Different membrane order measurement techniques are not mutually consistent.

"Membrane order" is a term commonly used to describe the elastic and mechanical properties of the lipid bilayer, though its exact meaning is somewhat context- and method dependent. These mechanical properties of the membrane control many cellular functions and are measured using various biophysical techniques. Here, we ask if the results obtained from various techniques are mutually consistent. Such consistency cannot be assumed a priori because these techniques probe different spatial locations and different spatial and temporal scales. We evaluate the change of membrane order induced by serotonin using nine different techniques in lipid bilayers of three different compositions. Serotonin is an important neurotransmitter present at 100s of mM concentrations in neurotransmitter vesicles, and therefore its interaction with the lipid bilayer is biologically relevant. Our measurement tools include fluorescence of lipophilic dyes (Nile Red, Laurdan, TMA-DPH, DPH), whose properties are a function of membrane order; atomic force spectroscopy, which provides a measure of the force required to indent the lipid bilayer; 2H solid-state NMR spectroscopy, which measures the molecular order of the lipid acyl chain segments; fluorescence correlation spectroscopy, which provides a measure of the diffusivity of the probe in the membrane; and Raman spectroscopy, where spectral intensity ratios are affected by acyl chain order. We find that different measures often do not correlate with each other and sometimes even yield conflicting results. We conclude that no probe provides a general measure of membrane order and that any inference based on the change of membrane order measured by a particular probe may be unreliable.

Validity of water compartments estimated using bioimpedance spectroscopy in athletes differing in hydration status.

We aimed to validate bioelectrical impedance spectroscopy (BIS), compared with tracer dilution measurements, for assessing total body water (TBW), intracellular water (ICW), and extracellular water (ECW) in athletes differing in hydration status. A total of 201 athletes participated. Reference TBW and ECW were determined by deuterium and bromide dilution methods, respectively; ICW was calculated as TBW-ECW. Water compartments were estimated by BIS. Urine specific gravity (USG) classified athletes into well-hydrated (WH) (USG < 1.023), euhydrated (EH) (USG:1.024-1.026), and dehydrated (DH) (USG>1.027). No significant differences were found between BIS and the reference methods for WH, EH, and DH athletes for TBW, ICW nor ECW (p>0.05). Concordance of TBW and its compartments by method was significant (p < 0.001) with coefficients of determination ranging by hydration classification [EH:52-96%;DH:56-98%;WH:71-96%]. Bland-Altman analyses showed no trend for TBW and its compartments with the exception of ICW in the WH athletes. The 95% confidence BIS intervals for the WH group ranged from -3.08 to 2.68 kg for TBW, -4.28 to 4.14 kg for ICW, and -3.29 to 3.02 kg for ECW. For the EH athletes, the 95% confidence intervals ranged from -2.78 to 2.24 kg for TBW, -4.10 to 3.94 kg for ICW, and -3.44 to 3.06 kg for ECW. In DH group, TBW ranged between -1.99 and 2.01 kg, ICW between -3.78 and 6.34 kg, and ECW between -6.22 and 3.74 kg. These findings show that BIS is useful at a group level in assessing water compartments in athletes differing in hydration status. However, the usefulness of BIS is limited at an individual level, especially in dehydrated athletes.

Fourier Transform Infrared Spectroscopy in Oral Cancer Diagnosis.

Oral cancer is one of the most common cancers worldwide. Despite easy access to the oral cavity and significant advances in treatment, the morbidity and mortality rates for oral cancer patients are still very high, mainly due to late-stage diagnosis when treatment is less successful. Oral cancer has also been found to be the most expensive cancer to treat in the United States. Early diagnosis of oral cancer can significantly improve patient survival rate and reduce medical costs. There is an urgent unmet need for an accurate and sensitive molecular-based diagnostic tool for early oral cancer detection. Fourier transform infrared spectroscopy has gained increasing attention in cancer research due to its ability to elucidate qualitative and quantitative information of biochemical content and molecular-level structural changes in complex biological systems. The diagnosis of a disease is based on biochemical changes underlying the disease pathology rather than morphological changes of the tissue. It is a versatile method that can work with tissues, cells, or body fluids. In this review article, we aim to summarize the studies of infrared spectroscopy in oral cancer research and detection. It provides early evidence to support the potential application of infrared spectroscopy as a diagnostic tool for oral potentially malignant and malignant lesions. The challenges and opportunities in clinical translation are also discussed.

