In this study, we collected Raman spectra from bone specimens using two RS systems: (i) confocal Horiba RS (Xplora, Horiba Jobin Yvon, Edison, NJ) with a 785 nm diode laser and with a 1200 lines/mm grating providing ~1.25 cm−1 spectral resolution and (ii) portable fiber optic probe-based RS. The probe-based RS involved: (i) an imaging spectrograph (Holospec f/1.8i, Kaiser Optical Systems, Ann Arbor, MI) coupled to a thermoelectrically cooled CCD camera (PIXIS: 256BR, Princeton. Instruments, Princeton, NJ), providing ~3.50 cm−1 spectral resolution, (ii) a 785 nm diode laser (Innovative Photonic Solutions, Monmouth Junction, NJ), and (iii) a custom-made fiber optic probe (EmVision, Loxahatchee, FL) consisting of one excitation and six collection fibers (each 300 µm in diameter) configured as a ring shape (Fig. 1 ). Wavelength calibration of the portable probe-based RS system was done using a neon-argon lamp. Naphthalene and acetaminophen standards were also used to determine the exact excitation wavelength for subsequent Raman shift calculations. The spectral response of the system was further corrected using a tungsten lamp calibrated by the National Institute of Standards and Technology.
For Raman micro-spectroscopy, the long axis of each specimen was aligned parallel to the axis of the primary laser polarization, and thirty-two Raman spectra per specimen were each obtained as the average of 12 consecutive spectra per spot with a 5-second acquisition using a 20x objective (NA = 0.40). Laser power was ~35 mW. For fiber-optic RS with a larger laser spot size than a 20x objective (~300 µm vs. ~2.5 µm), ten spectra per sample were each obtained as the average of 10 consecutive spectra per spot with 3-second acquisition, and laser power was set up at ~80 mW. The long axis was not specifically aligned with the polarization axis of the laser because fiber optics scramble the orientation of the light (Supp. Mater. Fig.4 ). Raman data collection were randomly distributed throughout the entire two longitudinal surfaces of bone specimens (sixteen Raman spectra and five Raman spectra per surface for research-grade RS and fiber optic RS, respectively). Since the bone specimens were not immersed in PBS during the acquisition of the multiple spectra, some dehydration occurred. To verify that this does not affect the spectra, we collected spectra from 6 bone specimens before and after 20 min in air which is the maximum time for total spectra collection in this study. We found that there were no apparent differences in the RS measures between these two time points (Supp. Mater. Fig. 5 ) indicating partial air-drying for 20 minutes did not affect significantly the RS properties.
For Raman micro-spectroscopy, the long axis of each specimen was aligned parallel to the axis of the primary laser polarization, and thirty-two Raman spectra per specimen were each obtained as the average of 12 consecutive spectra per spot with a 5-second acquisition using a 20x objective (NA = 0.40). Laser power was ~35 mW. For fiber-optic RS with a larger laser spot size than a 20x objective (~300 µm vs. ~2.5 µm), ten spectra per sample were each obtained as the average of 10 consecutive spectra per spot with 3-second acquisition, and laser power was set up at ~80 mW. The long axis was not specifically aligned with the polarization axis of the laser because fiber optics scramble the orientation of the light (Supp. Mater. Fig.
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