⁻¹. Instrument response (laser power and wavenumber) was checked by recording the spectrum of Si.

In order to characterize the microscopic appearance of the hydrogel beads, the samples were placed on a microscope slide and investigated under a Leica DMLM microscope (Leica Microsystems GmbH, Wetzlar, Germany) up to a 500-fold magnification. Digital photographs were taken with a Leica MC 170 HD microscope digital camera (Leica Microsystems GmbH, Wetzlar, Germany).

Polarized Raman Spectroscopy were recorded following published methods^{12 (link),58 (link)–60 (link)}. Specifically fiber samples were fixed by tape on a glass slide. Raman spectra were acquired with a Renishaw RM1000 InVia confocal Raman spectrometer (Renishaw, Wotton-under-Edge, United Kingdom) coupled to a Leica DM LM microscope with rotating stage (Leica Microsystems, Wetzlar, Germany). Fibers were initially oriented along the x-axis and were irradiated using a 514 nm line argon laser with polarization fixed along the x-axis and focused through a 50x objective (NA = 0.75). Fibers were then aligned to the y-axis, followed by the same data acquisition process. Spectra were recorded from 1150 to 1750 cm⁻¹ with an 1800 lines/mm grating. For each acquisition, a total of 16 spectra were accumulated, each for 10 s. All fibers remained intact after acquisition with no visual sign of degradation under the incident laser. Spectra collected were analyzed with Fityk 1.3.1^{61 (link)}. Baseline subtraction is accomplished using a built-in Fityk automatic convex hull algorithm. All spectra were normalized to the intensity of the 1447 cm⁻¹ peaks, which arises from CH₃ asymmetric stretching and CH₂ bending^{59 (link)} and is insensitive to protein conformation.