Recent progress in surface-enhanced Raman spectroscopy for biological and biomedical applications: from cells to clinics.

The application of surface-enhanced Raman spectroscopy (SERS) in biological and biomedical detection schemes is feasible due to its excellent molecular specificity and high sensitivity as well as the capability of SERS to be performed in complex biological compositions. SERS-based investigation of cells, which are the basic structure and functional unit of organisms, represents the starting point of this review. It is demonstrated that SERS provides a deep understanding of living cells as well as their microenvironment which is needed to assess the development of diseases. The clinical relevance of SERS is proved by its application for the detection of cancer cells and tumour margins under in vivo conditions and examples for theranostic approaches are discussed. This review article provides a comprehensive overview of the recent progress within the last 3 years.

Surface-enhanced Raman spectroscopy and microfluidic platforms: challenges, solutions and potential applications.

The exhaustive body of literature published in the last four years on the development and application of systems based on surface-enhanced Raman spectroscopy (SERS) combined with microfluidic devices demonstrates that this research field is a current hot topic. This synergy, also referred to as lab-on-a-chip SERS (LoC-SERS) or nano/micro-optofluidics SERS, has opened the door for new opportunities where both techniques can profit. On the one hand, SERS measurements are considerably improved because the processes previously performed on a large scale in the laboratory and prone to human error can now be carried out in nanoliter volumes in an automatic and reproducible manner; on the other hand, microfluidic platforms need detection methods able to sense in small volumes and therefore, SERS is ideal for this task. The present review not only aims to provide the reader an overview of the recent developments and advancements in this field, but it also addresses the key aspects of fundamental SERS theory that influence the interpretation of SERS spectra, as well as the challenges brought about by the experimental conditions and chemometric data analysis.

Developments in spontaneous and coherent Raman scattering microscopic imaging for biomedical applications.

First, the potential role of Raman-based techniques in biomedicine is introduced. Second, an overview about the instrumentation for spontaneous and coherent Raman scattering microscopic imaging is given with a focus of recent developments. Third, imaging strategies are summarized including sequential registration with laser scanning microscopes, line imaging and global or wide-field imaging. Finally, examples of biomedical applications are presented in the context of single cells, laser tweezers, tissue sections, biopsies and whole animals.

Recognition of tumor cells by immuno-SERS-markers in a microfluidic chip at continuous flow.

SERS active nanoparticles were labeled with a reporter molecule and conjugated with anti-EpCAM antibodies. These immuno SERS markers were mixed with leukocytes, MCF-7 breast cancer cells and polystyrene beads, and the mixture was injected into a microfluidic quartz chip. Raman spectra were acquired at 785 nm excitation with 25 milliseconds exposure time in a continuous flow regime. Spectral unmixing by N-FINDR identified spectral signatures of SERS-labelled cells and polystyrene beads. This approach demonstrated rapid and reproducible SERS-assisted cell detection. Strategies are discussed to further increase the throughput for cell sorting.

Molecular self-healing mechanisms between C60-fullerene and anthracene unveiled by Raman and two-dimensional correlation spectroscopy.

The self-healing polymer P(LMA-co-MeAMMA) crosslinked with C60-fullerene has been studied by FT-Raman spectroscopy in combination with two-dimensional (2D) correlation analysis and density functional theory calculations. To unveil the molecular changes during the self-healing process mediated by the Diels-Alder equilibrium between 10-methyl-9-anthracenyl groups and C60-fullerene different anthracene-C60-fullerene adducts have been synthesized and characterized by time-, concentration- and temperature-dependent FT-Raman measurements. The self-healing process could be monitored via the C60-fullerene vibrations at 270, 432 and 1469 cm(-1). Furthermore, the detailed analysis of the concentration-dependent FT-Raman spectra point towards the formation of anthracene-C60-fullerene adducts with an unusual high amount of anthracene bound to C60-fullerene in the polymer film, while the 2D correlation analysis of the temperature-dependent Raman spectra suggests a stepwise dissociation of anthracene-C60-fullerene adducts, which are responsible for the self-healing of the polymer.

Seamless stitching of tile scan microscope images.

