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

Photon migration of Raman signal in bone as measured with spatially offset Raman spectroscopy

Spatially offset Raman spectroscopy (SORS) is currently being developed as an in vivo tool for bone disease detection, but to date, information about the interrogated volume as influenced by the light propagation and scattering characteristics of the bone matrix is still limited. This paper seeks to develop our general understanding of the sampling depths of SORS in bone specimens as a function of the applied spatial offset. Equine metacarpal bone was selected as a suitable specimen of compact cortical bone large enough to allow several thin slices (600 μm) to be cut from the dorsal surface. Photon migration at 830-nm excitation was studied with five bone slices and a 380 μm-thin polytetrafluoroethylene (PTFE) slice placed consecutively between the layers. To optimize Raman signal recovery of the PTFE with increasing depth within the bone stack required a corresponding increase in spatial offset. For example, to sample effectively at 2.2-mm depth within the bone required an optimal SORS offset of 7mm. However, with a 7-mm offset, the maximum accessible penetration depth from which the PTFE signal could be still recovered was 3.7mm. These results provide essential basic information for developing SORS technology for medical diagnostics in general and optimizing sampling through bone tissue, permitting a better understanding of the relationship between the offset and depth of bone assessed, in particular. Potential applications include the detection of chemically specific markers for changes in bone matrix chemistry localized within the tissue and not present in healthy bone

Multispectral, diode laser based Raman investigations for in situ analysis of selected meats

Das Ziel der vorliegenden Arbeit ist die Demonstration der Eignung Raman-spektroskopischer In-situ-Verfahren zur Detektion des mikrobiellen Verderbs von Fleisch sowie der nicht-invasiven Unterscheidung ausgewählter Tierarten. Hierzu wurde ein innovatives, portables Prototyp-Messsystem für die In-situ-Ramanspektroskopie an Fleischproben realisiert. Alle aufeinander abgestimmten Komponenten des Systems, inklusive Mikrosystem-Diodenlaser, optischer Elemente und Miniaturspektrometer sind dabei speziell im Hinblick auf die Fleischanalytik entwickelt und für diesen Einsatzzweck optimiert worden. Umfangreiche Untersuchungen an Schweinefleisch konnten die Ramanspektroskopie als effizientes Verfahren für die nicht-invasive Verderbnisdetektion verifizieren, wobei mit Hilfe statistischer Verfahren eine Quantifizierung der mikrobiellen Kontamination hinsichtlich des Keimzahl-Grenzwertes von 106 KbE/cm2 erreicht werden konnte. Der Nachweis des Verderbs basiert dabei auf dem Signal-zu-Untergrund-Verhältnis der Spektren. Während anhand der Raman-spektroskopischen Signatur Veränderungen in den Proteinbanden deutlich werden, zeigt sich ca. 10 Tage nach der Schlachtung eine einsetzende Fluoreszenz von Porphyrinen. Mit Hilfe des portablen Ramansystems kann damit erstmalig, im Gegensatz zu teuren, arbeits- und zeitaufwändigen Laboruntersuchungen, das Überschreiten der kritischen Keimzahlbelastung bereits in wenigen Sekunden und in situ erfasst werden. Ein multispektrales Anregungskonzept konnte die Eignung der Anregungswellenlängen 785 nm und 671 nm zeigen, während bei 488 nm ein hochempfindlicher Nachweis von Carotinoiden demonstriert wurde. Als Zielwellenlänge für das portable System erwies sich 671nm als besonders vorteilhaft, da hier bereits Messzeiten von lediglich 1 s eine Klassifizierung des Fleisches ermöglichten, so dass On-line-Messungen realisierbar sind. Anhand ausgewählter Verpackungstypen wurde das Messverfahren als geeignete Technik für Untersuchungen durch die Verpackungsfolie hindurch verifiziert. Dies stellt im Vergleich zu anderen Detektionsverfahren des mikrobiellen Verderbs einen entscheidenden Vorteil dar und qualifiziert das System für den zukünftigen Einsatz durch Lebensmittelkontrolleure. Ebenso konnte die Übertragbarkeit auf Rind, Huhn und Pute experimentell bestätigt werden, was die universelle Eignung des Detektionskonzeptes unterstreicht. Darauf aufbauend erfolgte die Entwicklung von zwei neuartigen, miniaturisierten Prototyp-Messköpfen mit jeweils zwei spektral leicht gegeneinander verschobenen Anregungswellenlängen (671,0 nm und 671,6 nm bzw. 782,65 nm und 783,15 nm) für die Shifted Excitation Raman Difference Spectroscopy (SERDS). Hiermit konnte bei Messzeiten von nur 10 s mit Hilfe der Hauptkomponentenanalyse erstmals eine nicht-invasive Differenzierung des Fleisches sowie Fleischsaftes von Schwein, Rind, Huhn und Pute realisiert werden. Als Unterscheidungskriterien wurden dabei der Gehalt des Muskelfarbstoffes Myoglobin sowie Unterschiede in der Proteinzusammensetzung identifiziert. Damit weist SERDS ein großes Potential als Schnelltest-Methode zur In-situ-Identifikation ausgewählter Tierarten auf.This thesis aims to demonstrate the suitability of in situ Raman spectroscopy for the detection of microbial spoilage of meat and the non-invasive discrimination of selected animal species. For that purpose, an innovative portable prototype measurement system for Raman spectroscopic in situ investigations of meat was realized. All components of the system, including microsystem diode laser, optical elements, and a miniature spectrometer, were specially tailored and optimized for meat analyses. Extensive studies with pork meat were able to verify Raman spectroscopy as efficient method for the non-invasive spoilage detection. Applying statistical data analyses, a quantification of the microbial contamination with respect to the relevant limit of 106 cfu/cm2 could be achieved. Here, the verification of the spoilage status is based on the signal to background ratio of the spectra. While the Raman spectroscopic signature reveals changes in protein bands, the onset of a fluorescence emission caused by porphyrins becomes evident around 10 days after slaughter. Hence, in contrast to costly, labor-intensive, and time-consuming laboratory analyses, for the first time exceeding of the critical microbial load can be gathered within a few seconds and in situ using the portable Raman system. A multispectral excitation concept demonstrated the applicability of 785 nm and 671 nm as excitation wavelengths, whereas 488 nm is well suited for the sensitive detection of carotenoids. As target wavelength for the portable system, 671nm was identified as being particularly advantageous. Here, measurement times of only 1 s were sufficient to enable for a classification of the meat samples and thus making on-line investigations feasible. By means of selected packaging types, the measurement process could be verified as suitable technique for investigations even through the packaging foil. Compared to other detection methods for microbial spoilage, this option is a crucial advantage and qualifies the system for future usage by food inspectors. Furthermore, the transferability to beef as well as chicken and turkey meat could be experimentally verified and underlines the universal appropriateness of the applied detection concept. Based on the previous results, two novel miniaturized prototype measurement heads each comprising two slightly shifted emission wavelengths (671.0 nm and 671.6 nm or 782.65 nm and 783.15 nm) for shifted excitation Raman difference spectroscopy (SERDS) were developed. Using these devices, for the first time a non-invasive differentiation of meat and meat juice from pork, cow, chicken, and turkey could be realized using principal components analysis and applying integration times of 10 s only. Here, the content of the muscle pigment myoglobin as well as differences in the protein composition were identified as distinctive features. These findings demonstrate the large potential of SERDS as rapid test method for the in situ identification of selected animal species

