Raman spectroscopy is an increasingly popular technique in many areas including biology and medicine.It is based on Raman scattering, a phenomenon in which incident photons lose or gain energy via interactions with vibrating molecules in a sample. These energy shifts can be used to obtain information regarding molecular composition of the sample with very high accuracy. Applications of Raman spectroscopy in the life sciences have included quantification of biomolecules, hyperspectral molecular imaging of cells and tissue, medical diagnosis, and others. This review briefly presents the physical origin of Raman scattering explaining the key classical and quantum mechanical concepts. Variations of the Raman effect will also be considered, including resonance, coherent, and enhanced Raman scattering. We discuss the molecular origins of prominent bands often found in the Raman spectra of biological samples. Finally, we examine several variations of Raman spectroscopy techniques in practice, looking at their applications, strengths, and challenges. This review is intended to be a starting resource for scientists new to Raman spectroscopy, providing theoretical background and practical examples as the foundation for further study and exploration.

Spectral depth-profiling of optically turbid samples is of high interest to a broad range of applications. We present a method for measuring spatially-offset Raman spectroscopy (SORS) over a range of length scales by incorporating a digital micro-mirror device (DMD) into a sample-conjugate plane in the detection optical path. The DMD can be arbitrarily programmed to collect/reject light at spatial positions in the 2D sample-conjugate plane, allowing spatially offset Raman measurements. We demonstrate several detection geometries, including annular and simultaneous multi-offset modalities, for both macro- and micro-SORS measurements, all on the same instrument. Compared to other SORS modalities, DMD-based SORS provides more flexibility with only minimal additional experimental complexity for subsurface Raman collection.

We describe a multifocal Raman micro-spectroscopy detection method based on a digital micromirror device, which allows for simultaneous “power-sharing” acquisition of Raman spectra from ad hoc sampling points. As the locations of the points can be rapidly updated in real-time via software control of a liquid-crystal spatial light modulator (LC-SLM), this technique is compatible with automated adaptive- and selective-sampling Raman spectroscopy techniques, the latter of which has previously been demonstrated for fast diagnosis of skin cancer tissue resections. We describe the performance of this instrument and show examples of multiplexed measurements on a range of test samples. Following this, we show the feasibility of reducing measurement time for power-shared multifocal Raman measurements combined with confocal auto-fluorescence imaging to provide guided diagnosis of tumours in human skin samples.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

Various toxic compounds disrupt bacterial physiology. While bacteria harbor defense mechanisms to mitigate the toxicity, these mechanisms are often coupled to the physiological state of the cells and become ineffective when the physiology is severely disrupted.

We investigated the feasibility of using spatially offset Raman spectroscopy (SORS) for nondestructive characterization of bone tissue engineering scaffolds. The deep regions of these scaffolds, or scaffolds implanted subcutaneously in live animals, are typically difficult to measure by confocal Raman spectroscopy techniques because of the limited depth penetration of light caused by the high level of light scattering. Layered samples consisting of bioactive glass foams (IEIC16), three-dimensional (3D)-printed biodegradable poly­(lactic-co-glycolic acid) scaffolds (PLGA), and hydroxyapatite powder (HA) were used to mimic nondestructive detection of biomineralization for intact real-size 3D tissue engineering constructs. SORS spectra were measured with a new SORS instrument using a digital micromirror device (DMD) to allow software selection of the spatial offsets. The results show that HA can be reliably detected at depths of 0–2.3 mm, which corresponds to the maximum accessible spatial offset of the current instrument. The intensity ratio of Raman bands associated with the scaffolds and HA with the spatial offset depended on the depth at which HA was located. Furthermore, we show the feasibility for in vivo monitoring mineralization of scaffold implanted subcutaneously by demonstrating the ability to measure transcutaneously Raman signals of the scaffolds and HA (fresh chicken skin used as a top layer). The ability to measure spectral depth profiles at high speed (5 s acquisition time) and the ease of implementation make SORS a promising approach for noninvasive characterization of cell/tissue development in vitro, and for long-term in vivo monitoring the mineralization in 3D scaffolds subcutaneously implanted in small animals.

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Using phantom samples, we investigated the feasibility of spatially-offset Raman spectroscopy (SORS) as a tool for monitoring non-invasively the mineralization of bone tissue engineering scaffold in-vivo . The phantom samples consisted of 3D-printed scaffolds of poly-caprolactone (PCL) and hydroxyapatite (HA) blends, with varying concentrations of HA, to mimic the mineralisation process. The scaffolds were covered by a 4 mm layer of skin to simulate the real in-vivo measurement conditions. At a concentration of HA approximately 1/3 that of bone (~0.6 g/cm 3 ), the characteristic Raman band of HA (960 cm −1 ) was detectable when the PCL:HA layer was located at 4 mm depth within the scaffold (i.e. 8 mm below the skin surface). For the layers of the PCL:HA immediately under the skin (i.e. top of the scaffold), the detection limit of HA was 0.18 g/cm 3 , which is approximately one order of magnitude lower than that of bone. Similar results were also found for the phantoms simulating uniform and inward gradual mineralisation of the scaffold, indicating the suitability of SORS to detect early stages of mineralisation. Nevertheless, the results also show that the contribution of the materials surrounding the scaffold can be significant and methods for subtraction need to be investigated in the future. In conclusion, these results indicate that spatially-offset Raman spectroscopy is a promising technique for in-vivo longitudinal monitoring scaffold mineralization and bone re-growth.