Shifted Excitation Raman Difference Spectroscopy Combined with Wide Area Illumination and Sample Rotation for Wood Species Classification.

Raman spectroscopy has found its way into a wide range of applications and is successfully applied for qualitative and quantitative studies. Despite significant technical progress over the last few decades, there are still some challenges that limit its more widespread usage. This paper presents a holistic approach to addressing simultaneously the problems of fluorescence interference, sample heterogeneity, and laser-induced sample heating. Long wavelength shifted excitation Raman difference spectroscopy (SERDS) at 830 nm excitation combined with wide-area illumination and sample rotation is presented as a suitable approach for the investigation of selected wood species. Wood as a natural specimen represents a well-suited model system for our study as it is fluorescent, heterogeneous, and susceptible to laser-induced modifications. Two different subacquisition times (50 and 100 ms) and two sample rotation speeds (12 and 60 r/min) were exemplarily assessed. Results demonstrate that SERDS can effectively separate the Raman spectroscopic fingerprints of the wood species balsa, beech, birch, hickory, and pine from intense fluorescence interference. Sample rotation in conjunction with 1 mm-diameter wide-area illumination was suitable to obtain representative SERDS spectra of the wood species within 4.6 s. Using partial least squares discriminant analysis, a classification accuracy of 99.4% for the five investigated wood species was realized. This study highlights the large potential of SERDS combined with wide-area illumination and sample rotation for the effective analysis of fluorescent, heterogeneous, and thermally sensitive specimens in a wide range of application areas.

10†kHz Shifted-Excitation Raman Difference Spectroscopy with Charge-Shifting Charge-Coupled Device Read-Out for Effective Mitigation of Dynamic Interfering Backgrounds.

In this work we demonstrate an advanced concept of a charge-shifting charge-coupled device (CCD) read-out combined with shifted excitation Raman difference spectroscopy (SERDS) capable of operating at up to 10 kHz acquisition rates for the effective mitigation of fast-evolving interfering backgrounds in Raman spectroscopy. This rate is 10-fold faster than that achievable with an instrument we described previously and is overall 1000-fold faster than possible with conventional spectroscopic CCDs capable of operating at up to ∼10 Hz rates. The speed enhancement was realized by incorporating a periodic mask at the internal slit of an imaging spectrometer permitting a smaller shift of the charge on the CCD (8 pixels) to be required during the cyclic shifting process compared with the earlier design which employed an 80-pixel shift. The higher acquisition speed enables the more accurate sampling of the two SERDS spectral channels, enabling it to effectively tackle highly challenging situations with rapidly evolving interfering fluorescence backgrounds. The performance of the instrument is evaluated for heterogeneous fluorescent samples which are moved rapidly in front of the detection system aiming at the differentiation of chemical species and their quantification. The performance of the system is compared with that of the earlier 1 kHz design and a conventional CCD operated at its maximum rate of 5.4 Hz as previously. In all situations tested, the newly developed 10 kHz system outperformed the earlier variants. The 10 kHz instrument can benefit a number of prospective applications including: disease diagnosis where high sensitivity mapping of complex biological matrices in the presence of natural fluorescence bleaching restricts achievable limits of detection; accurate data acquisition from moving heterogeneous samples (or moving a handheld instrument in front of the sample during data acquisition) or data acquisition under varying ambient light conditions (e.g., due to casting shadows, sample or instrument movement). Other beneficial scenarios include monitoring rapidly evolving Raman signals in the presence of largely static background signals such as in situations where a heterogeneous sample is moving rapidly in front of a detection system (e.g., a conveyor belt) in the presence of static ambient light.

Evaluation of Modern Approaches for the Assessment of Dietary Carotenoids as Markers for Fruit and Vegetable Consumption.

