Polarized Light Spectroscopy Research Articles

Nanostructure silver absorbance with polarized light spectroscopy (PLS) and mobile imaging

This study presents a novel interdisciplinary approach for characterizing the optical properties of nanostructured silver films, combining methodologies from polarized light spectroscopy (PLS) and mobile app-based spectroscopic image detection. Silver nanofilms were deposited using electron beam evaporation (EBE) and subsequently analyzed for their absorbance across the visible light spectrum through PLS spectroscopy. The innovative aspect of this study lies in the validation of PLS spectral data using a mobile application developed with MIT App Inventor, which analyzes the intensity of light beams captured in images taken by a smartphone. The findings indicate that the silver nanofilm exhibits peak absorbance at approximately 400 nm within the violet region, corroborated by the mobile app’s data, which shows the lowest light intensity. However, discrepancies were observed from 500 to 600 nm, likely due to ambient light interferences and inherent optical phenomena such as diffraction, reflection, and refraction. Despite these deviations, the app’s readings closely align with the PLS data, supporting the film’s selective absorption properties. This study demonstrates the potential of this multidisciplinary technique in nanomaterial research, highlighting the application of nanotechnology in material science and the transformative potential of integrating software engineering for in-depth material characterization. These findings open avenues for advancements in both academic and practical applications.

Discriminating between Absorption and Scattering Effects in Complex Turbid Media by Coupling Polarized Light Spectroscopy with the Mueller Matrix Concept

The separation of the combined effects of absorption and scattering in complex media is a major issue for better characterization and prediction of media properties. In this study, an approach coupling polarized light spectroscopy and the Mueller matrix concept were evaluated to address this issue. A set of 50 turbid liquid optical phantoms with different levels of scattering and absorption properties were made and measured at various orientations of polarizers and analyzers to obtain the 16 elements of the complete Mueller matrix in the VIS-NIR region. Partial least square (PLS) was performed to build calibration models from diffuse reflectance spectra in order to evaluate the potential of polarization spectroscopy through the elements of the Mueller matrix to predict physical and chemical parameters and hence, to discriminate scattering and absorption effects, respectively. In particular, it was demonstrated that absorption and scattering effects can be distinguished in the Rayleigh regime with linear and circular polarization from the M22 and M44 elements of the Mueller matrix, correspondingly.

Perfusion, Stance and Plantar Pressure Asymmetries on the Human Foot in the Absence of Disease—A Pilot Study

Physiological perfusion asymmetries in the lower limb are known, although poorly understood, as are asymmetries reported in plantar pressure and stance. This preliminary study aims to explore potential relationships between perfusion and pressure variables in humans. A convenience sample of eight healthy individuals (25.25 ± 5.37 years old) of both sexes, was selected. Chosen variables were perfusion, plantar pressure, and stance. Perfusion was measured in both feet by laser Doppler flowmetry (LDF) and polarized light spectroscopy (PSp), and plantar pressure and stance obtained by a pressure plate. These were measured in baseline (Phase I) in a repeated squatting (Phase II), and in recovery (Phase III). A 95% confidence interval was adopted. Intraindividual significant perfusion asymmetries between both feet were detected by LDF in Phase I. These disappeared in Phase II and returned in Phase III. PSp did not detect any asymmetries. Plantar pressure was also asymmetric and differently distributed along both feet with no statistical significance except in the hindfoot. Significant correlations were found between BMI and mean Plantar Pressure in Phase I and Phase III, and an inverse correlation between LDF perfusion and Plantar Pressure in Phase I. These results seem to suggest an interesting direction for exploration and study of these asymmetries in the absence of disease.

Identification of Thioflavin T Binding Modes to DNA: A Structure-Specific Molecular Probe for Lasing Applications.

The binding mechanism of thioflavin T (ThT) to DNA was studied using polarized light spectroscopy and fluorescence-based techniques in solutions and in solid films. Linear dichroism measurements showed that ThT binds to DNA duplex by intercalation. Time-resolved fluorescence studies revealed a second binding mode which is the external binding to the DNA phosphate groups. Both binding modes represent the nonspecific type of interactions. The studies were complemented with the analysis of short oligonucleotides having DNA cavities. The results indicate that the interplay between three binding modes—intercalation, external binding, and binding inside DNA cavities—determines the effective fluorescence quantum yield of the dye in the DNA structures. External binding was found to be responsible for fluorescence quenching because of energy transfer between intercalated and externally bound molecules. Finally, amplified spontaneous emission (ASE) was successfully generated in the ThT-stained films and used for detecting different DNA structures. ASE measurements show that ThT-stained DNA structures can be used for designing bioderived microlasers.

