Jürgen Helfmann

Optical properties of platelets and blood plasma and their influence on the optical behavior of whole blood in the visible to near infrared wavelength range

The optical parameters absorption coefficient, scattering coefficient, and the anisotropy factor of platelets (PLTs) suspended in plasma and cell-free blood plasma are determined by measuring the diffuse reflectance, total and diffuse transmission, and subsequently by inverse Monte Carlo simulation. Furthermore, the optical behavior of PLTs and red blood cells suspended in plasma are compared with those suspended in saline solution. Cell-free plasma shows a higher scattering coefficient and anisotropy factor than expected for Rayleigh scattering by plasma proteins. The scattering coefficient of PLTs increases linearly with the PLT concentration. The existence of physiological concentrations of leukocytes has no measurable influence on the absorption and scattering properties of whole blood. In summary, red blood cells predominate over the other blood components by two to three orders of magnitude with regard to absorption and effective scattering. However, substituting saline solution for plasma leads to a significant increase in the effective scattering coefficient and therefore should be taken into consideration.

Empirical model functions to calculate hematocrit-dependent optical properties of human blood

The absorption coefficient, scattering coefficient, and effective scattering phase function of human red blood cells (RBCs) in saline solution were determined for eight different hematocrits (Hcts) between 0.84% and 42.1% in the wavelength range of 250-1100 nm using integrating sphere measurements and inverse Monte Carlo simulation. To allow for biological variability, averaged optical parameters were determined under flow conditions for ten different human blood samples. Based on this standard blood, empirical model functions are presented for the calculation of Hct-dependent optical properties for the RBCs. Changes in the optical properties when saline solution is replaced by blood plasma as the suspension medium were also investigated.

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%.

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.

Influence of osmolarity on the optical properties of human erythrocytes

Plasma osmolarity influences the volume and shape of red blood cells (RBCs). The volume change is inversely related to the hemoglobin concentration and as a consequence to the complex refractive index within the cell. These morphological changes can be linked to changes in the optical behavior of the cells. The optical parameters, absorption coefficient μa, scattering coefficient μs, and effective scattering phase function of red blood cells are investigated in dependence on osmolarity in the spectral range from 250 to 1100 nm. Integrating sphere measurements of light transmittance and reflectance in combination with inverse Monte-Carlo simulations are carried out for osmolarities from 225 to 400 mosmol/L. Osmolarity changes have a significant influence on the optical parameters, which can in part be explained by changes in the complex refractive index, cell shape, and cell volume. Spherical forms of RBCs induced by low osmolarity show reduced scattering effects compared to the normal RBC biconcave disk shape. Spinocytes, which are crenated erythrocytes induced by high osmolarity, show the highest scattering effects. Even only a 10% change in osmolarity has a drastic influence on the optical parameters, which appears to be of the same order as for 10% hematocrit and oxygen saturation changes.

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.

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.

Raman spectroscopy for the discrimination of cancerous and normal skin

Abstract: Various studies have shown promising results in using Raman spectroscopy (RS) for the detection of skin cancers. In-vivo evaluations showed similar results to those found by trained dermatologists using dermoscopy, the current clinical practice for skin cancer diagnosis. However, dermoscopy is highly subjective which would make an objective, non-invasive diagnostic method useful. Although successful results were achieved, RS is barely applied in clinical routine yet. This review summarizes studies of Raman spectroscopy for skin cancer diagnosis ex vivo and in vivo. The latter has special demands that often lead to a tradeoff between applicability and classification performance. The necessary steps are explained for instrumentation design, handling, data analysis and clinical testing on groups with a sufficient amount of subjects in order to promote the application of RS in a routine clinical setting. A number of methods are summarized which attempt to overcome the ongoing challenge of reducing large background signals. Modifications of RS by combination with other diagnostic methods are summarized that can give a new perspective to future developments in RS.