For diagnostic purposes, optical imaging techniques need to obtain high-resolution images of extended biological specimens in reasonable time. The field of view of an objective lens, however, is often smaller than the sample size. To image the whole sample, laser scanning microscopes acquire tile scans that are stitched into larger mosaics. The appearance of such image mosaics is affected by visible edge artefacts that arise from various optical aberrations which manifest in grey level jumps across tile boundaries. In this contribution, a technique for stitching tiles into a seamless mosaic is presented. The stitching algorithm operates by equilibrating neighbouring edges and forcing the brightness at corners to a common value. The corrected image mosaics appear to be free from stitching artefacts and are, therefore, suited for further image analysis procedures. The contribution presents a novel method to seamlessly stitch tiles captured by a laser scanning microscope into a large mosaic. The motivation for the work is the failure of currently existing methods for stitching nonlinear, multimodal images captured by our microscopic setups. Our method eliminates the visible edge artefacts that appear between neighbouring tiles by taking into account the overall illumination differences among tiles in such mosaics. The algorithm first corrects the nonuniform brightness that exists within each of the tiles. It then compensates for grey level differences across tile boundaries by equilibrating neighbouring edges and forcing the brightness at the corners to a common value. After these artefacts have been removed further image analysis procedures can be applied on the microscopic images. Even though the solution presented here is tailored for the aforementioned specific case, it could be easily adapted to other contexts where image tiles are assembled into mosaics such as in astronomical or satellite photos.

Droplet based microfluidics: spectroscopic characterization of levofloxacin and its SERS detection.

Levofloxacin (Levo), a second generation fluoroquinolone, has both clinical and environmental relevance. Therefore, the implementation of fast, robust and cost effective techniques for its monitoring is required. Here, its spectroscopic characterization and its detection in aqueous environment were carried out using surface enhanced Raman spectroscopy combined with droplet based microfluidics. The Levo molecule interacts with the silver nanoparticles via the carboxylate group and it adopts an upright or slightly tilted orientation. Furthermore, it is shown that the presence of Cl(-) ions has a strong influence on the enhancement efficiency of the Raman signal of the target molecule. Thus, for the determination of the limit of detection (LOD) the measurements were carried out in the absence of any electrolytes. The estimated LOD is ∼0.8 µM and the linear dynamic window ranges between 1-15 µM. These results were achieved after the normalization of the SERS signal to the Raman mode at 230 cm(-1). This band was attributed to the ν(Ag-O) stretching and it accounts for the Levo molecules in the first layer on the Ag nanoparticles.

Two-dimensional Raman correlation spectroscopy reveals molecular structural changes during temperature-induced self-healing in polymers based on the Diels-Alder reaction.

The thermally healable polymer P(LMA-co-FMA-co-MIMA) has been studied by temperature-dependent FT-Raman spectroscopy, two-dimensional Raman correlation analysis and density functional theory (DFT) calculations. To the best of our knowledge this study reports for the first time on the investigation of a self-healing polymer by means of two-dimensional correlation techniques. The synchronous correlation spectrum reveals that the spectrally overlapping C[double bond, length as m-dash]C stretching vibrations at 1501, 1575, 1585 and 1600 cm(-1) are perfect marker bands to monitor the healing process which is based on a Diels-Alder reaction of furan and maleimide. The comparison between experimental and calculated Raman spectra as well as their correlation spectra showed a good agreement between experiment and theory. The data presented within this study nicely demonstrate that Raman correlation analysis combined with a band assignment based on DFT calculations presents a powerful tool to study the healing process of self-healing polymers.

Discrimination and classification of liver cancer cells and proliferation states by Raman spectroscopic imaging.

Discrimination of nodular lesions in cirrhotic liver is a challenge in the histopathologic diagnostics. For this reason, there is an urgent need for new detection methods to improve the accuracy of the diagnosis of liver cancer. Raman imaging allows to determine the spatial distribution of a variety of molecules in cells or tissue label-free and to correlate this molecular information with the morphological structures at the same sample location. This study reports investigations of two liver cancer cell lines, - HepG2 and SK-Hep1, - as well as HepG2 cells in different cellular growth phases using Raman micro-spectroscopic imaging. Spectral data of all cells were recorded as a color-coded image and subsequentially analyzed by hierarchical cluster and principal component analysis. A support vector machine-based classification algorithm reliably predicts previously unknown cancer cells and cell cycle phases. By including selectively the Raman spectra of the cytoplasmic lipids in the classifier, the accuracy has been improved. The main spectral differences that were found in the comparative analysis can be attributed to a higher expression of unsaturated fatty acids in the hepatocellular carcinoma cells and during the proliferation phase. This corresponds to the already examined de novo lipogenesis in cells of liver cancer.

The effect of silver thickness on the enhancement of polymer based SERS substrates.