Microvascular effects of microneedles with subsequent histamin application in the skin prick test.

Since decades, histamin applications are routinely performed in skin prick tests using a lancet. However, this technique is associated with various drawbacks. In healthy human subjects, we investigated the effects of microneedle-enhanced histamin delivery (wheal size, erythema size) in the skin microvasculature using polarized light spectroscopy imaging (Tissue Viability imaging, TiVi). Histamin was applied on microneedle-pretreated skin or on -untreated skin, to assess the microvascular response in the local skin. In our results, histamin was delivered more rapidly into the skin after microneedle pretreatment compared to passive diffusion, visible as wheal and erythema. The here presented technique might be useful for a personalized drug-testing system in the future.

Studying the impact of different body positioning, squatting, and unipodal flexion on perfusion in the lower limb – an exploratory approach complemented with optical spectroscopy (TiVi)

Body posture and movement seem to be responsible for multiple variations of microcirculation in the foot, although difficulties associated with measurement during movement have limited their characterization. This pilot study sought to explore a new non-invasive instrument based on polarized light spectroscopy – the TiVi system, under orthostatic and dynamic conditions. Five healthy participants (duly informed, 26.0 ± 6.5 years of age) of both genders were selected. The protocol involved sequential posture changes (orthostatic variation) and dynamic movements (squatting and unipodal flexion). Perfusion variations were evaluated in both feet by laser-Doppler flowmetry (LDF) and polarized spectroscopy (TiVi). Both systems revealed the same type of modification of these flow-related variables. The most pronounced changes were obtained with exercise, especially with squatting, which caused a marked increase in foot perfusion. Less dramatic but in the same direction, the increased perfusion with unipodal flexion was also observed in the contralateral limb. This study confirmed the interest in using the TiVi system in these domains, as well as the appropriateness of this experimental design to look deeper into the impact of lower limb movement on the functional dynamics of the foot.

Objective assessment of skin microcirculation using a smartphone camera.

Existing techniques for assessment of microcirculation are limited by their large size and high costs and are often not so easy to use. Advances in mobile technology have enabled great improvements in smartphone sensor technology. In this study, we used SkinSight, an app for iPhone and iPad, to measure changes in skin microcirculation during physiological provocations. The system estimates changes in the concentration of hemoglobin in the skin by analyzing the reflected light emitted from the built-in light-emitting diode and detected by the camera of the smartphone. A relative hemoglobin (Hb) index was measured during a 5-min arterial occlusion, post-occlusive reactive hyperemia, and a 5-min venous occlusion in 10 healthy subjects, on two separate days. The index was calculated in an area of the skin from the color information in the images acquired by the phone camera. Polarized light spectroscopy imaging was used to measure changes in red blood cell concentration for comparison. During arterial occlusion, relative Hb index was unchanged compared to baseline (P = .40). After release of the cuff, a sudden 60%-75% increase in Hb index was observed (P < .001) followed by a gradual return to baseline. During venous occlusion, Hb index increased by 80% (P < .001) followed by a gradual decrease to baseline after reperfusion. Day-to-day reproducibility of the relative Hb index was excellent (ICC: 0.92, r = 0.94), although relative Hb index was consistently higher during the second day, possibly as a result of changed lighting conditions or calibration issues. Microvascular responses to physiological provocations in the skin can be accurately and reproducibly measured using a smartphone application. Although the system offers a handheld, easy to use and flexible technique for skin microvascular assessment, the effects of lighting on the measured values and need for calibration need to be further investigated.

Microvascular effects of microneedle application.

