Ingo Gersonde

Chemometric Determination of Blood Parameters Using Visible–Near-Infrared Spectra

Visible and near-infrared (NIR) integrating sphere spectroscopy and chemometric multivariate linear regression were applied to determine hematocrit (HCT) and oxygen saturation (SatO2) of circulating human blood. Diffuse transmission, total transmission, and diffuse reflectance were measured and the partial least squares method (PLS) was used for calibration considering different wavelength ranges and selected optical measurement parameters. HCT and SatO2 were changed independently. Each parameter was adjusted to different levels and four designs with blood from different donors were carried out for the calibration with PLS. The calibration included the changes in hemolysis as well as inter-individual differences in cell dimensions and hemoglobin content. At a sample thickness of 0.1 mm the HCT and SatO2 were predicted with a root mean square error (PRMSE) of 1.4% and 2.5%, respectively, using transmission and reflectance spectra and the full Vis-NIR range. Using only diffuse NIR reflectance spectroscopy and a sample thickness of 1 mm, HCT and SatO2 could be predicted with a PRMSE of 1.9% and 2.8%, respectively. Prediction of hemolysis was also possible for one blood sample with a PRMSE of 0.8% and keeping HCT and SatO2 stable with a PRMSE of 0.03%.

Radical Protection by Sunscreens in the Infrared Spectral Range

One essential reason for skin ageing is the formation of free radicals by excessive or unprotected sun exposure. Recently, free radical generation in skin has been shown to appear not only after irradiation in the UV wavelength range but also in the infrared (IR) spectral range. Sunscreens are known to protect against radicals generated by UV radiation; however, no data exist for those generated by IR radiation. This paper has investigated four different, commercially available sunscreens and one COLIPA standard with regard to radical formation in the skin after IR irradiation, using electron paramagnetic resonance spectroscopy. The use of sunscreens has led to reduced amounts of radicals compared to untreated skin. Furthermore, absorption and scattering properties and the radical protection factor of the formulations were determined to investigate their influence on the radical protection of the skin. None of these formulations contained an optical absorber in the IR range. The protection efficiency of the sunscreens was shown as being induced by the high scattering properties of the sunscreens, as well as the antioxidants contained in the formulations.

Radical Protection by Differently Composed Creams in the UV/VIS and IR Spectral Ranges

Modern sunscreens are well suited to provide sufficient protection in the UV range because the filter substances absorb or scatter UV radiation. Although up to 50% of radicals are formed in the visible and infrared spectral range during solar radiation protection strategies are not provided in this range. Previous investigations of commercially available products have shown that in addition to physical filters, antioxidants (AO) are necessary to provide protective effects in the infrared range by neutralizing already formed radicals. In this study, the efficacy of filter substances and AO to reduce radical formation in both spectral ranges was investigated after UV/VIS or IR irradiation. Optical properties and radical protection were determined for the investigated creams. It was found that organic UV filters lower radical formation in the UV/VIS range to 35% compared to untreated skin, independent of the presence of AO. Further reduction to 14% was reached by addition of 2% physical filters, whereas physical filters alone were ineffective in the UV/VIS range due to the low concentration. In contrast, this filter type reduced radical formation in the IR range significantly to 65%; similar effects were aroused after application of AO. Sunscreens which contain organic UV filters, physical filters and AO ensure protection in the complete solar spectrum.

Influence of tissue absorption and scattering on the depth dependent sensitivity of Raman fiber probes investigated by Monte Carlo simulations

We present a Monte Carlo model, which we use to calculate the depth dependent sensitivity or sampling volume of different single fiber and multi-fiber Raman probes. A two-layer skin model is employed to investigate the dependency of the sampling volume on the absorption and reduced scattering coefficients in the near infrared wavelength range (NIR). The shape of the sampling volume is mainly determined by the scattering coefficient and the wavelength dependency of absorption and scattering has only a small effect on the sampling volume of a typical fingerprint spectrum. An increase in the sampling depth in nonmelanoma skin cancer, compared to normal skin, is obtained.

In vivo study for the discrimination of cancerous and normal skin using fibre probe-based Raman spectroscopy.

Raman spectroscopy has proved its capability as an objective, non‐invasive tool for the detection of various melanoma and non‐melanoma skin cancers (NMSC) in a number of studies. Most publications are based on a Raman microspectroscopic ex vivo approach. In this in vivo clinical evaluation, we apply Raman spectroscopy using a fibre‐coupled probe that allows access to a multitude of affected body sites. The probe design is optimized for epithelial sensitivity, whereby a large part of the detected signal originates from within the epidermal layers depth down to the basal membrane where early stages of skin cancer develop. Data analysis was performed on measurements of 104 subjects scheduled for excision of lesions suspected of being malignant melanoma (MM) (n = 36), basal cell carcinoma (BCC) (n = 39) and squamous cell carcinoma (SCC) (n = 29). NMSC were discriminated from normal skin with a balanced accuracy of 73% (BCC) and 85% (SCC) using partial least squares discriminant analysis (PLS‐DA). Discriminating MM and pigmented nevi (PN) resulted in a balanced accuracy of 91%. These results lie within the range of comparable in vivo studies and the accuracies achieved by trained dermatologists using dermoscopy. Discrimination proved to be unsuccessful between cancerous lesions and suspicious lesions that had been histopathologically verified as benign by dermoscopy.

