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 layer's 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.

Photothermomechanical Nanopump: A Flow-Through Plasmonic Sensor at the Fiber Tip.

Optical fibers equipped with plasmonic flow sensors at their tips are fabricated and investigated as photothermomechanical nanopumps for the active transport of target analytes to the sensor surface. The nanopumps are prepared using a bottom-up strategy: i.e., by sequentially stacking a monolayer of a thermoresponsive polymer and a plasmonic nanohole array on an optical fiber tip. The temperature-dependent collapse and swelling of the polymer is used to create a flow-through pumping mechanism. The heat required for pumping is generated by exploiting the photothermal effect in the plasmonic nanohole array upon irradiation with laser light (405 nm). Simultaneous detection of analytes by the plasmonic sensor is achieved by monitoring changes in its optical response at longer wavelengths (∼500-800 nm). Active mass transport by pumping through the holes of the plasmonic nanohole array is visualized by particle imaging velocimetry. Finally, the performance of the photothermomechanical nanopumps is investigated for two types of analytes, namely nanoscale objects (gold nanoparticles) and molecules (11-mercaptoundecanoic acid). In the presence of the pumping mechanism, a 4-fold increase in sensitivity was observed compared to the purely photothermal effect, demonstrating the potential of the presented photothermomechanical nanopumps for sensing applications.

Noninvasive measurement of the 308 nm LED-based UVB protection factor of sunscreens.

The current method for determining the sun protection factor (SPF) requires erythema formation. Noninvasive alternatives have recently been suggested by several groups. Our group previously developed a functional sensor based on diffuse reflectance measurements with one UVB LED, which was previously evaluated on pig ear skin. Here we present the results of a systematic in vivo study using 12 sunscreens on 10 volunteers (skin types [ST] I-III). The relationship of the UVB-LED reflectance of unprotected skin and melanin index was determined for each ST. The spatial variation of the reflectance signal of different positions was analyzed and seems to be mainly influenced by sample inhomogeneity except for high-protection factors (PFs) where signal levels are close to detection noise. Despite the low-signal levels, a correlation of the measured LED-based UVB PF with SPF reference values from test institutes with R2 = 0.57 is obtained, suggesting a strong relationship of SPF and LED-based UVB-PF. Measured PFs tend to be lower for increasing skin pigmentation. The sensor design seems to be suitable for investigations where a fast measurement of relative changes of PFs, such as due to inhomogeneous application, bathing and sweating, is of interest.

Evaluation of detection distance-dependent reflectance spectroscopy for the determination of the sun protection factor using pig ear skin.

Determination of sun protection factors (SPFs) is currently an invasive method, which is based on erythema formation (phototest). Here we describe an optical setup and measurement methodology for the determination of SPFs based on diffuse reflectance spectroscopy, which measures UV-reflectance spectra at 4 distances from the point of illumination. Due to a high spatial variation of the reflectance data, most likely due to inhomogeneities of the sunscreen distribution, data of 50 measurement positions are averaged. A dependence of the measured SPF on detection distance is significant for 3 sunscreens, while being inconclusive for 2 sunscreens due to high inter-sample variations. Using pig ear skin samples (n=6), the obtained SPF of 5 different commercial sunscreens corresponds to the SPF values of certified test institutes in 3 cases and is lower for 2 sunscreens of the same manufacturer, suggesting a formulation specific reason for the discrepancy. The results demonstrate that the measurement can be performed with a UV dose below the minimal erythema dose. We conclude the method may be considered as a potential noninvasive in vivo alternative to the invasive in vivo phototest, but further tests on different sunscreen formulations are still necessary.

Nonscanning large-area Raman imaging for ex vivo/in vivo skin cancer discrimination.

