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Near-infrared (NIR) fluorescence imaging using exogenous fluorescent agents provides whole-field images in real-time to assist the surgeon in the excision of a tumor. Although the method has high sensitivity, the specificity can sometimes be lower than expected. Raman spectroscopy can detect tumors with high specificity. Therefore, a combination of both techniques can be advantageous. A complication that must be addressed is that the NIR spectral region is favored by both techniques for (in vivo) tissue analysis. When fluorescence and Raman emissions spectrally overlap, it becomes challenging or impossible to detect the Raman signal. In this paper, by avoiding this overlap, we describe a Raman spectroscopy setup capable of recording high-quality Raman spectra from tissue containing NIR exogenous fluorescent agents. We identify an optimal wavelength interval (900-915 nm) for Raman excitation, which avoids both excitation of fluorescent dyes and Raman signal self-absorption by the tissue. In this way, Raman spectroscopy can be combined with the currently most-used NIR fluorescent dyes. This combined novel setup could pave the way for clinical trials benefiting from both fluorescence imaging and Raman spectroscopy to avoid positive margins in cancer surgery.

Mean LD score for the validation data set for Stratum Corneum skin layer for MEK inhibitors (A), EGFR inhibitors (B) and BRAF inhibitors (C) and for Viable Epidermis skin layer for MEK inhibitors (D), EGFR inhibitors (E) and BRAF inhibitors (F). The p-value between the two groups is reported. In the 6 conditions, the p-value is lower than 0.001%.

Summary of cutaneous adverse events at the different time point for each patient included in this study. n/a: not available

Revised Supplementary Information

Raman spectroscopy is a noninvasive and label-free optical technique that provides detailed information about the molecular composition of a sample. In this study, we evaluated the potential of Raman spectroscopy to predict skin toxicity due to tyrosine kinase inhibitors treatment. We acquired Raman spectra of skin of patients undergoing treatment with MEK, EGFR, or BRAF inhibitors, which are known to induce severe skin toxicity; for this pilot study, three patients were included for each inhibitor. Our algorithm, based on partial least squares-discriminant analysis (PLS-DA) and cross-validation by bootstrapping, discriminated to variable degrees spectra from patient suffering and not suffering cutaneous adverse events. For MEK and EGFR inhibitors, discriminative power was more than 90% in the viable epidermis skin layer; whereas for BRAF inhibitors, discriminative power was 71%. There was a 81.5% correlation between blood drug concentration and Raman signature of skin in the case of EGFR inhibitors and viable epidermis skin layer. Our results demonstrate the power of Raman spectroscopy to detect apparition of skin toxicity in patients treated with tyrosine kinase inhibitors at levels not detectable via dermatological inspection and histological evaluation. Cancer Res; 77(2); 557–65. ©2016 AACR.

This study reports on the effects of insertion velocity, needle tip geometry and needle diameter on tissue deformation and maximum insertion force. Moreover, the effect of multiple insertions with the same needle on the maximum insertion force is reported. The tissue deformation and maximum insertion force strongly depend on the insertion velocity and the tip geometry. No correlation was found between the outer diameter and the maximum insertion force for small needles (30G – 32G). The endurance experiments showed no remarkable difference in the maximum insertion force during 100 insertions.

The goal of head and neck oncological surgery is complete tumor resection with adequate resection margins while preserving acceptable function and appearance. For oral cavity squamous cell carcinoma (OCSCC), different studies showed that only 15%-26% of all resections are adequate. A major reason for the low number of adequate resections is the lack of information during surgery; the margin status is only available after the final histopathologic assessment, days after surgery. The surgeons and pathologists at the Erasmus MC University Medical Center in Rotterdam started the implementation of specimen-driven intraoperative assessment of resection margins (IOARM) in 2013, which became the standard of care in 2015. This method enables the surgeon to turn an inadequate resection into an adequate resection by performing an additional resection during the initial surgery. Intraoperative assessment is supported by a relocation method procedure that allows accurate identification of inadequate margins (found on the specimen) in the wound bed. The implementation of this protocol resulted in an improvement of adequate resections from 15%-40%. However, the specimen-driven IOARM is not widely adopted because grossing fresh tissue is counter-intuitive for pathologists. The fear exists that grossing fresh tissue will deteriorate the anatomical orientation, shape, and size of the specimen and therefore will affect the final histopathologic assessment. These possible negative effects are countered by the described protocol. Here, the protocol for specimen-driven IOARM is presented in detail, as performed at the institute.

Confocal Raman spectroscopy (CRS) enables the real-time profiling of substances penetrating to the skin without sample pre-treatment or labelling. Until now, CRS had been used as a semi-quantitative method, which posed challenges for evaluating topical formulations and assessing bioequivalence. We present a novel approach of CRS for quantitative analysis of skin delivery. The quantitative CRS in vivo has been correlated with the well-established in vitro Franz-diffusion experiments, indicating the potential of CRS for determining skin delivery. We anticipate CRS providing a rapid and non-invasive method that will be an attractive alternative to the clinical studies currently used in bioequivalence testing.

