Non-destructive Optical Technique Research Articles

RAMAN SPECTROSCOPY FOR INTRAOPERATIVE ASSESSMENT OF BONE RESECTION MARGINS IN ORAL CAVITY SQUAMOUS CELL CARCINOMA

<h3>Background</h3> 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. <h3>Objective</h3> 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. <h3>Methods</h3> 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. <h3>Results</h3> Results show that RS can detect OCSCC in bone resection surfaces with a high sensitivity (96%) and specificity (83%; 26 mapping experiments, 22 patients). <h3>Conclusions</h3> These results form the basis for further development of an RS tool as an objective method for intraoperative assessment of BRMs.

INTRAOPERATIVE ASSESSMENT OF RESECTION MARGINS BASED ON RAMAN SPECTROSCOPY IN OCSCC SURGERY

<h3>Background</h3> 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. <h3>Objective</h3> Our aim is to develop an objective method for fast and reliable IOARM based on Raman spectroscopy (RS). <h3>Methods</h3> 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. <h3>Results</h3> First tests of the method show that the instrument accurately predicts the achieved resection margins. Per location the measurement and assessment takes 5 seconds. <h3>Conclusions</h3> 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.

Monitoring Changes in Biochemical and Biomechanical Properties of Collagenous Tissues Using Label-Free and Nondestructive Optical Imaging Techniques

We demonstrate the ability of nondestructive optical imaging techniques such as second-harmonic generation (SHG), two-photon fluorescence (TPF), fluorescence lifetime imaging (FLIM), and Raman spectroscopy (RS) to monitor biochemical and mechanical alterations in tissues upon collagen degradation. Decellularized equine pericardium (EP) was treated with 50 μg/mL bacterial collagenase at 37 °C for 8, 16, 24, and 32 h. The SHG ratio (defined as the normalized ratio between SHG and TPF signals) remained unchanged for untreated EP (stored in phosphate-buffered solution (PBS)), whereas treated EP showed a trend of a decreasing SHG ratio with increasing collagen degradation. In the fluorescence domain, treated EP experienced a red-shifted emission and the fluorescence lifetime had a trend of decreasing lifetime with increasing collagen digestion. RS monitors collagen degradation, the spectra had less intense Raman bands at 814, 852, 938, 1242, and 1270 cm-1. Non-negative least-squares (NNLS) modeling quantifies collagen loss and relative increase of elastin. The Young's modulus, derived from atomic force microscope-based nanoindentation experiments, showed a rapid decrease within the first 8 h of collagen degradation, whereas more gradual changes were observed for optical modalities. We conclude that optical imaging techniques like SHG, RS, and FLIM can monitor collagen degradation in a label-free manner and coarsely access mechanical properties in a nondestructive manner.

Optimizing Model Calibrations for Natural Product Chemical Compositions with Absorbance-Transmittance Excitation-Emission (A-TEEM) Spectroscopy

The food, beverage and natural supplements industries face many challenges relating to quality control as influenced by natural product variation, formulation errors and product adulteration. Conventional chromatographic and spectrophotometric assays can be time-consuming, costly and insensitive to key quality parameters and adulterants. Thus, there is a recognized need for a rapid, sensitive, non-destructive optical technique. This study investigates optimization of multivariate calibrations of chemical product compositions analyzed using A-TEEM spectroscopy. The A-TEEM method provides rapid (s-min) analyses of all chromophoric and fluorescent compounds in the UV-VIS range with micro- to sub-microgram/L sensitivity to many aromatic compounds. Conventional fluorescence EEM analyses primarily focus only on the inner-filter-effect corrected fluorescence data. In this paper, we systematically investigated the statistical significance of evaluating the separate absorbance, EEM and combined multi-block absorbance and EEM data variable sets. We report that there is a consistent improvement for the majority of A-TEEM models using ‘multi-block’ data organization compared to the separate absorbance or EEM variable data sets. We attribute the increased statistical advantage to the fact that most chemicals, even with similar structures, in a given solvent exhibit unique molar extinction coefficients, fluorescence quantum efficiencies as well as absorbance- and fluorescence excitation and emission spectral shapes. The A-TEEM data analyses further compared the Extreme Gradient Boosting (XGB) algorithm for both discrimination and regression to other comparable methods including Partial-Least Squares and Support Vector Machine. The conclusion, based on a multi-instrument comparison, was that the XGB analyses of the multi-block A-TEEM data lead to the most effective validation for both discrimination and regression models of food, beverage and natural supplements chemical composition and adulteration.