Imaging Raman spectroscopy can be used to identify cancerous tissue. Traditionally, a step-by-step scanning of the sample is applied to generate a Raman image, which, however, is too slow for routine examination of patients. By transferring the technique of integral field spectroscopy (IFS) from astronomy to Raman imaging, it becomes possible to record entire Raman images quickly within a single exposure, without the need for a tedious scanning procedure. An IFS-based Raman imaging setup is presented, which is capable of measuring skin ex vivo or in vivo. It is demonstrated how Raman images of healthy and cancerous skin biopsies were recorded and analyzed.

Determination of oxygen saturation and hematocrit of flowing human blood using two different spectrally resolving sensors.

The blood parameters oxygen saturation and hematocrit were determined by two different spectral sensors using reflectance spectra from 550 to 900 nm and partial transmission spectra centered at 660 nm. The spectra were analyzed by the method of partial least squares. One sensor consists of a miniature integrating sphere, while the other was fiber-guided. The results show that the geometry of the sensors and different blood flows do not influence the spectral analysis significantly. Independent of the sensor geometry, both hematocrit and oxygen saturation could be determined with an absolute predicted root mean square error of less than 3%. Furthermore, the analysis showed that hematocrit prediction requires eight wavelength regions and oxygen saturation prediction requires four wavelength regions using reflectance spectroscopy. This implies that if the measurement is restricted to reflectance, a spectrometer is indispensable for determining both blood parameters. Hematocrit determination could be improved using reflectance measurements in combination with transmission.

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 non-invasive 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 misclassification in tumor discrimination.

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

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.

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.

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.

Evaluation of a novel noncontact spectrally and spatially resolved reflectance setup with continuously variable source-detector separation using silicone phantoms.

We present a new variant of a noncontact, oblique incidence spatially resolved reflectance setup. The continuously variable source detector separation enables adaptation to high and low albedo samples. Absorption (µ(a)) and reduced scattering coefficients (µ(') (s)) are determined in the wavelength range of 400-1000 nm using a lookup table, calculated by a Monte Carlo simulation of the light transport. The method is characterized by an silicone phantom study covering a wide parameter range 0.01 mm(-1) ≤ µ(a) ≤ 2.5 mm(-1) and 0.2 mm(-1) ≤ µ(') (s) ≤ 10 mm(-1), which includes the optical parameters of tissue in the visible and near infrared. The influence of the incident angle and the detection aperture on the simulated remission was examined. Using perpendicular incidence and 90-deg detection aperture in the Monte Carlo simulation in contrast to the experimental situation with 30-deg incidence and 4.6-deg detection aperture is shown to be valid for the parameter range µ(') (s) > 1 mm(-1) and µ(a) < 1.2 mm(-1). A Mie calculation is presented, showing that a decreasing reduced scattering coefficient for increasing absorption can be the consequence of real physics instead of cross talk.

Quantitative Raman spectroscopy in turbid media.

Intrinsic Raman spectra of biological tissue are distorted by the influences of tissue absorption and scattering, which significantly challenge signal quantification. A combined Raman and spatially resolved reflectance setup is introduced to measure the absorption coefficient micro(a) and the reduced scattering coefficient micro(s) (') of the tissue, together with the Raman signals. The influence of micro(a) and micro(s) (') on the resonance Raman signal of beta-carotene is measured at 1524 cm(-1) by tissue phantom measurements and Monte Carlo simulations for micro(a)=0.01 to 10 mm(-1) and micro(s) (')=0.1 to 10 mm(-1). Both methods show that the Raman signal drops roughly proportional to 1 micro(a) for micro(a)>0.2 mm(-1) in the measurement geometry and that the influence of micro(s) (') is weaker, but not negligible. Possible correction functions dependent on the elastic diffuse reflectance are investigated to correct the Raman signal for the influence of micro(a) and micro(s) ('), provided that micro(a) and micro(s) (') are measured as well. A correction function based on the Monte Carlo simulation of Raman signals is suggested as an alternative. Both approaches strongly reduce the turbidity-induced variation of the Raman signals and allow absolute Raman scattering coefficients to be determined.