Enhancement of skin radical scavenging activity and stratum corneum lipids after the application of a hyperforin-rich cream

Hyperforin is well-known for its anti-inflammatory, anti-tumor, anti-bacterial, and antioxidant properties. The application of a hyperforin-rich verum cream could strengthen the skin barrier function by reducing radical formation and stabilizing stratum corneum lipids. Here, it was investigated whether topical treatment with a hyperforin-rich cream increases the radical protection of the skin during VIS/NIR irradiation. Skin lipid profile was investigated applying HPTLC on skin lipid extracts. Furthermore, the absorption- and scattering coefficients, which influence radical formation, were determined. 11 volunteers were included in this study. After a single cream application, VIS/NIR-induced radical formation could be completely inhibited by both verum and placebo showing an immediate protection. After an application period of 4weeks, radical formation could be significantly reduced by 45% following placebo application and 78% after verum application showing a long-term protection. Furthermore, the skin lipids in both verum and placebo groups increased directly after a single cream application but only significantly for ceramide [AP], [NP1], and squalene. After long-term cream application, concentration of cholesterol and the ceramides increased, but no significance was observed. These results indicate that regular application of the hyperforin-rich cream can reduce radical formation and can stabilize skin lipids, which are responsible for the barrier function.

Perturbation Factors in the Clinical Handling of a Fiber-Coupled Raman Probe for Cutaneous in Vivo Diagnostic Raman Spectroscopy:

The application of fiber-coupled Raman probes for the discrimination of cancerous and normal skin has the advantage of a noninvasive in vivo application, easy clinical handling, and access to the majority of body sites, which would otherwise be limited by stationary Raman microscopes. Nevertheless, including optical fibers and miniaturizing optical components, as well as measuring in vivo, involves the sensibility to external perturbation factors that could introduce artifacts to the acquired Raman spectra and thereby potentially reduce classification performance. In this study, typical perturbation factors of Raman measurements with a Raman fiber probe, optimized for clinical in vivo discrimination of skin cancer, were investigated experimentally. Measurements were performed under standardized conditions in clinical settings in vivo on human skin, as well as ex vivo on porcine ears. Raman spectra were analyzed in the fingerprint region between 1150 and 1730 cm−1 using principal component analysis. The largest artifacts in the Raman spectra were found in measurements performed under the influence of strong ambient light conditions as well as after miscellaneous pre-treatments to the skin, such as use of a permanent marker or a sunscreen. Minor influences were also found in measurements using H2O immersion and when varying the probe contact force. The effect of reasonable variation of the fiber-bending radius was found to be of negligible impact. The influence of measurements on hairy or sun-exposed body sites, as well as inter-subject variation, was also investigated. The presented results may serve as a guide to avoid negative effects during the process of data acquisition and so avoid misclassifcation in tumor discrimination.

Evaluation of Raman spectroscopic macro raster scans of native cervical cone biopsies using histopathological mapping

Abstract. Raman spectroscopy based discrimination of cervical precancer and normal tissue has been shown previously in vivo with fiber probe based measurements of colposcopically selected sites. With a view to developing in vivo large area imaging, macro raster scans of native cervical cone biopsies with an average of 200 spectra per sample are implemented (n=16). The diagnostic performance is evaluated using histopathological mapping of the cervix surface. Different data reduction and classification methods (principal component analysis, wavelets, k-nearest neighbors, logistic regression, partial least squares discriminant analysis) are compared. Using bootstrapping to estimate confidence intervals for sensitivity and specificity, it is concluded that differences among different spectra classification procedures are not significant. The classification performance is evaluated depending on the tissue pathologies included in the analysis using the average performance of different classification procedures. The highest sensitivity (91%) and specificity (81%) is obtained for the discrimination of normal squamous epithelium and high-grade precancer. When other non-high-grade tissue sites, such as columnar epithelium, metaplasia, and inflammation, are included, the diagnostic performance decreases.

Fibre-optic IR-spectroscopy for biomedical diagnostics

The use of microscopy is a valuable means of gaining vital information for medical diagnostics. Due to a number of recent technological developments advances have been made in IR microscopy and in particular, rapid detection methods. Microscopic examination methods usually involve sampling followed by a method of sample purification or preparation. The advantages of the IR analytical method are that it is based on a direct, non‒destructive measurement of sample material and that the resulting IR spectra provide extensive and specific information about the sample composition and structure. The course of a disease can lead to either formation or loss of organic compounds in metabolism as well as changes within the biological matrix. Corresponding changes can also be expected in the IR‒signature in view to the grading of alteration. Our preliminary IR microscopic investigations compared diseased and healthy tissue samples individually and basic information was obtained about the tissue specific spectral signature, taking account of biological variance. Human tissue samples taken from the colon were used for these studies. Given the number of endoscopic applications used in minimally invasive medicine, we hope to establish the IR fibre based procedure as an optical biopsy method for tissue diagnostics. The aqueous environment as well as the IR radiation source, signal detection and the flexible wave guide type will be a limiting factor for an IR system. The hygiene requirements are particularly high for a fibre based system to be used for in vivo applications.

Correction of Raman signals for tissue optical properties

Raman signals of biological tissues are distorted by the influence of tissue absorption and scattering properties, which significantly challenges signal quantification. We investigated the influence of the tissue optical properties on the resonance Raman signal of β-carotene with tissue phantom measurements and Monte Carlo simulations. Both methods show that the Raman signal drops roughly proportional to 1/μa for absorption coefficients (μa) in the range of 0.1-4mm-1. The influence of the reduced scattering coefficient in the range of 0.1-8mm-1 is less strong, but not negligible. We introduce combined Raman and spatially resolved reflectance measurements to measure both Raman signals and tissue optical properties. The optical properties were deduced from the spatially resolved reflectance measurements by a Monte Carlo-based lookup table (LUT). For the signal correction we applied correction functions derived from the Monte Carlo simulations. We corrected in vivo resonance Raman measurements of carotenoids in caucasian skin (n=9) with respect to the mean optical properties of the group. The magnitude of the average correction effect was found to be 18±11% on the minimal pigmented palm.