Background In head and neck oncological surgery the goal is to achieve a complete tumor resection with acceptable remaining function and appearance. For oral cavity squamous cell carcinoma (OCSCC) only 15% of the resections are reported as adequate. Since 2013, we have performed intraoperative assessment of resection margins (IOARM) in our institute, based on palpation and visual inspection of the resected specimens by pathologist and surgeon. This has resulted in an improvement of adequate resection margins from 15% to 50%, underlining the importance of IOARM. However, this method is subjective, labor intensive, and logistically challenging. Objective Our aim is to develop an objective method for fast and reliable IOARM based on Raman spectroscopy (RS). Methods RS is a non-destructive objective optical technique that provides information about the molecular composition of tissues. It can discriminate between healthy tissue and tumors. We developed a prototype Raman instrument employing a fiber-optic needle probe. The fiber-optic needle is driven into the OCSCC specimen, from the resection surface towards the tumor. Based on the Raman spectra collected along the insertion path, the location of the tumor border can be determined. From this the resection margin can be determined. Results First tests of the method show that the instrument accurately predicts the achieved resection margins. Per location the measurement and assessment takes 5 seconds. Conclusions This development signifies an important step towards a fast and objective IOARM. The fast measurement time enables an objective inspection of the margins achieved at a large number of locations of the resection surface.

Previously, we reported the use of Confocal Raman Spectroscopy (CRS) to investigate the topical delivery of actives and excipients. We have also correlated the results from CRS with findings from in vitro diffusion studies in human skin. However, until now CRS has only been used as a semi-quantitative method of determining the skin uptake of molecules, with results expressed as arbitrary units of signal intensity. Clearly, this posed challenges for using CRS to determine skin delivery and to assess the drug bioavailability and bioequivalence of topical formulations. In the present work, the permeation of niacinamide (NIA) from various formulations in human skin was studied in vitro using conventional Franz cells and in vivo using a quantitative CRS method under finite dose conditions. The selection of NIA was based on its wide use in pharmaceutical and personal care formulations for many years. This is the first fully quantitative study to compare these methods. The vehicles investigated were neat Transcutol® P (TC); binary combinations of propylene glycol (PG) with propylene glycol monolaurate (PGML); and ternary mixtures of PG, PGML, and isopropyl myristate (IPM). These solvents were selected to encompass a range of physicochemical properties. NIA permeation was evident from all formulations in vitro and in vivo. The vehicles PG:PGML and PG:PGML:IPM delivered comparable amounts across the skin in vitro at 24 h (100.3–106.7 µg/cm2, p > 0.05) that were significantly higher compared with those of TC (1.3 µg/cm2, p < 0.05). An excellent in vitro in vivo correlation (R2 = 0.98) was found following the linear regression of the cumulative amounts of NIA permeated in vitro and the amounts of NIA at 2 μm in the skin measured with CRS. A very good correlation between the cumulative permeation of NIA in vitro and the total amount of NIA that penetrated the stratum corneum (SC) per unit of surface area (μg/cm2) in vivo was also observed, with a Pearson correlation coefficient (R2) of 0.94. The findings support the use of CRS for the quantitative measurement of actives delivered to the skin in vivo. Future studies will focus on exploring the reproducibility and reliability of the method by investigating the delivery of different actives from a wider range of vehicles. Additionally, quantitative CRS will be evaluated further as a method for assessing the bioequivalence of topical formulations.

Carriers of loss-of-function mutations in the filaggrin gene (LoF FLG) have less natural moisturizing factor (NMF) in their stratum corneum (SC) and an increased risk of atopic dermatitis (AD). Natural moisturizing factor can be measured noninvasively by Raman spectroscopy. The use of Raman-derived NMF at birth to screen for FLG genotype could inform targeted AD prevention, but values in neonatal populations are largely unexplored.To examine the associations between Raman-derived neonatal NMF measurements and FLG genotype.Natural moisturizing factor was measured by Raman spectroscopy in the SC of the thenar eminence within 4 days of birth in 139 term neonates. Filaggrin genotyping was performed for 117 neonates (84%).The mean (SD) NMF was 0.37 (0.11) g/g protein, with values increasing across the first 3 days (day 1 vs 3: 0.29 [0.09] vs 0.43 [0.08, P.001]). Twelve infants (10.3%) were carriers of LoF FLG, all heterozygous. Natural moisturizing factor was lower in LoF FLG carriers compared with wild-type (0.27 [0.08] vs 0.38 [0.11] g/g protein, P ≤ .001). Natural moisturizing factor had good discriminatory power for FLG genotype (area under the receiver operating curve [AUROC]: 0.79; 95% CI: 0.66, 0.91; P ≤ .001). This improved after correcting day 1 and 2 measurements to day 3 (AUROC: 0.83; 95% CI: 0.75, 0.92; P.001).This study suggests that Raman-derived NMF measured in the early postnatal period may have the potential to classify by FLG genotype. The full translational value of this needs to be determined.

