Chemical composition and fibrillar organization are the major determinants of osteonal bone mechanics. However, prominent methodologies commonly applied to investigate mechanical properties of bone on the micro scale are usually not able to concurrently describe both factors. In this study, we used polarized Raman spectroscopy (PRS) to simultaneously analyze structural and chemical information of collagen fibrils in human osteonal bone in a single experiment. Specifically, the three-dimensional arrangement of collagen fibrils in osteonal lamellae was assessed. By analyzing the anisotropic intensity of the amide I Raman band of collagen as a function of the orientation of the incident laser polarization, different parameters related to the orientation of the collagen fibrils and the degree of alignment of the fibrils were derived. Based on the analysis of several osteons, two major fibrillar organization patterns were identified, one with a monotonic and another with a periodically changing twist direction. These results confirm earlier reported twisted and oscillating plywood arrangements, respectively. Furthermore, indicators of the degree of alignment suggested the presence of disordered collagen within the lamellar organization of the osteon. The results show the versatility of the analytical PRS approach and demonstrate its capability in providing not only compositional, but also 3D structural information in a complex hierarchically structured biological material. The concurrent assessment of chemical and structural features may contribute to a comprehensive characterization of the microstructure of bone and other collagen-based tissues.

Collagen is a versatile structural molecule in nature and is used as a building block in many highly organized tissues, such as bone, skin, and cornea. The functionality and performance of these tissues are controlled by their hierarchical organization ranging from the molecular up to macroscopic length scales. In the present study, polarized Raman microspectroscopic and imaging analyses were used to elucidate collagen fibril orientation at various levels of structure in native rat tail tendon under mechanical load. In situ humidity-controlled uniaxial tensile tests have been performed concurrently with Raman confocal microscopy to evaluate strain-induced chemical and structural changes of collagen in tendon. The methodology is based on the sensitivity of specific Raman scattering bands (associated with distinct molecular vibrations, such as the amide I) to the orientation and the polarization direction of the incident laser light. Our results, based on the changing intensity of Raman lines as a function of orientation and polarization, support a model where the crimp and gap regions of collagen hierarchical structure are straightened at the tissue and molecular level, respectively. However, the lack of measurable changes in Raman peak positions throughout the whole range of strains investigated indicates that no significant changes of the collagen backbone occurs with tensing and suggests that deformation is rather redistributed through other levels of the hierarchical structure.

In this work, we utilize a short-wavelength, 532-nm picosecond pulsed laser coupled with a time-gated complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector to acquire Raman spectra of several drugs of interest. With this approach, we are able to reveal previously unseen Raman features and suppress the fluorescence background of these drugs. Compared to traditional Raman setups, the present time-resolved technique has two major improvements. First, it is possible to overcome the strong fluorescence background that usually interferes with the much weaker Raman spectra. Second, using the high photon energy excitation light source, we are able to generate a stronger Raman signal compared to traditional instruments. In addition, observations in the time domain can be performed, thus enabling new capabilities in the field of Raman and fluorescence spectroscopy. With this system, we demonstrate for the first time the possibility of recording fluorescence-suppressed Raman spectra of solid, amorphous and crystalline, and non-photoluminescent and photoluminescent drugs such as caffeine, ranitidine hydrochloride, and indomethacin (amorphous and crystalline forms). The raw data acquired by utilizing only the picosecond pulsed laser and a CMOS SPAD detector could be used for identifying the compounds directly without any data processing. Moreover, to validate the accuracy of this time-resolved technique, we present density functional theory (DFT) calculations for a widely used gastric acid inhibitor, ranitidine hydrochloride. The obtained time-resolved Raman peaks were identified based on the calculations and existing literature. Raman spectra using non-time-resolved setups with continuous-wave 785- and 532-nm excitation lasers were used as reference data. Overall, this demonstration of time-resolved Raman and fluorescence measurements with a CMOS SPAD detector shows promise in diverse areas, including fundamental chemical research, the pharmaceutical setting, process analytical technology (PAT), and the life sciences.Graphical abstractTime-resolved Raman measurement of a pharmaceutical sample using the complementary metal-oxide semiconductor (CMOS) single photon avalanche diode (SPAD) detector technologyElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-9156-6) contains supplementary material, which is available to authorized users.