Triple-isotope calibration of in-house water standards supplemented by determination of 17O content of USGS49-50 reference materials using cavity ring-down laser spectrometry.

The procedure of calibrating in-house water standards suitable for routine analyses of triple-isotope composition of water samples using Picarro L2140-i CRDS analyser is presented and discussed. Such standards are indispensable for achieving and maintaining high quality of isotope analyses of water in terms of their precision and accuracy. A set of seven different water standards consisting of three in-house standards and four secondary standards commercially available was calibrated against VSMOW2/SLAP2 primary reference materials. The calibrated standards cover a wide range of isotopic composition, with δ values ranging from close to zero to the values comparable with SLAP2. The apparent consistency of the calibrated values of δ2H, δ18O and d-excess with corresponding certified values for commercially available USGS47-50 standards and the consistency of the calibrated values of δ17O and Δ17O with its literature values for USGS47-48 standards confirm the high quality of the performed calibration. Moreover, the calibration exercise allowed to obtain δ17O and Δ17O values for USGS49 and USGS50 standards, not reported so far.

Imaging and quantifying drug delivery in skin - Part 2: Fluorescence andvibrational spectroscopic imaging methods.

Understanding the delivery and diffusion of topically-applied drugs on human skin is of paramount importance in both pharmaceutical and cosmetics research. This information is critical in early stages of drug development and allows the identification of the most promising ingredients delivered at optimal concentrations to their target skin compartments. Different skin imaging methods, invasive and non-invasive, are available to characterize and quantify the spatiotemporal distribution of a drug within ex vivo and in vivo human skin. The first part of this review detailed invasive imaging methods (autoradiography, MALDI and SIMS). This second part reviews non-invasive imaging methods that can be applied in vivo: i) fluorescence (conventional, confocal, and multiphoton) and second harmonic generation microscopies and ii) vibrational spectroscopic imaging methods (infrared, confocal Raman, and coherent Raman scattering microscopies). Finally, a flow chart for the selection of imaging methods is presented to guide human skin ex vivo and in vivo drug delivery studies.

Factors influencing the accuracy for tissue classification in multi spectral in-vivo endoscopy for the upper gastro-internal tract.

Hyper spectral imaging is a possible way for disease detection. However, for carcinoma detection most of the results are ex-vivo. However, in-vivo results of endoscopic studies still show fairly low accuracies in contrast to the good results of many ex-vivo studies. To overcome this problem and to provide a reasonable explanation, Monte-Carlo simulations of photon trajectories are proposed as a tool to generate multi spectral images including inter patient variations to simulate 40 patients. Furthermore, these simulations have the huge advantage that the position of the carcinoma is known. Due to this, the effect of mislabelled data can be studied. As shown in this study, a percentage of 30-35% of mislabelled data might lead to significant decrease of the accuracy from around 90% to around 70-75%. Therefore, the main focus of hyper spectral imaging has to be the exact characterization of the training data in the future.

Assessing the internal standardization of the direct multi-element determination in beer samples through microwave-induced plasma optical emission spectrometry.

An evaluation of different elements (Be, Ga, In, Sc, and Y) as internal standards was performed to determine the content of Al, Ba, Co, Cr, Cu, Fe, Mn, Ni, Sr, Zn, Ca, Mg, Na, K in beer samples through microwave induced plasma optical emission spectrometry. The analytes were determined after simple dilutions of the samples with a 1.0 M nitric acid solution at a 1:4 ratio (sample: acid solution) with the addition of the IS. The analytical performance for each potential IS was established based on the limit of detection, limit of quantification, addition and recovery tests and accuracy obtained in the determination of each analyte. Each analyte responded differently when internal standardization was applied, and as such, the evaluation of each IS is important in the development of the method. In the presence of the recommended internal standard, the limit of detection varied, in µg L-1, from 0.23 to 4.6 for the microelements and between 10 and 620 for the macroelements. The limit of quantification, in µg L-1, was between 0.78 and 15.4 and between 30 and 970 for the microelements and macroelements, respectively. The precisions of the measurements, expressed as the relative standard deviation (n = 10; 0.50 and 3.0 mg L-1 of each analyte), were lower than 1.0% for all analytes. The proposed method was applied for the multi-element determination in commercial beer samples and the results were compared with those obtained by inductively coupled plasma optical emission spectrometry after sample digestion.

Response Standardization for Drift Correction and Multivariate Calibration Transfer in "Electronic Tongue" Studies.