Nanoscale quantitative surface roughness measurement of articular cartilage using second-order statistical-based biospeckle.

Quantitative measurement of nanoscale surface roughness of articular cartilage tissue is significant to assess the surface topography for early treatment of osteoarthritis, the most common joint disease worldwide. Since it was not established by clinical diagnostic tools, the current studies have been suggesting the use of alternative diagnostic tools using pre-clinical methods. This study aims to measure the nanoscale surface roughness of articular cartilage tissue utilizing biospeckle which is used as a non-destructive and non-contact optical imaging technique. An experimental setup was implemented to capture biospeckle images from twelve cross-section areas of articular cartilage tissue gathered from bovine knee joints at 632 nm wavelength laser radiation. Then, to analyze the biospeckle image, a second-order statistical-based method was proposed through the combination of 308 highly correlated statistical features extracted from implemented gray-level co-occurrence matrices by employing principal component analysis. The result indicated that the measurement of the nanoscale surface roughness based on the first principal component only is able to provide accurate and precise quantitative measurement of early signs of articular cartilage degeneration up to 2500 nm.

Spontaneous Raman and Surface-Enhanced Raman Scattering Bioimaging.

Raman spectroscopy is a specific, noninvasive and nondestructive optical technique and is able to obtain chemical information from molecules. Optical imaging based on Raman spectroscopy has been a powerful technique for monitoring minute chemical changes of biological samples and generating images through direct or indirect strategies. Two widely applied Raman imaging techniques include spontaneous Raman and surface-enhanced Raman scattering (SERS) imaging. In this chapter, we introduce the basic principles including the physics behind Raman and SERS imaging, design of Raman/SERS labels or probes, and current strategies for further improvements. The progress in the use of spontaneous Raman and SERS spectroscopy for bioimaging is discussed, either in fundamental studies or inbiomedical theranostics. In addition, we give insights into the challenges and opportunities for improving Raman imaging performance.

On the application of multi-parametric optical phenotyping of bacterial colonies for multipurpose microbiological diagnostics

The development of new diagnostics techniques and modalities is critical for early detection of microbial contamination. In this study, the novel integrated system for multi-parametric optical phenotyping and characterization of bacterial colonies, is presented. The system combines Mach-Zehnder interferometer with a spectral imaging system for capturing multispectral diffraction patterns and multispectral two-dimensional transmission maps of bacterial colonies, along with the simultaneous interferometric profilometry. The herein presented investigation was carried out on five representative bacteria species and nearly 3000 registered multispectral optical signatures. The interferograms were analyzed by four-step phase shift algorithm to reconstruct the colony profile to enable the obtaining of the comparable optical signatures. The dedicated image processing algorithms were used for extraction of quantitative features of these signatures. The random forest algorithm was applied for selection of the most predictive set of features, which were used in classification model based on Support-Vector Machine. Obtained results have shown that the use of multiple multispectral optical signatures provide a multi-parametric bacteria identification at an exceptionally high accuracy (99.4-100%), significantly better than in case of classification based on each of these signatures (multispectral diffraction patterns, two-dimensional transmission coefficient maps), separately. Obtained results revealed that analysis of multispectral signatures can also be applied for characterisation of physical, physicochemical and chemical properties of the bacterial colonies in the presence of the antimicrobial factors. Therefore, the proposed label-free, non-destructive optical technique has perspectives to be exploited in the multipurpose diagnostics and it can be used as a pre-screening tool in microbiological laboratories.