The efficiency of transdermal drug delivery may be increased by pretreating the skin with microneedles, but distinct effects of microneedles and the microneedle-enhanced delivery of vasoactive drugs on the skin microvasculature are still not well investigated. In eight healthy human subjects, we measured the microvascular response to microneedle-induced microtraumas in the skin microvasculature using polarized light spectroscopy imaging (Tissue Viability imaging, TiVi). The microvascular response was assessed for up to 48hours for three microneedle sizes (300µm, 500µm, and 750µm) and for different pressures and application times. In our results, microneedle application increased the local red blood cell (RBC) concentration for up to 24hours dependent on the needle lengths, applied time, and force. Optimization of microneedles size, pressure, and application time should be taken into account for future protocols for drug delivery and experimental provocations.

Assessment of oxygenation with polarized light spectroscopy enables new means for detecting vascular events in the skin

IntroductionImpaired oxygenation in the skin may occur in disease states and after reconstructive surgery. We used tissue viability imaging (TiVi) to measure changes in oxygenation and deoxygenation of haemoglobin in an in vitro model and in the dermal microcirculation of healthy individuals. Materials and methodsOxygenation was measured in human whole blood with different levels of oxygenation. In healthy subjects, changes in red blood cell concentration (CRBC,TiVi), oxygenation (ΔCOH,TiVi) and deoxygenation (ΔCDOH,TiVi) of haemoglobin were measured during and after arterial and venous occlusion using TiVi and were compared with measurements from the enhanced perfusion and oxygen saturation system (EPOS). ResultsDuring arterial occlusion, CRBC,TiVi remained unchanged while ΔCOH,TiVi decreased to −44.2 (10.4) AU (p = 0.04), as compared to baseline. After release, CRBC,TiVi increased to 39.2 (18.8) AU (p < 0.001), ΔCOH,TiVi increased to 38.5. During venous occlusion, CRBC,TiVi increased to 28.9 (11.2) AU (p < 0.001), ΔCOH,TiVi decreased to −52.2 (46.1) AU (p < 0.001) compared to baseline after 5 min of venous occlusion. There was a significant correlation between the TiVi Oxygen Mapper and EPOS, for arterial (r = 0.92, p < 0.001) and venous occlusion (r = 0.87, p < 0.001), respectively. ConclusionThis study shows that TiVi can measure trends in oxygenation and deoxygenation of haemoglobin during arterial and venous stasis in healthy individuals.

Development of a polarized hyperspectral imaging system for investigation of absorption and scattering properties

This study was carried out to investigate the feasibility of an original polarized hyperspectral imaging setup in the spectral range of 400–1100 nm for enhancement of absorbance signal measurement on highly scattering samples. Spatial response and spectral calibration have been verified, indicating the consistency of this system and reliability of the acquired data. Model samples consisting of layered sand were prepared and used to uncover the hidden spectral information. In the model matrix, sand worked as scattering particle and dye E141 as absorbing material. Cross ([Formula: see text]) and parallel ([Formula: see text]) reflectance signals, along with the back-scattered reflectance, [Formula: see text]([Formula: see text]+[Formula: see text]) and the weakly scattered reflectance [Formula: see text] ([Formula: see text]−[Formula: see text]) spectra were computed and compared. Results demonstrated that cross-polarized images showed more subsurface information from the second layer due to the rejection of the superficial reflectance, while weakly scattered reflectance ([Formula: see text]) preserved only the surface information from the first layer. In addition, polarized light spectroscopy absorbance based on Dahm's equation in the frame of the representative layer theory and standard normal variate preprocessing [Formula: see text] spectra were also obtained from the prepared model matrix. The visual inspection of spectral curves revealed that [Formula: see text] and PoLiS absorbance showed two narrow peaks at 405 nm and 630 nm that were less impacted by multi-scattering effects. Partial least squares regression models were developed to predict dye concentration in the mixture sample. Consistent with the spectral profiles, [Formula: see text] and PoLiS absorbance presented the best model performances with determination of coefficients of prediction ([Formula: see text]) equal to 0.96 and 0.95, respectively. The resulting distribution maps of S1/S2 sand sample again confirmed the superior performance of [Formula: see text] and PoLiS absorbance, manifesting their better ability to reveal chemically related information. The overall results obtained in this research showed that the developed polarized-hyperspectral imaging system coupled with scattering correction methods has great potential for the analysis of powdered or turbid samples.

The tissue viability imaging system-Suitable method for discovering minimal skin changes in occupational screenings? Results of a cross-sectional field study.