The procedures for response standardization in "electronic tongue" (ET) studies are described. The construction of reliable multivariate calibration for "electronic tongue" requires the analysis of a large number of representative samples both with ET and reference techniques. This is a laborious and expensive process. Long-term sensor array operation leads to the changes in sensor response characteristics and thus invalidates the multivariate predictive models. Moreover, due to the individual parameters of each sensor in different sensor arrays, it is not possible to use the calibration model for one system together with the data acquired by another system, even if they have the same sensors. Both of these issues lead to the necessity of frequent sensor array calibration which would be ideal to avoid. Instead of recalibration, these two problems can be handled using mathematical methods intended for sensor response standardization. This chapter describes two popular methods of standardization which can be used for both drift correction and calibration transfer. Thus, significant efforts on measuring representative sample sets for sensor array recalibration can be avoided.

Preliminary Assessment of Visible, Near-Infrared, and Short-Wavelength-Infrared Spectroscopy with a Portable Instrument for the Detection of Staphylococcus aureus Biofilms on Surfaces.

Bacterial biofilms constitute a major source of sanitary problems and economic losses in the food industry. Indeed, biofilm removal may require intense mechanical cleaning procedures or very high concentrations of disinfectants or both, which can be damaging to the environment and human health. This study assessed the efficacy of a technique based on spectroscopy in the visible, near-infrared, and short-wavelength infrared range for the quick detection of biofilms formed on polystyrene by the pathogenic bacterium Staphylococcus aureus. To do that, biofilms corresponding to three S. aureus strains, which differed in biofilm-forming ability and composition of the extracellular matrix, were allowed to develop for 5 or 24 h, representing an active formation stage and mature biofilms, respectively. Spectral analysis of the samples, corresponding to three biological replicates of each condition, was then performed by using a portable device. The results of these experiments showed that partial least-squares discriminant analysis of the spectral profile could discriminate between surfaces containing attached bacterial biomass and noninoculated ones. In this model, the two first principal components accounted for 39 and 19% of the variance and the estimated error rate stabilized after four components. Cross-validation accuracy of this assessment was 100%. This work lays the foundation for subsequent development of a spectroscopy-based protocol that allows biofilm detection on food industrial surfaces.

Requirements for Portable Instrument Suites during Human Scientific Exploration of Mars.

Human explorers on the surface of Mars will have access to a far wider array of scientific tools than previous crewed planetary exploration missions, but not every tool will be compatible with the restrictions of this exploration. Spectrometers on flyby, orbital, and landed missions are currently used to determine the composition and mineralogy of geological materials of various types and sizes, from small fragments to celestial bodies in the solar system. Handheld spectrometers that are capable of in situ analyses are already used for geological exploration on Earth; however, their usefulness for human exploration missions and how data from multiple handheld instruments could be combined to enhance scientific return must be further evaluated. As part of the Biologic Analog Science Associated with Lava Terrains (BASALT) research project, we incorporated two handheld instruments, a visible-near infrared spectrometer and an X-Ray Fluorescence spectrometer, into simulated Mars exploration missions conducted on basaltic terrains in Idaho and Hawai'i. To understand the data quality provided by these handheld spectrometers, we evaluated their performance under varying conditions of measurement time, distance, angle, atmosphere, and sample matrix, and we compared data quality between handheld instruments and laboratory techniques. Here, we summarize these findings, provide guidelines and requirements on how to effectively incorporate these instruments into human exploration missions to Mars, and posit that future iterations of these instruments will be beneficial for enhancing science returned from human exploration missions.

Validation of optical properties quantification with a dual-step technique for biological tissue analysis.

To approach wide-field optical properties quantification in real heterogeneous biological tissue, we developed a Dual-Step setup that couples a punctual diffuse reflectance spectroscopy (DRS) technique with multispectral imaging (MSI). The setup achieves wide-field optical properties assessment through an initial estimation of scattering with DRS, which is used to estimate absorption with MSI. The absolute quantification of optical properties is based on the ACA-Pro algorithm that has been adapted both for DRS and for MSI. This paper validates the Dual-Step system not only on homogeneous Intralipid phantoms but also on a heterogeneous gelatine phantom with different scattering and absorbing properties.

Transducer impulse response correction for a deconvolution derived ultrasound transit time spectrum.