Shifted excitation Raman difference spectroscopy as enabling technique for the analysis of animal feedstuff

To achieve the best performance and health in farm animals, high-quality pellets should be applied for feeding. Raw materials used for pellet production can have a significant influence on the nutritive and physical characteristics of the final product. A comprehensive quality control of raw materials and pellets is therefore essential. Optical inspection techniques show great promise as they enable fast, simple, and non-destructive analysis. This study demonstrates the potential of shifted excitation Raman difference spectroscopy (SERDS) for inspection of intact feed pellets and their constituents. SERDS combines the ability of conventional Raman spectroscopy to obtain chemically specific information from the sample with efficient fluorescence background rejection capabilities. The latter is an essential prerequisite for the application to highly fluorescent natural samples, for example, feedstuffs. A custom dual-wavelength diode laser with two slightly shifted emission wavelengths (785.2 and 784.6 nm) as required for SERDS is used as excitation light source. Results demonstrate that Raman signals can efficiently be separated from unwanted background contributions allowing for qualitative spatially resolved analysis of chicken feed pellets. Individual constituents present at levels down to 10 g/kg were successfully detected by means of their characteristic spectral signature. This highlights the large potential of SERDS and could pave the way for future inspection of raw materials and pellets at selected points along the process chain

Assessment of photon migration for subsurface probing in selected types of bone using spatially offset Raman spectroscopy

Bone diseases and disorders are a growing challenge in aging populations; so effective diagnostic and therapeutic solutions are now essential to manage the demands of healthcare sectors effectively. Spatially offset Raman spectroscopy (SORS) allows for chemically specific sub-surface probing and has a great potential to become an in vivo tool for early non-invasive detection of bone conditions. Bone is a complex hierarchical material and the volume probed by SORS is dependent on its optical properties. Understanding and taking into account the variations in diffuse scattering properties of light in various bone types is essential for the effective development and optimization of SORS as a diagnostic in vivo tool for characterizing bone disease. This study presents SORS investigations at 830 nm excitation on two specific types of bone with differing mineralization levels. Thin slices of bone from horse metacarpal cortex (0.6 mm thick) and whale bulla (1.0 mm thick) were cut and stacked on top of each other (4-7 layers with a total thickness of 4.1 mm). To investigate the depth origin of the detected Raman signal inside the bone a 0.38 mm thin Teflon slice was used as test sample and inserted in between the layers of stacked bone slices. For both types of bone it could be demonstrated that chemically specific Raman signatures different from those of normal bone can be retrieved through 3.8-4.0 mm of overlying bone material with a spatial offset of 7-8 mm. The determined penetration depths can be correlated with the mechanical and optical properties of the specimens. The findings of this study increase our understanding of SORS analysis of bone and thus have impact for medical diagnostic applications e.g. enabling the non-invasive detection of spectral changes caused by degeneration, infection or cancer deep inside the bone matrix

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), β-carotene (r = 0.71, p < 0.0001), and total plasma carotenoids (r = 0.65, p < 0.0001); weak correlations with plasma lutein/zeaxanthin and β-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 β-carotene (r = 0.79–0.71, p < 0.0001); moderately with plasma lutein/zeaxanthin (both r = 0.51, p < 0.0001); weakly with plasma β-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

sj-docx-1-asp-10.1177_00037028231167441 - Supplemental material for 10 kHz Shifted-Excitation Raman Difference Spectroscopy with Charge-Shifting Charge-Coupled Device Read-Out for Effective Mitigation of Dynamic Interfering Backgrounds

Supplemental material, sj-docx-1-asp-10.1177_00037028231167441 for 10 kHz Shifted-Excitation Raman Difference Spectroscopy with Charge-Shifting Charge-Coupled Device Read-Out for Effective Mitigation of Dynamic Interfering Backgrounds by Sara Mosca, Kay Sowoidnich, Megha Mehta, William H. Skinner, Benjamin Gardner, Francesca Palombo, Nicholas Stone and Pavel Matousek in Applied Spectroscopy</p

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 p p p p p p p 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