The assessment of dietary carotenoids via blood measurements has been widely used as a marker for fruit and vegetable consumption. In the present study, modern, non-invasive approaches to assess dietary carotenoids, such as skin measurements and an app-based short dietary record (ASDR), were compared with conventional methods such as plasma status and handwritten 3-day dietary records. In an 8-week observational study, 21 healthy participants aged 50-65 years recorded their daily consumption of carotenoid-rich fruits and vegetables via a specially developed ASDR. Anthropometry, blood samplings and assessment of skin carotenoids via Raman and reflection spectroscopy were performed at baseline, after four weeks and at the end of the study. App-based intake data showed good correlations with plasma α-carotene (r = 0.74, p < 0.0001), ß-carotene (r = 0.71, p < 0.0001), and total plasma carotenoids (r = 0.65, p < 0.0001); weak correlations with plasma lutein/zeaxanthin and ß-cryptoxanthin (both r = 0.34, p < 0.05); and no correlation with plasma lycopene. Skin measurements via reflection and Raman spectroscopy correlated well with total plasma carotenoids (r = 0.81 and 0.72, respectively; both p < 0.0001), α-carotene (r = 0.75-0.62, p < 0.0001), and ß-carotene (r = 0.79-0.71, p < 0.0001); moderately with plasma lutein/zeaxanthin (both r = 0.51, p < 0.0001); weakly with plasma ß-cryptoxanthin (r = 0.40-0.31, p < 0.05); and showed no correlation with plasma lycopene. Skin measurements could provide a more convenient and noninvasive approach of estimating a person's fruit and vegetable consumption compared to traditional methods, especially in studies that do not intend blood sampling. ASDR records might function as a suitable, convenient tool for dietary assessment in nutritional intervention studies.

Determination of Soil Constituents Using Shifted Excitation Raman Difference Spectroscopy.

Soil analysis to estimate soil fertility parameters is of great importance for precision agriculture but nowadays it still relies mainly on complex and time-consuming laboratory methods. Optical measurement techniques can provide a suitable alternative. Raman spectroscopy is of particular interest due to its ability to provide a molecular fingerprint of individual soil components. To overcome the major issue of strong fluorescence interference inherent to soil, we applied shifted excitation Raman difference spectroscopy (SERDS) using an in-house-developed dual-wavelength diode laser emitting at 785.2 and 784.6 nm. To account for the intrinsic heterogeneity of soil components at the millimeter scale, a raster scan with 100 individual measurement positions has been applied. Characteristic Raman signals of inorganic (quartz, feldspar, anatase, and calcite) and organic (amorphous carbon) constituents within the soil could be recovered from intense background interference. For the first time, the molecule-specific information derived by SERDS combined with partial least squares regression was demonstrated for the prediction of the soil organic matter content (coefficient of determination R2 = 0.82 and root mean square error of cross validation RMSECV = 0.41%) as important soil fertility parameter within a set of 33 soil specimens collected from an agricultural field in northeast Germany.

Shifted Excitation Raman Difference Spectroscopy with Charge-Shifting Charge-Coupled Device (CCD) Lock-In Detection.

Shifted excitation Raman difference spectroscopy (SERDS) can provide effective, chemically specific information on fluorescent samples. However, the restricted ability for fast alternating detection (usually < 10 Hz) of spectra excited at two shifted laser wavelengths can limit its effectiveness when rapidly varying emission backgrounds are present. This paper presents a novel charge-shifting lock-in approach permitting fast SERDS operation (exemplarily demonstrated at 1000 Hz) using a specialized dual-wavelength diode laser (emitting at 829.40 nm and 828.85 nm) and a custom-built charge-coupled device (CCD) enabling charge retention and shifting back and forth on the CCD chip. For six selected mineral samples (moved irregularly during spectral acquisition), results demonstrate superior reproducibility of the fast charge-shifting read-out over the conventional read-out (operated at 5.4 Hz). Partial least squares discriminant analysis revealed improved classification performance of charge-shifting (four latent variables, sensitivity: 99%, specificity: 94%) versus conventional read-out (six latent variables, sensitivity: 90%, specificity: 92%). The charge-shifting concept was also successfully translated to sub-surface analysis using spatially offset Raman spectroscopy (SORS). Charge-shifting SERDS-SORS spectra recorded from a polytetrafluoroethylene layer, concealed behind a 0.25 mm thick, opaque, heterogeneous layer, matched reference spectra much more closely and exhibited a signal-to-background-noise (S/NB) ratio two times higher than that achieved with conventional CCD read-out SERDS-SORS. The novel approach overcomes fundamental limitations of conventional CCDs. In conjunction with the inherent capability of the charge-shifting lock-in technique to suppress rapidly varying ambient light interference demonstrated by us earlier it is expected to be particularly beneficial with heterogeneous fluorescent samples in field applications.