Real-time in situ optical tracking of oxygen vacancy migration in memristors

Resistive switches, which are also known as memristors, are low-power, nanosecond-response devices that are used in a range of memory-centric technologies. Driven by an externally applied potential, the switching mechanism of valence change resistive memories involves the migration, accumulation and rearrangement of oxygen vacancies within a dielectric medium, leading to a change in electrical conductivity. The ability to look inside these devices and understand how morphological changes characterize their function has been vital in their development. However, current technologies are often destructive and invasive. Here, we report a non-destructive optical spectroscopy technique that can detect the motion of a few hundred oxygen vacancies with nanometre-scale sensitivity. Resistive switches are arranged in a nanoparticle-on-mirror geometry to exploit the high optical sensitivity to morphological changes occurring in tightly confined plasmonic hotspots within the switching material. Using this approach, we find that nanoscale oxygen bubbles form at the surface of a strontium titanate memristor film, leading ultimately to device breakdown on cycling. A nanoplasmonic technique was used to investigate in operando the switching properties of materials used in redox random access memories, providing insight into the operation and potential breakdown mechanisms of the devices.

Nanometer accuracy statistical interferometric technique in monitoring the short-term effects of exogenous plant hormones, auxin and gibberellic acid, on rice plants.

Statistical interferometric technique (SIT) is a highly sensitive, high speed non-contact, and non-destructive optical technique developed by our group capable of measuring instantaeoues sub-nanometer displacements. SIT applied to plant leaf elongation revealed nanometric intrinsic fluctuaitons (NIF) that are robust and sensitive to variations in the environment making NIF as a measure of healthiness of the plants. In this study, exogenous plant hormones, auxin (2,4-dichlorophenoxyacetic acid-2,4-D), and gibberellic acid (GA3), along with an auxin transport inhibitor 2,3,5-triiodobenzoic acid-TIBA, that affect plant growth were used to investigate their effects on NIF. Rice (Oriza sativa) seedlings were used, and their roots were exposed to 1, 2, and 4 µM 2,4-D, and the auxin transport inhibitor, TIBA, of 10, and 20 µM for 22 h and GA3 solution of different concentrations of 10, 40, and 100 µM for 5 h. Results showed significant increment in NIF for 1 µM and reduction for 4 µM 2,4-D while applicaiton of both 10, and 20 µM TIBA led to reduction in NIF. On the other hand, significant increment in NIF for 40 µM, and a significant reduction at a higher concentration of 100 µM for 5 hours of GA3 were also observed in comparison to those of control. Our results indicate that NIF as revealed by SIT could show both the positive and negative effects depending on the concentration of exogenous hormones, and transport inhibitors. Results suggest that SIT could be a valuable tool being sensitive enough to speedily assess the effects of plant growth hormones.

Recent Development and Challenges in Spectroscopy and Machine Vision Technologies for Crop Nitrogen Diagnosis: A Review

Recent development of non-destructive optical techniques, such as spectroscopy and machine vision technologies, have laid a good foundation for real-time monitoring and precise management of crop N status. However, their advantages and disadvantages have not been systematically summarized and evaluated. Here, we reviewed the state-of-the-art of non-destructive optical methods for monitoring the N status of crops, and summarized their advantages and disadvantages. We mainly focused on the contribution of spectral and machine vision technology to the accurate diagnosis of crop N status from three aspects: system selection, data processing, and estimation methods. Finally, we discussed the opportunities and challenges of the application of these technologies, followed by recommendations for future work to address the challenges.

Combining Mass Spectrometry of Picoliter Samples with a Multicompartment Electrodynamic Trap for Probing the Chemistry of Droplet Arrays.