Raman spectroscopy in the high-wavenumber spectral region (HWR) is particularly suited for fiber-optic in vivo medical applications. The most-used fiber-optic materials have negligible Raman signal in the HWR. This enables the use of simple and cheap single-fiber-optic probes that can be fitted in endoscopes and needles. The HWR generally shows less tissue luminescence than the fingerprint region. However, the luminescence can still be stronger than the Raman signal. Hardware- and software-based strategies have been developed to correct for these luminescence signals. Typically, hardware-based strategies are more complex and expensive than software-based solutions. Effective software strategies have almost exclusively been developed for the fingerprint region. First-order polynomial baseline fitting (PBF) is the most common background/luminescence estimation employed for the HWR. The goal of this study was to characterize the luminescence background signals of HW spectra of human oral tissue and compare the performance of two algorithms for correction of these background signals: PBF and multiple regression fitting (MRF). In the MRF method, we introduce here, prior knowledge of the range of Raman signals that can be obtained from the tissues of interest is explicitly used. MRF is more robust than PBF because it does not require an a priori choice of the polynomial order for fitting the background signal. This is important because, as we show, no single polynomial order can optimally characterize all backgrounds that are encountered in HW tissue spectra. We conclude that MRF should be the preferred method for background subtraction in the HWR.

Background Oral cavity squamous cell carcinoma (OCSCC) is the most frequent head and neck cancer. Surgery is the mainstay treatment for patients with OCSCC. In case of bone involvement, the affected bone needs to be resected. Cancer-free bone resection margins (BRMs) are of crucial importance: patients with cancer-free BRMs have a 2 times higher chance of survival. Currently, there is no standard method for intraoperative assessment of BRMs. Bone margin status is only known after tissue decalcification, which takes 1 to 2 weeks. After that time reoperations are highly undesirable, because the surgical defect has healed. Therefore, it is crucial to achieve tumor-free resection surfaces, which requires the possibility of intraoperative assessment of BRMs. Objective The aim of this study was to investigate the potential of Raman spectroscopy (RS) for detection of OCSCC in bone resection surfaces during mandibulectomy. RS is a nondestructive objective optical technique that provides information about the molecular composition of tissues. Methods Raman mapping experiments were performed on fresh mandible resection specimens from patients treated with mandibulectomy for OCSCC. A tumor detection algorithm was created based on water concentration and the high-wavenumber range (2800 cm−1-3050 cm−1) of the Raman spectra. Results Results show that RS can detect OCSCC in bone resection surfaces with a high sensitivity (96%) and specificity (83%; 26 mapping experiments, 22 patients). Conclusions These results form the basis for further development of an RS tool as an objective method for intraoperative assessment of BRMs.

We present the first clinical integration of a prototype device based on integrated auto-fluorescence imaging and Raman spectroscopy (Fast Raman device) for intra-operative assessment of surgical margins during Mohs micrographic surgery of basal cell carcinoma (BCC). Fresh skin specimens from 112 patients were used to optimise the tissue pre-processing and the Fast Raman algorithms to enable an analysis of complete Mohs layers within 30 minutes. The optimisation allowed >95% of the resection surface area to be investigated (including the deep and epidermal margins). The Fast Raman device was then used to analyse skin layers excised from the most relevant anatomical sites (nose, temple, eyelid, cheek, forehead, eyebrow and lip) and to detect the three main types of BCC (nodular, superficial and infiltrative). These results suggest that the Fast Raman technique is a promising tool to provide an objective diagnosis “tumour clear yes/no” during Mohs surgery of BCC. This clinical integration study is a key step towards a larger scale diagnosis test accuracy study to reliably determine the sensitivity and specificity in a clinical setting.

Melanoma is a pigmented type of skin cancer, which has the highest mortality of all skin cancers. Because of the low clinical diagnostic accuracy for melanoma, an objective tool is needed to assist clinical assessment of skin lesions that are suspected of (early) melanoma. The aim of this study was to identify spectral differences in the CH region of HWVN (high-wavenumber) Raman spectra between melanoma and benign melanocytic lesions clinically suspected of melanoma. We used these spectral differences to explore preliminary classification models to distinguish melanoma from benign melanocytic lesions. Data from 82 freshly excised melanocytic lesions clinically suspected of melanoma were measured using an in-house built Raman spectrometer, which has been optimized for measurements on pigmented skin lesions (excitation wavelength 976 nm and a wavelength range of the Raman signal 1340-1540 nm). Clear spectral differences were observed between melanoma and benign melanocytic lesions. These differences can be assigned mainly to the symmetric CH2, stretching vibrations of lipids. Our results show that the Raman bands between 2840 and 2930 cm(-1) have increased intensity for melanoma when compared to benign melanocytic lesions, suggesting an increase in lipid content in melanoma. These results demonstrate that spectroscopic information in the CH-stretching region of HWVN Raman spectra can discriminate melanoma from benign melanocytic lesions that are often clinically misdiagnosed as melanoma and that Raman spectroscopy has the potential to provide an objective clinical tool to improve the clinical diagnostic accuracy of skin lesions suspected of melanoma.