For early detection of initial skin changes in occupational screenings, only few objective assessment systems are available. With the aim of assessing an objective measurement method for hand eczema, we trialed the application of the tissue viability imaging (TiVi) system, quantifying erythema non-invasively by polarized light spectroscopy. In a field study with 625 employees of a semiconductor production company, 411 were exposed to prolonged wearing of occlusive gloves in the clean room. TiVi system and Hand Eczema Score for Occupational Screenings (HEROS), a quantitative skin score for the hands, were used, supplemented by a standardized personal interview. TiVi values of 65 up to 246 for each hand (palm or back), for each participant summed to overall 289 up to 848 (median 389), were measured. Higher TiVi values were noted for men, smokers, and with increasing age. Correlation between TiVi and HEROS was only weak. Several factors like skin pigmentation, thickness of the skin, or tattoos seem to influence TiVi results. The practical relevance of one-time measurements with the TiVi system in occupational screenings seems to be limited. Specifically, the TiVi system cannot replace dermatological examinations at the workplace. Notwithstanding, the application for other scientific purposes might be useful.

Binding mode of proflavine to DNA probed by polarized light spectroscopy

It is well known that proflavine binds to DNA. Here we investigate the binding mode of proflavine to native DNA using absorption, circular dichroism (CD), and linear dichroism (LD) spectroscopy as well as by fluorescence techniques. The observed changes of proflavine upon complexation with DNA can be summarized as a red shift and hypochromism in the absorption spectrum. The negative LDr in the proflavine absorption region has a magnitude comparable to or larger than that of the DNA absorption region, confirming the intercalative binding mode of proflavine to DNA. Saturation of the LD spectrum in the proflavine absorption region at R = 0.25 and a decrease in the fluorescence intensity provide further evidence of intercalation. Furthermore, the coupling of electric transition of intercalated proflavine is observed. Although proflavine has been reported to position itself along the DNA stem at high [proflavine]/[DNA base] ratios, the spectral characteristics, which include a clear isosbestic point in the absorption spectrum and proportionality in the LD magnitude in the proflavine absorption region, do not show any possibility of exterior binding.

Flow Alignment of Extracellular Vesicles: Structure and Orientation of Membrane-Associated Bio-macromolecules Studied with Polarized Light.

Extracellular vesicles (EVs) are currently in scientific focus, as they have great potential to revolutionize the diagnosis and therapy of various diseases. However, numerous aspects of these species are still poorly understood, and thus, additional insight into their molecular-level properties, membrane-protein interactions, and membrane rigidity is still needed. We here demonstrate the use of red-blood-cell-derived EVs (REVs) that polarized light spectroscopy techniques, linear and circular dichroism, can provide molecular-level structural information on these systems. Flow-linear dichroism (flow-LD) measurements show that EVs can be oriented by shear force and indicate that hemoglobin molecules are associated to the lipid bilayer in freshly released REVs. During storage, this interaction ceases; this is coupled to major protein conformational changes relative to the initial state. Further on, the degree of orientation gives insight into vesicle rigidity, which decreases in time parallel to changes in protein conformation. Overall, we propose that both linear dichroism and circular dichroism spectroscopy can provide simple, rapid, yet efficient ways to track changes in the membrane-protein interactions of EV components at the molecular level, which may also give insight into processes occurring during vesiculation.

Probing Microscopic Orientation in Membranes by Linear Dichroism.

The cell membrane is an ordered environment, which anisotropically affects the structure and interactions of all of its molecules. Monitoring membrane orientation at a local level is rather challenging but could reward crucial information on protein conformation and interactions in the lipid bilayer. We monitored local lipid ordering changes upon varying the cholesterol concentration using polarized light spectroscopy and pyrene as a membrane probe. Pyrene, with a shape intermediate between a disc and a rod, can detect microscopic orientation variations at the level of its size. The global membrane orientation was determined using curcumin, a probe with nonoverlapping absorption relative to that of pyrene. While the macroscopic orientation of a liquid-phase bilayer decreases with increasing cholesterol concentration, the local orientation is improved. Pyrene is found to be sensitive to the local effects induced by cholesterol and temperature on the bilayer. Disentangling local and global orientation effects in membranes could provide new insights into functionally significant interactions of membrane proteins.