Single droplet levitation provides contactless access to the microphysical and chemical properties of micrometer-sized samples. Most applications of droplet levitation to chemical and biological systems use nondestructive optical techniques to probe droplet properties. To provide improved chemical specificity, we coupled a multicompartment quadrupole electrodynamic trap (QET) with single droplet mass spectrometry. Our QET continuously traps a monodisperse droplet population (tens to hundreds of droplets) and allows for the simultaneous sizing of a single droplet using its Mie scattering pattern. Single droplets are subsequently ejected into the ionization region of an ambient pressure inlet mass spectrometer. We optimized two complementary soft ionization techniques for picoliter aqueous droplets: (1) paper spray (PS) ionization and (2) thermal desorption glow discharge (TDGD) ionization. Both techniques detect oxygenated organic acids in single droplets, with signal-to-noise ratios >100 and detection limits on the order of 10 pg. Sensitivity and reproducibility across single droplets are driven by the droplet deposition location and spray stability in PS-MS and the ionization region humidity and analyte evaporation rate in TDGD-MS. Importantly, the analyte evaporation rate can control the TDGD-MS quantitative capability because high evaporation rates result in significant ion suppression. This effect is mitigated by optimizing the vaporization temperature, droplet size range, and analyte volatility. We demonstrate quantitative and reproducible measurements with a droplet internal standard (<10% RSD) and compare the sensitivity of PS-MS and TDGD-MS. Finally, we demonstrate the application of QET-MS to the study of heterogeneous chemical kinetics with the reaction of gas phase O3 and aqueous maleic acid droplets.

Using 2D digital image correlation to investigate the flexural behavior of continuous composite beams

This paper aims at studying the flexural behavior of continuous composite beams casting from normal concrete and Slurry Infiltrated Fiber Concrete (SIFCON) by using DIC (Digital Image Correlation) technique. The DIC results are compared with the standard methods measurement. DIC is a new non-contacting and non-destructive optical technique based on digital photographs for the tested area to compare them at different stages of loading. Tests were performed on three full-scale T-section continuous beam specimens subjected to two-point loads. In this paper, the effect of using SIFCON at compression or tension parts of plastic hinge locations on the flexural capacity, moment redistribution and crack width are investigated experimentally. The results indicate that the DIC technique gives very good correspondence comparing with the results recorded by standard methods measurement. In addition, it was found that the enhancement in moment redistribution ratio for the beam with SIFCON at compression parts of plastic hinge zones was significantly higher by about 38% comparing with the beam with SIFCON at tension parts of plastic hinge zones.

Laser fluorescence spectroscopy in predicting the formation of a keloid scar: preliminary results and the role of lipopigments.

Keloid scars, in contrast to other scar types, significantly reduce the patient's quality of life. To develop a nondestructive optical diagnostic technique predicting the keloid scars formation in vivo, laser-induced fluorescence spectroscopy (LFS) was used to study the autofluorescence in skin of patients with various types of head and neck cicatricial deformities. The unexpected results were obtained for the endogenous fluorescence of lipofuscin. Significantly reduced autofluorescence of lipofuscin was registered both in the intact and in the keloid scar tissues in comparison with the intact and scar tissues in patients with hypertrophic and normotrophic scars. Sensitivity and specificity achieved by LFS in keloid diagnosis are 81.8% and 93.9% respectively. It could take place due to the changes in the reductive-oxidative balance in cells, as well as due to the proteolysis processes violation. Therefore, we suppose that the evaluation of the lipofuscin autofluorescence in skin before any surgical intervention could predict the probability of the subsequent keloid scars formation.

Measurement of junctional tension in epithelial cells at the onset of primitive streak formation in the chick embryo via non-destructive optical manipulation.

ABSTRACTDirectional cell intercalations of epithelial cells during gastrulation has, in several organisms, been shown to be associated with a planar cell polarity in the organisation of the actin-myosin cytoskeleton and is postulated to reflect directional tension that drives oriented cell intercalations. We have characterised and applied a recently introduced non-destructive optical manipulation technique to measure the tension in individual epithelial cell junctions of cells in various locations and orientations in the epiblast of chick embryos in the early stages of primitive streak formation. Junctional tension of mesendoderm precursors in the epiblast is higher in junctions oriented in the direction of intercalation than in junctions oriented perpendicular to the direction of intercalation and higher than in junctions of other cells in the epiblast. The kinetic data fit best with a simple viscoelastic Maxwell model, and we find that junctional tension, and to a lesser extent viscoelastic relaxation time, are dependent on myosin activity.