A Raman tissue spectrum is a quantitative representation of the overall molecular composition of that tissue. Raman spectra are often used as tissue fingerprints without further interpretation of the specific information that they contain about the tissue's molecular composition. In this study, we analyzed the differences in molecular composition between oral cavity squamous cell carcinoma (OCSCC) and healthy tissue structures in tongue, based on their Raman spectra. A total of 1087 histopathologically annotated spectra (142 OCSCC, 202 surface squamous epithelium, 61 muscle, 65 adipose tissue, 581 connective tissue, 26 gland, and 10 nerve) were obtained from Raman maps of 44 tongue samples from 21 patients. A characteristic, average spectrum of each tissue structure was fitted with a set of 55 pure-compound reference spectra, to define the best library of fit-spectra. Reference spectra represented proteins, lipids, nucleic acids, carbohydrates, amino acids and other miscellaneous molecules. A non-negative least-squares algorithm was used for fitting. Individual spectra per histopathological annotation were then fitted with this selected library in order to determine the molecular composition per tissue structure. The spectral contribution per chemical class was calculated. The results show that all characteristic tissue-type spectra could be fitted with a low residual of

We present a fully-automated device combining auto-fluorescence imaging and Raman spectroscopy capable of investigating skin resection margins in 30 minutes. The table top-device can easily fit in a surgery theatre.

Pigmented tissues are inaccessible to Raman spectroscopy using visible laser light because of the high level of laser-induced tissue fluorescence. The fluorescence contribution to the acquired Raman signal can be reduced by using an excitation wavelength in the near infrared range around 1000 nm. This will shift the Raman spectrum above 1100 nm, which is the principal upper detection limit for silicon-based CCD detectors. For wavelengths above 1100 nm indium gallium arsenide detectors can be used. However, InGaAs detectors have not yet demonstrated satisfactory noise level characteristics for demanding Raman applications. We have tested and implemented for the first time a novel sensitive InGaAs imaging camera with extremely low readout noise for multichannel Raman spectroscopy in the short-wave infrared (SWIR) region. The effective readout noise of two electrons is comparable to that of high quality CCDs and two orders of magnitude lower than that of other commercially available InGaAs detector arrays. With an in-house built Raman system we demonstrate detection of shot-noise limited high quality Raman spectra of pigmented samples in the high wavenumber region, whereas a more traditional excitation laser wavelength (671 nm) could not generate a useful Raman signal because of high fluorescence. Our Raman instrument makes it possible to substantially decrease fluorescence background and to obtain high quality Raman spectra from pigmented biological samples in integration times well below 20 s. Copyright © 2015 John Wiley & Sons, Ltd.

Oncological applications of Raman spectroscopy have been contemplated, pursued, and developed at academic level for at least 25 years. Published studies aim to detect pre-malignant lesions, detect cancer in less invasive stages, reduce the number of unnecessary biopsies and guide surgery towards the complete removal of the tumour with adequate tumour resection margins. This review summarizes actual clinical needs in oncology that can be addressed by spontaneous Raman spectroscopy and it provides an overview over the results that have been published between 2007 and 2017. An analysis is made of the current status of translation of these results into clinical practice. Despite many promising results, most of the applications addressed in scientific studies are still far from clinical adoption and commercialization. The main hurdles are identified, which need to be overcome to ensure that in the near future we will see the first Raman spectroscopy-based solutions being used in routine oncologic diagnostic and surgical procedures.

Background/Aims: Sensitive skin (SS), a frequently reported condition in the Western world, has been suggested to be underlined by an impaired skin barrier. The aim of this study was to investigate the skin barrier molecular composition in SS subjects using confocal Raman microspectroscopy (CRS), and to compare it with that of non-SS (NSS) individuals as well as atopic dermatitis (AD) and allergic rhinoconjunctivitis (AR) subjects, who frequently report SS. Methods: Subjects with SS (n = 29), NSS (n = 30), AD (n = 11), and AR (n = 27) were included. Stratum corneum (SC) thickness, water, ceramides/fatty acids, and natural moisturizing factor (NMF) were measured by CRS along with transepidermal water loss and capacitance on the ventral forearm, thenar, and cheek. Sebum levels were additionally measured on the forearm and cheek. Results: No differences between SS and NSS subjects were found regarding SC thickness, water, and NMF content, yet a trend towards lower ceramides/fatty acids was observed in the cheek. Compared to AD subjects, the SS group showed higher ceramides/fatty acid content in the forearm, whereas no differences emerged with AR. The correlation of macroscopic biophysical techniques and CRS was weak, yet CRS confirmed the well-known lower content of NMF and water, and thinner SC in subjects with filaggrin mutations. Conclusion: The skin barrier in SS is not impaired in terms of SC thickness, water, NMF, and ceramides/fatty acid content. The failure of biophysical techniques to follow alterations in the molecular composition of the skin barrier revealed by CRS emphasizes a strong need in sensitive and specific tools for in vivo skin barrier analysis.