We investigated silver-covered polymer based nanogratings as substrates for surface-enhanced Raman spectroscopy (SERS), in particular with respect to the thickness of the plasmonically active silver film. In order to obtain accurate geometrical input data for the simulation process, we inspected cross sections of the gratings prepared by breaking at cryogenic temperature. We noticed a strong dependence of the simulation results on geometrical variations of the structures. Measurements revealed that an increasing silver film thickness on top of the nanogratings leads to a blue shift of the plasmonic resonance, as predicted by numerical simulations, as well as to an increased field enhancement for an excitation at 488 nm. We found a clear deviation of the experimental data compared to the simulated results for very thin silver films due to an island-like growth at a silver thickness below 20 nm. In order to investigate the SERS activity. we carried out measurements with crystal violet as a model analyte at an excitation wavelength of 488 nm. The SERS enhancement increases up to a silver thickness of about 30 nm, whereas it remains nearly constant for thicker silver films.

Deeper understanding of biological tissue: quantitative correlation of MALDI-TOF and Raman imaging.

In order to achieve a comprehensive description of biological tissue, spectral information about proteins, lipids, nucleic acids, and other biochemical components need to be obtained concurrently. Different analytical techniques may be combined to record complementary information of the same sample. Established techniques, which can be utilized to elucidate the biochemistry of tissue samples are, for instance, MALDI-TOF-MS and Raman microscopic imaging. With this contribution, we combine these two techniques for the first time. The combination of both techniques allows the utilization and interpretation of complementary information (i.e., the information about the protein composition derived from the Raman spectra with data of the lipids analyzed by the MALDI-TOF measurements). Furthermore, we demonstrate how spectral information from MALDI-TOF experiments can be utilized to interpret Raman spectra.

Multimodal mapping of human skin.

BACKGROUND: The combination of coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) imaging--referred to as multimodal imaging--provides complementary contrast based on molecular vibrations, the structure of various tissue components and endogenous fluorophores, respectively. OBJECTIVES: To present a comprehensive overview of the appearance of human skin in multimodal imaging. METHODS: Multimodal imaging of unstained skin cross-sections of 32 individuals was performed using a laser scanning microscope and picosecond laser pulse for excitation. RESULTS: The epidermis, dermis and subcutis are distinguishable in all three applied modalities, but are unveiled best in multimodal images. While the subcutis is dominated by the CARS signal, predominately SHG and the secondary TPEF signal detect the dermis. In contrast, no SHG signal is detected in the epidermis, whereas CARS and TPEF show equal contributions. Additionally, the appearance of the major skin appendages is described, i.e. the hair follicle, sebaceous and sweat glands, and blood vessels belonging to the vascular system. All four investigated functional units show a characteristic morphochemistry in TPEF and CARS, allowing identification of further subunits, e.g. the major components of the hair follicle, while the SHG signal delineates the localization of the functional units. CONCLUSIONS: Multimodal imaging is a powerful tool to investigate human skin by providing high contrast based on the molecular constitution. It is therefore suggested that multimodal imaging has a high potential in application to dermatological research and clinical diagnostics of various skin alterations.

The evolution of personality in patients with mild cognitive impairment.

AIMS: To describe personality traits and their changes in mild cognitive impairment (MCI) and control subjects. METHODS: Sixty-three MCI and 90 control subjects were asked to describe their current personality traits by the Structured Interview for the Five-Factor Model (SIFFM). For each subject, a close relative retrospectively assessed these descriptions both as to the previous and current personality traits, using the Revised NEO Personality Inventory, Form R (NEO-PI-R). RESULTS: Self-assessed MCI subjects reported significantly lower scores in the openness dimension than control subjects [F(1, 150) = 9.84, p = 0.002, ηp(2) = 0.06]. In current observer ratings, MCI subjects had higher scores on neuroticism [F(1, 137) = 7.55, p = 0.007, ηp(2) = 0.05] and lower ones on extraversion [F(1, 137) = 6.40, p = 0.013, ηp(2) = 0.04], openness [F(1, 137) = 9.93, p = 0.002, ηp(2) = 0.07], agreeableness [F(1, 137) = 10.18, p = 0.002, ηp(2) = 0.07] and conscientiousness [F(1, 137) = 25.96, p < 0.001, ηp(2) = 0.16]. Previous personality traits discriminated the groups as previous openness [odds ratio (OR) = 0.97, 95% confidence interval (CI) = 0.95-0.99, p = 0.014] and conscientiousness (OR = 0.96, 95% CI 0.94-0.98, p = 0.001) were negatively related to MCI group membership. In MCI subjects, conscientiousness [F(1, 137) = 19.20, p < 0.001, ηp(2) = 0.12] and extraversion [F(1, 137) = 22.27, p < 0.001, ηp(2) = 0.14] decreased between previous and current evaluations and neuroticism increased [F(1, 137) = 22.23, p < 0.001, ηp(2) = 0.14], whereas no significant change was found in control subjects. CONCLUSIONS: MCI subjects undergo significant personality changes. Thus, personality assessment may aid the early detection of dementia.