Spatially offset Raman spectroscopy for photon migration studies in bones with different mineralization levels.

The ability of Spatially Offset Raman Spectroscopy (SORS) to obtain chemically specific information from below the sample surface makes it a promising technique for non-invasive in vivo diagnosis of bone conditions by sampling bone through the skin. The depth below a surface interrogated by SORS depends on the system's optical properties and is difficult to estimate for complex bone material. This paper uses 830 nm laser excitation to investigate the influence of bone mineralization on photon migration properties in deer antler cortex, equine metacarpal cortex and whale tympanic bulla. Thin slices form each type of bone (thickness: 0.6-1.0 mm) were cut and put together on top of each other forming stacks with a total thickness of 4.1-4.7 mm. A 0.38 mm thin slice of polytetrafluoroethylene (PTFE) served as a test material for Raman signal recovery and was placed in between the individual bone slices within the stack. At SORS offsets of 8.0-9.5 mm Raman bands of materials not present in healthy bone (e.g. PTFE as an example) can be recovered through 4.4-4.7 mm of cortical bone tissue, depending on mineralization level and porosity. These findings significantly increase our understanding of SORS analysis through bones of different composition and provide information that is vital to determine the value of SORS as a medical diagnostic technique. The data serve to define which SORS offset is best deployed for the non-invasive detection of chemically specific markers associated with infection, degeneration and disease or cancer within bone.

Noninvasive analysis of thin turbid layers using microscale spatially offset Raman spectroscopy.

Here, we demonstrate, for the first time, the extension of applicability of recently developed microscale spatially offset Raman spectroscopy (SORS), micro-SORS, from the area of cultural heritage to a wider range of analytical problems involving thin, tens of micrometers thick diffusely scattering turbid layers. The method can be applied in situations where a high turbidity of layers prevents the deployment of conventional confocal Raman microscopy with its depth resolving capability. The method was applied successfully to detect noninvasively the presence of thin, highly turbid layers within polymers, wheat seeds, and paper. An invasive, cross sectional analysis confirmed the micro-SORS findings. Micro-SORS represents a new Raman imaging modality expanding the portfolio of noninvasive, chemically specific analytical tools.

A prototype hand-held Raman sensor for the in situ characterization of meat quality.

As a tool for the in situ characterization of meat quality, a hand-held Raman sensor head using an excitation wavelength of 671 nm was developed. A microsystem-based external cavity diode laser module was integrated into the sensor head and attached to a Raman probe, which is equipped with lens optics for excitation and signal collection as well as a Raman filter stage for Rayleigh rejection. The Raman signal was guided by an optical fiber to the detection unit, which was in the initial phase a laboratory spectrometer with a charge-coupled device (CCD) detector. The laser and the sensor head were characterized in terms of stability and performance for in situ Raman investigations. Raman spectra of meat were obtained with 35 mW within 5 seconds or less, ensuring short measuring times for the hand-held device. In a series of measurements with raw and packaged pork meat, the Raman sensor head was shown to detect microbial spoilage on the meat surface, even through the packaging foil.