Early detection of eggplant fruit stored at chilling temperature using different non-destructive optical techniques and supervised classification algorithms

Eggplant fruit is a chilling injury sensitive vegetable and should not be stored at lower than 12 °C postharvest, although fruit are often placed in temperatures as low as 0–5 °C. For this reason, a rapid early detection of eggplants previously stored at chilling temperatures would allow early removal of those fruit from the market. Eggplant fruit (cv. Fantasy) were stored either at 2 °C (chilling injurious temperature) or at 12 °C (safe storage temperature) for 10 days. Every 2 days, fruit from each group were sampled and left at room temperature, for one additional day. Color measurements in the CIE L*a*b* mode and reflectance data in the wavelength range 360–740 nm, Fourier Transform (FT)-NIR spectra (800–2777 nm) and hyperspectral images at the visible (400–1000 nm) and near infrared (900–1700 nm) part of the electromagnetic spectrum were also acquired on each fruit. Three supervised algorithms; partial least square (PLS), supervised vector machine (SVM) and k-nearest neighbor (kNN) were applied to classify fruit according to the storage temperature. Chilling injury (CI) was subjectively evaluated, according to the presence of black seeds or of brown discolored flesh area. According to the results, although chilling injury symptoms started being evident only after the 4th day of storage at 2 °C, it was possible to discriminate fruit earlier, since day 2, by processing the FT-NIR spectral data with the SVM classifier (100 and 92% non-error-rate (NER)) in calibration and cross validation, respectively) in the whole period data set. Color or FT-NIR spectral data classified with PLSDA permitted relatively good classification of fruit (>83% accuracy) since the 4th day of storage, while L, C, H° color measurements or Vis-NIR hyperspectral imaging data combined with PLSDA generate trustworthy models only after the 6th day of storage. On the other hand, NIR hyperspectral imaging technique and kNN classification algorithm were incapable to separate the fruit either accurately or consistently. These results indicate a good potential of adapting selected protocols, in terms of technique, processing of the raw data and supervised classification algorithm, in order to minimize postharvest losses induced by the improper temperature management of chilling sensitive fruit, such as the eggplants.

Molecularly imprinted polymer SPE sensor for analysis of CA-125 on serum

Considering the high incidence level and mortality rate of ovarian cancer, particularly among the European female population, the carbohydrate antigen 125 (CA-125) was selected as the protein target for this study for the development of a MIP-based biosensor.This work presents the development of molecular imprinting polymers (MIPs) on gold electrode surface for CA-125 biomarker recognition. The preparation of the CA-125 imprinting was obtained by electropolymerization of pyrrole (Py) monomer in a gold electrode using cyclic voltammetry (CV) in order to obtain highly selective materials with great molecular recognition capability. The quantification of CA-125 biomarker was made through the comparison of two methods: electrochemical (square wave voltammetry -SWV) and optical transduction (surface plasmon resonance -SPR). SWV has been widely used in biological molecules analysis since it is a fast and sensitive technique. In turn, SPR is a non-destructive optical technique that provides high-quality analytical data of CA-125 biomarker interactions with MIP.Several analytical parameters, such as sensitivity, linear response interval, and detection limit were determined to proceed to the performance evaluation of the electrochemical and optical transduction used in the development of the CA-125 biosensor.The biosensor based in the electrochemical transduction was the one that presented the best analytical parameters, yielding a good selectivity and a detection limit (LOD) of 0.01 U/mL, providing a linear concentration range between 0.01 and 500 U/mL. This electrochemical biosensor was selected for the study and it was successfully applied in the CA-125 analysis in artificial serum samples with recovery rates ranging from 91 to 105% with an average relative error of 5.8%.