Binding Geometry of Free Base and Mn(III)meso‐Tetrakis(N‐methylpyridium‐4‐yl)porphyrin to Various Duplex and Triplex DNAs

The binding mode of free base and Mn(III)meso‐tetrakis(N‐methylpyridium‐4‐yl)porphyrin (TMPyP and MnTMPyP) to various duplex and triplex synthetic polynucleotides was investigated by polarized light spectroscopy, specifically circular and linear dichroism (CD and LD) spectroscopy. Both TMPyP and MnTMPyP produced a similar positive CD spectrum in the Soret absorption region when bound to poly(dA)·poly(dT) duplex and poly(dA)·[poly(dT)]2 triplex. Considering that the third poly(dT) strand blocks the major groove, this result suggested that both porphyrins bound near the minor groove where the axial ligands of the central Mn(III) ion did not affect the binding mode. In contrast, TMPyP intercalated and MnTMPyP bound the exterior of poly(dG)·poly(dC) and poly(dI)·poly(dC) duplexes, as judged from the negative and positive CD signals, respectively. The CD signals for both intercalated TMPyP and externally bound MnTMPyP were greatly altered when bound to poly(dG)·poly(dC)·poly(dC)+ and poly(dI)·poly(dC)·poly(dC)+ triplexes, indicating that the binding modes of both porphyrins were affected by the presence of the third strand, poly(dC)+, which contrasted with the AT polynucleotides. This observation supported the conclusion that the external binding site of MnTMPyP at the GC and IC duplexes is the major groove. The reduced LD spectrum indicated that the angles of the two electric transition moments of both TMPyP and MnTMPyP in the Soret absorption region were 57° and 65° with respect to the local DNA helix axis when bound to the poly(dA)·[poly(dT)]2 triplex. On the other hand, a strong, wavelength‐dependent reduced LD was observed in the Soret region for both porphyrins when associated with CGC triplex, and the excessive tilt degree prevented any reasonable calculation of the angles.

Improvement of the chemical content prediction of a model powder system by reducing multiple scattering using polarized light spectroscopy.

Near-infrared spectroscopy (NIRS) is a powerful non-destructive analytical method used to analyze major compounds in bulk materials and products and requiring no sample preparation. It is widely used in routine analysis and also in line in industries, in vivo with biomedical applications, or in field for agricultural and environmental applications. However, highly scattering samples subvert Beer-Lambert law's linear relationship between spectral absorbance and the concentration. Instead of spectral pre-processing, which is commonly used by NIR spectroscopists to mitigate the scattering effect, we put forward an optical method, i.e., coupling polarized light with NIR spectrometry, to free spectra from scattering effect. This should allow us to retrieve linear and steady conditions for spectral analysis. When tested in visible-NIR (Vis-NIR) range (400-800 nm) on model media, mixtures of scattering and absorbing particles, the setup provided significant improvements in absorber concentration estimation precision as well as in the quality and robustness of the calibration model.

Orientation of aromatic residues in amyloid cores: structural insights into prion fiber diversity.

Structural conversion of one given protein sequence into different amyloid states, resulting in distinct phenotypes, is one of the most intriguing phenomena of protein biology. Despite great efforts the structural origin of prion diversity remains elusive, mainly because amyloids are insoluble yet noncrystalline and therefore not easily amenable to traditional structural-biology methods. We investigate two different phenotypic prion strains, weak and strong, of yeast translation termination factor Sup35 with respect to angular orientation of tyrosines using polarized light spectroscopy. By applying a combination of alignment methods the degree of fiber orientation can be assessed, which allows a relatively accurate determination of the aromatic ring angles. Surprisingly, the strains show identical average orientations of the tyrosines, which are evenly spread through the amyloid core. Small variations between the two strains are related to the local environment of a fraction of tyrosines outside the core, potentially reflecting differences in fibril packing.