Spectral in vivo signature of carotenoids in visible light diffuse reflectance from skin in comparison to ex vivo absorption spectra

Abstract Objective: To increase the carotenoid concentration in skin by drinking carrot juice to generate an optical response and to determine an in vivo absorption spectrum of the carotenoids and compare it to ex vivo absorption spectra. Material and methods: The first author of the presented study consumed carrot juice over a period of several weeks and during this time regularly performed optical reflection measurements on his palm. The spectroscopic measurements were carried out with a fiber-based sensor using a thermal light source. Absorption coefficients have been deduced from the diffuse reflectance data. Measurements were also performed on a β-carotene solution, on carrot juice and its extracted carotenoids. The correlation coefficient between carrot juice intake in liters and the optical skin signal was used to select an optimal wavelength for carotenoid detection (493 nm). Results: An in vivo signal of the carotenoids was found in the spectral range from 400 nm to 580 nm. A 1-l intake of carrot juice resulted in a 0.6% decrease of the diffuse reflectance. The absorption of hemoglobin interferes with the carotenoid signal even though the blood was pressed away. Consequently, a method was used that could lead to the elimination of this disturbance and an in vivo absorption spectrum from the carotenoids in skin was determined by way of trial. The in vivo carotenoid spectrum and that of the carrot juice were both found to a similar extent to be spectrally broader than the absorption spectrum of β-carotene dissolved in cyclohexane. Conclusion: Detection of carotenoids in skin is possible by diffuse reflectance measurements with a simple spectroscopic optical setup, as it is described in this article. As found by comparing different publications, several geometries for illumination and detection support carotenoid quantification. However, the fiber probe described here is not limited to carotenoid detection and it does not need arrangement of several optical elements such as lenses, mirrors or apertures and therefore requiring less effort for development. To eliminate the interference of hemoglobin, the authors suggest the combination of pressure and the described software method. Other publications have reported hemoglobin interference with respect to in vivo carotenoid measurement. As the carotenoids are mainly found in the bloodless epidermis, setups are suggested which use smaller sampling volumes. Zusammenfassung Ziel: Erhöhen der Karotinoidkonzentration in der Haut, um messbare Veränderungen der optischen Eigenschaften der Haut zu erzeugen. Bestimmen eines In-vivo-Absorptionsspektrums der Karotinoide und Vergleich mit Ex-vivo-Absorptionsspektren. Material und Methoden: Über einen Zeitraum von mehreren Wochen nahm der Erstautor der Studie Karottensaft zu sich und führte währenddessen regelmäßige optische Reflektionsmessungen am Handballen durch. Die spektroskopischen Messungen wurden mit einem Faser-Sensor und einer thermischen Lichtquelle durchgeführt. Aus den Reflektionsspektren wurden Absorptionskoeffizienten ermittelt. Weitere Messungen wurden an einer Beta-Carotin-Lösung, an Karottensaft und an dessen extrahierten Karotinoiden durchgeführt. Es wurde eine optimale Wellenlänge für die Karotinoiddetektion (493 nm) bestimmt, bei der die Korrelation zwischen der diffusen Reflektion der Haut und der Menge an getrunkenem Karottensaft am höchsten war. Ergebnisse: Im Wellenlängenbereich von 400 bis 580 nm wurde ein In-vivo-Signal der Karotinoide gemessen. Pro Aufnahme von 1 l Karottensaft verringerte sich die diffuse Reflektion um 0.6%. Allerdings interferiert die Absorption von Hämoglobin mit der Karotinoidmessung, obwohl das Blut durch Anpressen des Messkopfes weggedrückt wurde. Folglich wurde eine Methode verwendet, die eine Korrektur dieses Störeinflusses ermöglichen könnte. Versuchsweise wurde hiermit ein In-vivo-Absorptionsspektrum der Karotinoide ermittelt. Es wurde festgestellt, dass das Signal der Karotinoide aus dem Karottensaft und der Haut in einem ähnlichen Ausmaß spektral breiter ist als das in Cyclohexan gelöste Beta-Carotin. Fazit: Karotinoide in der Haut können mit dem in diesem Artikel beschriebenen, einfachen spektroskopischen Messaufbau detektiert werden. Durch Vergleich mit anderen Versuchen zeigte sich, dass für die Beleuchtung und Detektion verschiedene Geometrien möglich sind. Der hier beschriebene Fasermesskopf ist jedoch nicht auf die Karotinoiddetektion beschränkt und es werden keine zusätzlichen Teile wie Linsen, Spiegel oder Blenden benötigt, womit der Entwicklungsaufwand verringert wird. Um den Störeinfluss durch Hämoglobin zu eliminieren, bietet sich eine Kombination aus Wegdrücken des Blutes und einer softwaretechnischen Korrekturrechnung an. Alternativ sollte die Beleuchtungs- und Detektionsgeometrie weiter verkleinert werden, damit hauptsächlich in der blutfreien Epidermis gemessen wird, denn dort ist die Karotinoidkonzentration am höchsten.