Investigation of reflectometry for in situ process monitoring and characterization of co-evaporated and stacked Cu-Zn-Sn-S based thin films

During preparation of compound semiconductor thin films undesired secondary phases and inhomogeneities may occur. Therefore, process control and monitoring are important aspects towards thin film optimization. In this study, white light reflectometry (WLR) was investigated as an in situ non-destructive optical technique to monitor thin films during thermal vacuum evaporation (PVD) of kesterite-type Cu-Zn-Sn-S thin films.The impact of composition on optical properties was studied ex situ by Raman spectroscopy and reflectometry to identify possibilities for in situ detection of secondary phases. A WLR setup was designed and integrated with the PVD system. Four Cu-Zn-Sn-S films were prepared by evaporation and direct reflection was monitored in situ. Reflection spectra were analyzed to identify the influence of process parameters and phases such as CuS and ZnS. Transfer matrix simulations were performed to further explain experimental observations.It is shown that the occurrence of different phases, growth rate/thickness are well related to reflection spectra. Time-dependent reflection spectra reveal specific properties that could serve as characteristic fingerprints for process control and monitoring. Therefore, this study extends the possibilities for reflectometry as a straightforward, low cost method for in situ process control of Cu-Zn-Sn-S based films.

Non-destructive characterization of rare-earth-doped optical fiber preforms.

We present a non-destructive optical technique for rare-earth-doped optical fiber preform inspection, which combines luminescence spectroscopy measurements, analyzed through an optical tomography technique, and ray-deflection measurements for calculating the refractive-index profile (RIP) of the sample. We demonstrate the technique on an optical fiber preform sample with a Yb3+-doped aluminosilicate core. The spatial distribution of the photoluminescence signals originating from Yb3+-single ions and from Yb3+-Yb3+ cluster sites were obtained inside the core. By modifying the characterization system, we were able to concurrently evaluate the RIP of the core and, thus, establish with good accuracy the dopant distribution within the core region. This technique will be useful for quality evaluation and optimization of optical fiber preforms.

Cortical bone quality affectations and their strength impact analysis using holographic interferometry.

It is now accepted that bone strength is a complex property determined mainly by three factors: quantity, quality and turnover of the bone itself. Most of the patients who experience fractures due to fragility could never develop affectations related to bone mass density (i.e. osteoporosis). In this work, the effect of secondary bone strength affectations are analyzed by simulating the degradation of one or more principal components (organic and inorganic) while they are inspected with a nondestructive optical technique. From the results obtained, a strong correlation among the hydroxyapatite, collagen and water is found that determines the bone strength.

Crystalline, Phononic, and Electronic Properties of Heterostructured Polytypic Ge Nanowires by Raman Spectroscopy.

Semiconducting nanowires (NWs) offer the unprecedented opportunity to host different crystal phases in a nanostructure, which enables the formation of polytypic heterostructures where the material composition is unchanged. This characteristic boosts the potential of polytypic heterostructured NWs for optoelectronic and phononic applications. In this work, we investigate cubic Ge NWs where small (∼20 nm) hexagonal domains are formed due to a strain-induced phase transformation. By combining a nondestructive optical technique (Raman spectroscopy) with density-functional theory (DFT) calculations, we assess the phonon properties of hexagonal Ge, determine the crystal phase variations along the NW axis, and, quite remarkably, reconstruct the relative orientation of the two polytypes. Moreover, we provide information on the electronic band alignment of the heterostructure at points of the Brillouin zone different from the one (Γ) where the direct band gap recombination in hexagonal Ge takes place. We demonstrate the versatility of Raman spectroscopy and show that it can be used to determine the main crystalline, phononic, and electronic properties of the most challenging type of heterostructure (a polytypic, nanoscale heterostructure with constant material composition). The general procedure that we establish can be applied to several types of heterostructures.