Background The barrier function of the skin is primarily provided by the stratum corneum (SC), the outermost layer of the skin. Skin barrier impairment is thought to be a primary factor in the pathogenesis of atopic eczema (AE). Filaggrin is an epidermal barrier protein and common mutations in the filaggrin gene strongly predispose for AE. However, the role of filaggrin mutations in the decreased skin barrier in AE is not fully understood. It was recently shown that changes in SC lipid composition and organization play a role in the reduced skin barrier in AE. Objectives To determine whether the lipid/protein ratio and the total dry SC mass per surface area are related to the skin barrier function of controls and patients with AE. Methods A case-control study was performed to compare nonlesional and lesional skin of AE with skin of controls. The dry SC mass was determined by tape-stripping and Squamescan™. The ratio between lipid and protein bands in the Raman spectrum was used to determine the lipid/protein ratio. Skin barrier function was assessed by transepidermal water loss. Results The results show that the dry SC mass per skin area is altered only in lesional SC of patients with AE compared with control subjects. The observed reduction in the lipid/protein ratio in SC of patients with AE was more pronounced, both in lesional and nonlesional SC and correlated strongly with the skin barrier function and disease severity. Conclusions The lipid/protein ratio plays a role in the reduced skin barrier function in AE. What's already known about this topic? Filaggrin mutations are a predisposing factor for atopic eczema (AE). In nonlesional and lesional stratum corneum (SC) of patients with AE the intercellular lipid composition and organization are altered. Changes in the SC lipid properties correlate with a reduced skin barrier function, but are not associated with filaggrin mutations. What does this study add? The dry SC mass, being a measure for SC thickness, is only lower in lesional AE SC compared with SC from controls. The lipid/protein ratio in SC is reduced in both nonlesional and lesional SC of patients with AE and strongly correlates with the impaired skin barrier function and disease severity of patients with AE. The lipid/protein ratio plays a more important role in the impaired skin barrier of AE than the SC thickness.

Background: Natural moisturizing factor (NMF) is used as genotypic and phenotypic biomarker in diagnostics. This study is a side-to-side comparison of two different methods to determine NMF in atopic dermatitis patients: Raman microspectroscopy and stratum corneum tape stripping followed by HPLC.Results: Measured NMF values were significantly correlated (R2 = .61; p

Loss-of-function mutations in the gene coding for filaggrin are the single most important risk factor for development of atopic dermatitis and associated allergic rhinitis and asthma. Filaggrin is enzymatically degraded to natural moisturizing factor (NMF) in the stratum corneum (SC). In vivo Raman spectra of human skin can be used to quantify the NMF concentration in SC and thereby identify carriers of a loss-of-function mutation in the gene coding for filaggrin, which results in decreased NMF content. Here, we demonstrate that strongly reduced Raman spectral information is sufficient to make this differentiation. This is an important step towards development of a dedicated diagnostic device of reduced complexity, size and cost as compared to current state-of-the-art Raman equipment. A genetic algorithm was used to select the spectral regions needed to classify skin based on normal or reduced NMF content in SC. Using the NMF content based on full spectral information as gold standard, only four Raman regions were required to create a linear discriminant analysis model that can differentiate between skin with low NMF and skin with normal NMF with a prediction accuracy of 93 %. Copyright © 2013 John Wiley & Sons, Ltd.

__Background__ Penetration, autoxidation and _N_-acetylation of _p_-phenylenediamine (PPD) have been studied _in vitro_ and _ex vivo_. However, a clear understanding of _in vivo_ PPD penetration and the formation of PPD derivatives is lacking. __Objectives__ To obtain insights into the _in vivo_ penetration, clearance and formation of PPD derivatives in human skin. __Methods__ Patch test chambers containing PPD 1% pet. were applied to the forearms of two human volunteers, with increasing application times. Non-invasive Raman microspectroscopy was used for detection of PPD (derivatives) in skin at several follow-up times. __Results__ Application of a PPD 1% pet. patch for 30 min resulted in substantial amounts of PPD in the stratum corneum of 90mg PPD/g keratin. PPD contents were highest after three applications for 1 h each (330mgPPD/g keratin), followed by single applications for 2 h40 min,2 h, and23 h. The PPD half-time in the skinwas3 h.No spectral contributions of Bandrowski’s base, monoacetyl-PPD and diacetyl-PPD were detected. __Conclusions__ We have gained insights into the _in vivo_ penetration of PPD in human skin by using non-invasive Raman spectroscopy. Penetration into the skin was fast, and the PPD concentrations detected in the stratum corneum were high.PPDwas detected in both the stratum corneum and the viable epidermis. Oxidized or acetylated PPD derivatives could not be detected.