Physico-chemical and microbiological properties of raw fermented sausages are not influenced by color differences of turkey breast meat.

It has been suggested that the color of turkey breast meat influences both physico-chemical and microbiological properties of raw fermented sausages. In this study, raw fermented sausages were produced with turkey breast meat in 3 different colors (pale, normal, or dark), which were obtained from 2 fast-growing-genetic-line toms at 2 slaughterhouses. Prior to the sausage production, the breast muscles were sorted into color groups according to the lightness values determined at 24 h postmortem. This meat was subsequently processed to raw fermented sausages using 1.5 or 2.5% curing salt (CS). The pale meat had higher lightness, electrical conductivity, and drip loss, whereas the dark meat showed a darker color only. The physico-chemical (pH, water activity), visual (lightness, redness), and microbial (total plate count) properties of the sausages were not influenced by the color of the turkey breast meat. The sausage made with 2.5% CS had lower aw and higher ash and hardness values than the sausages produced with 1.5% CS. In conclusion, processing of differently colored turkey meat to raw fermented sausages does not influence the quality characteristics of the products. Based on these findings, there is no reason for the sausage producer to separate turkey breast muscles by color before producing raw fermented sausages.

[Traumatic dissection of the carotid artery: challenges for diagnostics and therapy illustrated by a case example]. / Traumatische Karotisdissektion : Herausforderungen der Diagnostik und Therapie anhand eines Fallbeispiels.

Traumatic dissection of the carotid artery is an easily overlooked consequence of trauma with notable morbidity and mortality which can be observed in up to 4% of cases involving multiple trauma. Certain mechanisms and patterns of injury as well as specific symptoms should serve as indicators of a dissection and should therefore result in further diagnostic measures. An early diagnosis is of major relevance. This report describes the case of a 45-year-old victim of a traffic accident who showed symptoms of a dissection which had initially not been diagnosed.

Identification of Bacillus anthracis via Raman spectroscopy and chemometric approaches.

Raman micro-spectroscopy was applied to compile a large-scale database of Raman spectra of single Bacillus endospores and to calculate classification functions, which were trained to discriminate between endospores of 66 strains from 13 Bacillus and Bacillus-related species including B. anthracis. The developed two-stage classification system comprising two support vector machines and one linear discriminant analysis classifier was then challenged by a test set of 27 samples to simulate the case of a real-world-scenario, when "unknown samples" are to be identified. In the end, all 27 test set samples including six B. anthracis strains were identified correctly. The samples thereby covered a diverse selection of species within the phylogenetically broad Bacillus genus and also included strains, which were not incorporated in the database before. All of them were correctly identified on the species level with accuracies between 88 and 100%. The sample analysis itself requires no biomass enrichment step prior to the analysis and qualifies the presented Raman spectroscopic approach to be a rapid analysis system in term of Bacillus endospore typing.

Spectroscopic detection and quantification of heme and heme degradation products.

Heme and heme degradation products play critical roles in numerous biological phenomena which until now have only been partially understood. One reason for this is the very low concentrations at which free heme, its complexes and the partly unstable degradation products occur in living cells. Therefore, powerful and specific detection methods are needed. In this contribution, the potential of nondestructive Raman spectroscopy for the detection, quantification and discrimination of heme and heme degradation products is investigated. Resonance Raman spectroscopy using different excitation wavelengths (413, 476, 532, and 752 nm) is employed to estimate the limit of detection for hemin, myoglobin, biliverdin, and bilirubin. Concentrations in the low micromolar range (down to 3 µmol/L) could be reliably detected when utilizing the resonance enhancement effect. Furthermore, a systematic study on the surface-enhanced Raman spectroscopy (SERS) detection of hemin in the presence of other cellular components, such as the highly similar cytochrome c, DNA, and the important antioxidant glutathione, is presented. A microfluidic device was used to reproducibly create a segmented flow of aqueous droplets and oil compartments. Those aqueous droplets acted as model chambers where the analytes have to compete for the colloid. With the help of statistical analysis, it was possible to detect and differentiate the pure substances as well as the binary mixtures and gain insights into their interaction.

Interaction effects of subjective memory impairment and ApoE4 genotype on episodic memory and hippocampal volume.