Combining linear polarization spectroscopy and the Representative Layer Theory to measure the Beer–Lambert law absorbance of highly scattering materials

Visible and Near Infrared (Vis–NIR) Spectroscopy is a powerful non destructive analytical method used to analyze major compounds in bulk materials and products and requiring no sample preparation. It is widely used in routine analysis and also in-line in industries, in-vivo with biomedical applications or in-field for agricultural and environmental applications. However, highly scattering samples subvert Beer–Lambert law's linear relationship between spectral absorbance and the concentrations. Instead of spectral pre-processing, which is commonly used by Vis–NIR spectroscopists to mitigate the scattering effect, we put forward an optical method, based on Polarized Light Spectroscopy to improve the absorbance signal measurement on highly scattering samples. This method selects part of the signal which is less impacted by scattering. The resulted signal is combined in the Absorption/Remission function defined in Dahm's Representative Layer Theory to compute an absorbance signal fulfilling Beer–Lambert's law, i.e. being linearly related to concentration of the chemicals composing the sample. The underpinning theories have been experimentally evaluated on scattering samples in liquid form and in powdered form. The method produced more accurate spectra and the Pearson's coefficient assessing the linearity between the absorbance spectra and the concentration of the added dye improved from 0.94 to 0.99 for liquid samples and 0.84–0.97 for powdered samples.

Automatic diagnosis of melanoma using machine learning methods on a spectroscopic system.

BackgroundEarly and accurate diagnosis of melanoma, the deadliest type of skin cancer, has the potential to reduce morbidity and mortality rate. However, early diagnosis of melanoma is not trivial even for experienced dermatologists, as it needs sampling and laboratory tests which can be extremely complex and subjective. The accuracy of clinical diagnosis of melanoma is also an issue especially in distinguishing between melanoma and mole. To solve these problems, this paper presents an approach that makes non-subjective judgements based on quantitative measures for automatic diagnosis of melanoma.MethodsOur approach involves image acquisition, image processing, feature extraction, and classification. 187 images (19 malignant melanoma and 168 benign lesions) were collected in a clinic by a spectroscopic device that combines single-scattered, polarized light spectroscopy with multiple-scattered, un-polarized light spectroscopy. After noise reduction and image normalization, features were extracted based on statistical measurements (i.e. mean, standard deviation, mean absolute deviation, L 1 norm, and L 2 norm) of image pixel intensities to characterize the pattern of melanoma. Finally, these features were fed into certain classifiers to train learning models for classification.ResultsWe adopted three classifiers – artificial neural network, naïve bayes, and k-nearest neighbour to evaluate our approach separately. The naive bayes classifier achieved the best performance - 89% accuracy, 89% sensitivity and 89% specificity, which was integrated with our approach in a desktop application running on the spectroscopic system for diagnosis of melanoma.ConclusionsOur work has two strengths. (1) We have used single scattered polarized light spectroscopy and multiple scattered unpolarized light spectroscopy to decipher the multilayered characteristics of human skin. (2) Our approach does not need image segmentation, as we directly probe tiny spots in the lesion skin and the image scans do not involve background skin. The desktop application for automatic diagnosis of melanoma can help dermatologists get a non-subjective second opinion for their diagnosis decision.

UV transition moments of tyrosine.

To assist polarized-light spectroscopy for protein-structure analysis, the UV spectrum of p-cresol, the chromophore of tyrosine, was studied with respect to transition moment directions and perturbation by solvent environment. From linear dichroism (LD) spectra of p-cresol aligned in stretched matrices of poly(vinyl alcohol) and polyethylene, the lowest π-π* transition (Lb) is found to have pure polarization over its entire absorption (250-300 nm) with a transition moment perpendicular to the symmetry axis (C1-C4), both in polar and nonpolar environments. For the second transition (La), polarized parallel with the symmetry axis, a certain admixture of intensity with orthogonal polarization is noticed, depending on the environment. While the Lb spectrum in cyclohexane shows a pronounced vibrational structure, it is blurred in methanol, which can be modeled as due to many microscopic polar environments. With the use of quantum mechanical (QM) calculations, the transition moments and solvent effects were analyzed with the B3LYP and ωB97X-D functionals in cyclohexane, water, and methanol using a combination of implicit and explicit solvent models. The blurred Lb band is explained by solvent hydrogen bonds, where both accepting and donating a hydrogen causes energy shifts. The inhomogeneous solvent-shift sensitivity in combination with robust polarization can be exploited for analyzing tyrosine orientation distributions in protein complexes using LD spectroscopy.