Adequate resection of oral cavity squamous cell carcinoma (OCSCC) means complete tumor removal with a clear margin of more than 5 mm. For OCSCC, 85% of the surgical resections appear inadequate. Raman spectroscopy is an objective and fast tool that can provide real-time information about the molecular composition of tissue and has the potential to provide an objective and fast intraoperative assessment of the entire resection surface. A previous study demonstrated that OCSCC can be discriminated from healthy surrounding tissue based on the higher water concentration in tumor. In this study, we investigated how the water concentration changes across the tumor border toward the healthy surrounding tissue on freshly excised specimens from the oral cavity. Experiments were performed on tissue sections from 20 patients undergoing surgery for OCSCC. A transition from a high to a lower water concentration, from tumor (76% ± 8% of water) toward healthy surrounding tissue (54% ± 24% of water), takes place over a distance of about 4 to 6 mm across the tumor border. This was accompanied by an increase of the heterogeneity of the water concentration in the surrounding healthy tissue. The water concentration distributions between the regions were significantly different (P < 0.0001). This new finding highlights the potential of Raman spectroscopy for objective intraoperative assessment of the resection margins. Cancer Res; 76(20); 5945–53. ©2016 AACR.

The stratum corneum (SC) is the main barrier of the skin, offering a first line of defence against external pathogens and insults. The SC of patients with atopic dermatitis (AD) has underlying barrier deficiencies, even in non-lesional sites. A defective SC barrier is more vulnerable to environmental insults, which trigger immune reactions, further deteriorating the barrier. [...]

This paper describes a new in vivo Raman probe that allows investigation of areas of the body that are otherwise difficult to access. It is coupled to a previously described commercially available in vivo Raman spectrometer that samples the skin through an optical flat. In the work presented here, the laser light emerges from a smaller pen-shaped probe. It thus works on the same principles as the original spectrometer, while its relative performance in terms of signal-to-noise ratio of the spectra and obtained spatial resolution is only slightly diminished. It allows the window to be placed against the subject in more curved and recessed areas of subject's body and also for them to be more comfortable while the measurements take place. Results from three areas of the body that have previously been very difficult to study are described, the mouth, axilla, and scalp. Results from the scalp and axilla strata cornea (SC) show significant differences from the “normal” SC of the volar forearm. For instance, the scalp is observed to have lower amounts of natural moisturizing factors (NMF) compared to the volar forearm within the same subjects. Also for both the axilla and scalp the lipids show a change in order as compared to the lipids in the volar forearm and also differences from each other. The potential significance of these observations is discussed. Further, we show how we can probe the mouth, in this case observing the presence of the astringent tea polyphenol epigallocatechin gallate within the oral mucosa.

An integrated arrayed-waveguide grating (AWG) fabricated in silicon oxynitride (SiON) technology is used as the wavelength separation and selection element for Raman spectroscopy. With a spectral resolution of 5.5 nm and a free spectral range of 215 nm, the AWG enables wavelength separation of the full Raman fingerprint region in a single diffraction order without order overlap. It is demonstrated that Raman signal detection of cyclohexane with the AWG is feasible and that the information on position and relative intensities of the Raman bands of cyclohexane are preserved. A detailed description of the signal processing methodology to obtain AWG-based Raman measurements corrected for instrument response is presented. The Raman measurement based on the integrated intensity output of the device corresponds well to the expected relative intensity channel output of the AWG. Raman information based on band intensity ratios from the AWG channels is also preserved compared with the spectral integration of bands, with a maximum relative error of 9% attributed to some light that might have leaked from the channels to the device substrate. These results are a step towards the realization of a technology suitable for dedicated, low-cost, miniaturized Raman devices.

A hallmark of atopic eczema (AE) is skin barrier dysfunction. Lipids in the stratum corneum (SC), primarily ceramides, fatty acids, and cholesterol, are crucial for the barrier function, but their role in relation to AE is indistinct. Filaggrin is an epithelial barrier protein with a central role in the pathogenesis of AE. Nevertheless, the precise causes of AE-associated barrier dysfunction are largely unknown. In this study, a comprehensive analysis of ceramide composition and lipid organization in nonlesional SC of AE patients and control subjects was performed by means of mass spectrometry, infrared spectroscopy, and X-ray diffraction. In addition, the skin barrier and clinical state of the disease were examined. The level of ceramides with an extreme short chain length is drastically increased in SC of AE patients, which leads to an aberrant lipid organization and a decreased skin barrier function. Changes in SC lipid properties correlate with disease severity but are independent of filaggrin mutations. jlr We demonstrate for the fi rst time that changes in ceramide chain length and lipid organization are directly correlated with the skin barrier defects in nonlesional skin of AE patients. We envisage that these insights will provide a new therapeutic entry in therapy and prevention of AE.-Janssens, M., J. van Smeden, G. S. Gooris, W. Bras, G. Portale, P. J. Caspers, R. J. Vreeken, T. Hankemeier, S. Kezic, R. Wolterbeek, A. P. Lavrijsen, and J. A. Bouwstra. Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients. J. Lipid Res. 2012. 53: 2755-2766.