BACKGROUND: The apolipoprotein E4 allele (ApoE4) is an established genetic risk factor for Alzheimer's disease (AD). However, its effects on cognitive performance and brain structure in healthy individuals are complex. We investigated the effect of ApoE4 on cognitive performance and medial temporal lobe volumetric measures in cognitively unimpaired young elderly with and without subjective memory impairment (SMI), which is an at-risk condition for dementia.MethodAltogether, 40 individuals with SMI and 62 without were tested on episodic memory and on tasks of speed and executive function. All participants were ApoE genotyped. 21 subjects with SMI and 47 without received additional structural magnetic resonance imaging. Volumetric measures of the hippocampus, the entorhinal cortex and the amygdala were obtained manually. RESULTS: In the SMI group, ApoE4 carriers performed worse on the episodic memory (p=0.049) and showed smaller left hippocampal volumes (p=0.030). In the individuals without SMI, the ApoE4 carriers performed better on episodic memory (p=0.018) and had larger right hippocampal volumes (p=0.039). The interaction of group (SMI/no SMI) and ApoE genotype was significant for episodic memory (p=0.005) and right and left hippocampal volumes (p=0.042; p=0.035). There were no within-group differences or interaction effects on speed and executive function composite measures or other volumetric measures. CONCLUSIONS: The negative effect of ApoE4 on episodic memory and hippocampal volume in SMI supports SMI as a prodromal condition of AD. The positive effects of ApoE4 in subjects without SMI adds to a number of reports on positive ApoE4 effects in young and very old individuals.

Soluble amyloid precursor proteins in the cerebrospinal fluid as novel potential biomarkers of Alzheimer's disease: a multicenter study.

In this report, we present the results of a multicenter study to test analytic and diagnostic performance of soluble forms of amyloid precursor proteins alpha and beta (sAPP alpha and sAPP beta) in the cerebrospinal fluid (CSF) of patients with different forms of dementing conditions. CSF samples were collected from 188 patients with early dementia (mini-mental state examination >or=20 in majority of cases) and mild cognitive impairment (MCI) in 12 gerontopsychiatric centers, and the clinical diagnoses were supported by neurochemical dementia diagnostic (NDD) tools: CSF amyloid beta peptides, Tau and phospho-Tau. sAPP alpha and sAPP beta were measured with multiplexing method based on electrochemiluminescence. sAPP alpha and sAPP beta CSF concentrations correlated with each other with very high correlation ratio (R=0.96, P<0.001). We observed highly significantly increased sAPP alpha and sAPP beta CSF concentrations in patients with NDD characteristic for Alzheimer's disease (AD) compared to those with NDD negative results. sAPP alpha and sAPP beta highly significantly separated patients with AD, whose diagnosis was supported by NDD findings (sAPP alpha: cutoff, 117.4 ng ml(-1), sensitivity, 68%, specificity, 85%, P<0.001; sAPP beta: cutoff, 181.8 ng ml(-1), sensitivity, 75%, specificity, 85%, P<0.001), from the patients clinically assessed as having other dementias and supported by NDD untypical for AD. We conclude sAPP alpha and sAPP beta might be regarded as novel promising biomarkers supporting the clinical diagnosis of AD.

The plant sterol brassicasterol as additional CSF biomarker in Alzheimer's disease.

OBJECTIVE: Plant sterols (sitosterol, campesterol, stigmasterol and brassicasterol) are solely dietary-derivable sterols that are structurally very similar to cholesterol. In contrast to peripheral cholesterol, plant sterols can cross the blood-brain barrier and accumulate within mammalian brain. As an impaired function of the cerebrospinal fluid (CSF)-blood barrier is linked to neurodegenerative disorders, i.e. Alzheimer's disease (AD), we investigated whether this results in altered plant sterol concentrations in CSF. METHOD: Applying gas chromatography/mass spectrometry analysis, plant sterol concentrations were measured in plasma and CSF of patients with AD (n = 67) and controls (n = 29). Age, gender, plasma-to-CSF albumin ratio, CSF Aß(42) , CSF pTau, APOE4 genotype, and serum creatinine were applied as covariates in the statistical analysis for individual plant sterols in order to compare plasma and CSF plant sterol concentrations between patients with AD and controls. RESULTS: Albumin quotient was a consistent predictor in CSF for cholesterol and methyl plant sterols campesterol and brassicasterol. Comparison of lipid parameters per diagnosis based on relevant predictors revealed significantly lower concentrations of brassicasterol (P < 0.001) in CSF of patients with AD. Binary logistic regression analysis revealed that brassicasterol improved the predictive value when added to pTau and Aß42 in a biomarker model. CONCLUSION: Brassicasterol might be a relevant additional biomarker in AD.