Caspase-14 is a protease that is mainly expressed in suprabasal epidermal layers and activated during keratinocyte cornification. Caspase-14-deficient mice display reduced epidermal barrier function and increased sensitivity to UVB radiation. In these mice, profilaggrin, a protein with a pivotal role in skin barrier function, is processed correctly to its functional filaggrin (FLG) repeat unit, but proteolytic FLG fragments accumulate in the epidermis. In wild-type stratum corneum, FLG is degraded into free amino acids, some of which contribute to generation of the natural moisturizing factors (NMFs) that maintain epidermal hydration. We found that caspase-14 cleaves the FLG repeat unit and identified two caspase-14 cleavage sites. These results indicate that accumulation of FLG fragments in caspase-14(-/-) mice is due to a defect in the terminal FLG degradation pathway. Consequently, we show that the defective FLG degradation in caspase-14-deficient skin results in substantial reduction in the amount of NMFs, such as urocanic acid and pyrrolidone carboxylic acid. Taken together, we identified caspase-14 as a crucial protease in FLG catabolism.

We demonstrate the feasibility of short wave infrared (SWIR) spectroscopy combined with tape stripping for depth profiling of lipids and water in the stratum corneum of human skin. The proposed spectroscopic technique relies on differential detection at three wavelengths of 1720, 1750, and 1770 nm, with varying ratio of the lipid-to-water absorption coefficient and an ‘isosbestic point’. Comparison of the data acquired using SWIR spectroscopy with that obtained by a gold standard for non-invasive quantitative molecularspecific skin measurements, namely confocal Raman spectroscopy (CRS), revealed specificity of the proposed modality for water and lipid quantification. At the same time, we provide evidence showing aberrant sensitivity of Corneometer hydration read-outs to the presence of skin surface lipids, and a lack of sensitivity of the Sebumeter when attempting to measure the lipids of the cornified lipid envelope and intracellular lipid layers. We conclude that a spectroscopic SWIR-based spectroscopic method combined with tape stripping has the potential for depth profiling of the stratum corneum water and lipids, due to superior measurement sensitivity and specificity compared to the Corneometer and Sebumeter.

The high-frequency electrical conductance of tape-stripped human skin in vivo can be used to evaluate the hydration profile of Stratum corneum (SC). Tape-stripping provides access to the underlying SC layers, and the conductance of these layers (as measured by the Skicon instrument) correlates well with their water content, as demonstrated by independent confocal Raman spectroscopic measurements. The correlation shows high inter-individual variance and is not linear over the full measurement range of the instrument, but is helpful to discriminate between dry, normal and highly hydrated SC. The depth profile of hydration in tape-stripped SC corresponds to the one in intact SC only if the barrier function of the skin is not impaired. Thus, conductometry of tape-stripped skin must be used in conjunction with a method that allows to estimate the barrier damage inflicted to SC during the tape-stripping procedure, for example, measurement of the trans-epidermal water loss. The methodology described here is simple, rapid and minimally invasive, and it employs commercially available instrumentation that is cheap, portable and easy to use. This approach is applicable to in vivo estimation of the SC hydration in studies in the areas of dermatology, skin care and transdermal drug delivery.

The purpose of this study is to monitor in vivo the effect of chemical penetration enhancers on the delivery of trans-retinol into human skin. Chemical penetration enhancers reversibly alter barrier properties of the SC by disruption of the membrane structures or maximising drug solubility with the skin. So far, most of permeation or penetration experiments are performed in vitro. Raman spectroscopy is uniquely placed to be able to measure biological processes in vivo and this paper shows for the first time that the effect of penetration enhancer on the delivery of trans-retinol can successfully be measured in vivo using this technique. Here, the volar forearm of volunteers was treated with four formulations. One formulation is a highly effective model delivery system identified from ex vivo experiments: trans-retinol in Propylene Glycol (PG)/ethanol, with PG being a well-known and efficient penetration enhancer. The other three formulations are based on 0.3% trans-retinol in Caprylic/Capric Acid Triglyceride (MYRITOL 318), an oil commonly used in skin creams but in two of them a specific penetration enhancer is added. One contains a lipid extractor, Triton X 100, whereas another formulation contains a lipid fluidiser, Oleic Acid. Solutions were applied once and measurements were performed up to 6 h after treatment. Remarkable differences in the delivery of trans-retinol between formulation with or without penetration enhancer can clearly be seen. Moreover, the type of penetration enhancer is also shown to influence the delivery. While using the Oleic Acid, which is a lipid fluidiser, a better delivery of trans-retinol in the skin can be detected. For the first time, the effect of penetration enhancer on the delivery of trans-retinol has been monitored, non invasively in vivo, with time.

In the present study, the distribution of the carotenoids as a marker for the complete antioxidative potential in human skin was investigated before and after the topical application of carotenoids by in vivo Raman spectroscopy with an excitation wavelength of 785 nm. The carotenoid profile was assessed after a short term topical application in 4 healthy volunteers. In the untreated skin, the highest concentration of natural carotenoids was detected in different layers of the stratum corneum (SC) close to the skin surface. After topical application of carotenoids, an increase in the antioxidative potential in the skin could be observed. Topically applied carotenoids penetrate deep into the epidermis down to approximately 24 μm. This study supports the hypothesis that antioxidative substances are secreted via eccrine sweat glands and/or sebaceous glands to the skin surface. Subsequently they penetrate into the different layers of the SC. A graph is presented. Typical Raman spectrum of untreated skin (forearm, 10 μm depth) under the excitation wavelength of 785 nm.

To demonstrate the “skin reservoir” of urea by confocal Raman microspectroscopy in vivo and to evaluate its impact on the non-invasive monitoring of the analyte by reverse iontophoresis. Urea was extracted iontophoretically over a 2-h period across the skin of adult volunteers and patients with chronic kidney disease. Confocal Raman microspectroscopic profiles of skin were recorded before and after 30 min of current application. Urea extraction was higher at the beginning of current passage, but then decreased to achieve stable values after 2 h of iontophoresis. After 30 min of iontophoresis, the Raman spectra highlighted a clear depletion of urea at the surface of the skin. Lactate distribution was also modified both at the surface and deeper into the skin. A source of urea in the skin, unrelated to the concentration circulating in the blood, was strongly suggested by extracted urea flux observed over time and by the Raman spectroscopy. This “urea reservoir” must be removed before systemic urea levels can be non-invasively monitored by reverse iontophoresis.

The objective of this in vitro study was to explore the applicability of Raman spectroscopy to distinguish basal cell carcinoma from its surrounding noncancerous tissue; therefore, identifying possibilities for the development of an in vivo diagnostic technique for tumor border demarcation. Raman spectra were obtained in a two-dimensional grid from unstained frozen sections of 15 basal cell carcinoma specimens. Pseudo-color Raman images were generated by multivariate statistical analysis and clustering analysis of spectra and compared with histopathology. In this way a direct link between histologically identifiable skin layers and structures and their Raman spectra was made. A tissue classification model was developed, which discriminates between basal cell carcinoma and surrounding nontumorous tissue, based on Raman spectra. The logistic regression model, shows a 100% sensitivity and 93% selectivity for basal cell carcinoma. The Raman spectra were, furthermore, used to obtain information about the differences in molecular composition between different skin layers and structures. An interesting finding was that in four samples of nodular basal cell carcinoma, the collagen signal contribution in spectra of dermis close to a basal cell carcinoma, was markedly reduced. The study demonstrates the sensitivity of Raman spectroscopy to biochemical changes in tissue accompanying malignancy, resulting in a high accuracy when discriminating between basal cell carcinoma and noncancerous tissue.

The stratum corneum (SC) barrier typically consists of layers of corneocytes embedded in a lipid continuum that regulates barrier function. The lipid domain containing ceramides, cholesterol, and free fatty acids provides the major pathway for most drugs permeating across SC. Penetration enhancers diminish the SC barrier function. The classic enhancer is dimethyl sulfoxide (DMSO). Its mechanisms of action remain unclear, although DMSO disrupts lipid organisation and may displace protein-bound water. Here we use confocal Raman spectroscopy to probe molecular interactions between a finite (depleting) dose of DMSO and SC, as functions of depth and time, providing novel information about residence time and location of DMSO in human SC in vivo

The application of in vivo Raman spectroscopy for clinical diagnosis demands dedicated software that can perform the necessary signal processing and subsequent (multivariate) data analysis, enabling clinically relevant parameters to be extracted and made available in real time. Here we describe the design and implementation of a software package that allows for real-time signal processing and data analysis of Raman spectra. The design is based on automatic data exchange between Grams, a spectroscopic data acquisition and analysis program, and Matlab, a program designed for array-based calculations. The data analysis software has a modular design providing great flexibility in developing custom data analysis routines for different applications. The implementation is illustrated by a computationally demanding application for the classification of skin spectra using principal component analysis and linear discriminant analysis. Copyright © 2002 John Wiley & Sons, Ltd.