Propagation of ultrasound through a complex composite sample may exhibit phase interference between two or more sonic-rays if differences in transit time are less than the pulse length. The transit time spectrum of a test sample, equivalent to its impulse response, was derived through active-set deconvolution of ultrasound signals with, and without, the test sample. The aim of this study was to test the hypothesis that in cases where only the transmit ultrasound transducer's digitally-coded excitation signal is available, hence not the input ultrasound signal without the test sample, incorporation of the transducer impulse response may increase both accuracy and precision of ultrasound transit time spectroscopy. A digital 1 MHz sinusoid signal was used to create an ultrasound pulse that was propagated through a 5 step-wedge acrylic sample immersed in water. Transit time spectra were obtained through deconvolution utilising an ultrasound input signal, along with a digital input signal, with and without incorporation of the transducer impulse response. Incorporation of the transducer impulse response reduced a quantitative measure of noise-to-signal ratio by a factor of 12. The paper has demonstrated the potential for increased accuracy and precision of transit time spectroscopy when the transducer impulse response is incorporated within active-set deconvolution analysis.

Are LOD and LOQ Reliable Parameters for Sensitivity Evaluation of Spectroscopic Methods?

The limit of detection (LOD) and the limit of quantification (LOQ) are common parameters to assess the sensitivity of analytical methods. In this study, the LOD and LOQ of previously reported terbium sensitized analysis methods were calculated by different methods, and the results were compared with sensitivity parameters [lower limit of quantification (LLOQ)] of U.S. Food and Drug Administration guidelines. The details of the calibration curve and standard deviation of blank samples of three different terbium-sensitized luminescence methods for the quantification of mycophenolic acid, enrofloxacin, and silibinin were used for the calculation of LOD and LOQ. A comparison of LOD and LOQ values calculated by various methods and LLOQ shows a considerable difference. The significant difference of the calculated LOD and LOQ with various methods and LLOQ should be considered in the sensitivity evaluation of spectroscopic methods.

Single fiber reflectance spectroscopy calibration.

To accurately determine sample optical properties using single fiber reflectance spectroscopy (SFR), an absolute calibration of the reflectance is required. We investigated two SFR calibration methods, using a calibrated mirror and using the Fresnel reflection at the fiber tip as a reference. We compared these to commonly used calibration methods, using either Intralipid-20% in combination with Monte Carlo simulations or Spectralon as a reference. The Fresnel reflection method demonstrated the best reproducibility and yielded the most reliable result. We therefore recommend the Fresnel reflection method for the measured absolute reflectance calibration of SFR.

Comparison of methods for determination of total oil sands-derived naphthenic acids in water samples.

There are several established methods for the determination of naphthenic acids (NAs) in waters associated with oil sands mining operations. Due to their highly complex nature, measured concentration and composition of NAs vary depending on the method used. This study compared different common sample preparation techniques, analytical instrument methods, and analytical standards to measure NAs in groundwater and process water samples collected from an active oil sands operation. In general, the high- and ultrahigh-resolution methods, namely high performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) and Orbitrap mass spectrometry (Orbitrap-MS), were within an order of magnitude of the Fourier transform infrared spectroscopy (FTIR) methods. The gas chromatography mass spectrometry (GC-MS) methods consistently had the highest NA concentrations and greatest standard error. Total NAs concentration was not statistically different between sample preparation of solid phase extraction and liquid-liquid extraction. Calibration standards influenced quantitation results. This work provided a comprehensive understanding of the inherent differences in the various techniques available to measure NAs and hence the potential differences in measured amounts of NAs in samples. Results from this study will contribute to the analytical method standardization for NA analysis in oil sands related water samples.

Measuring Spectral Inconsistency of Multispectral Images for Detection and Segmentation of Retinal Degenerative Changes.

Multispectral imaging (MSI) creates a series of en-face fundus spectral sections by leveraging an extensive range of discrete monochromatic light sources and allows for an examination of the retina's early morphologic changes that are not generally visible with traditional fundus imaging modalities. An Ophthalmologist's interpretation of MSI images is commonly conducted by qualitatively analyzing the spectral consistency between degenerated areas and normal ones, which characterizes the image variation across different spectra. Unfortunately, an ophthalmologist's interpretation is practically difficult considering the fact that human perception is limited to the RGB color space, while an MSI sequence contains typically more than ten spectra. In this paper, we propose a method for measuring the spectral inconsistency of MSI images without supervision, which yields quantitative information indicating the pathological property of the tissue. Specifically, we define mathematically the spectral consistency as an existence of a pixel-specific latent feature vector and a spectrum-specific projection matrix, which can be used to reconstruct the representative features of pixels. The spectral inconsistency is then measured using the number of latent feature vectors required to reconstruct the representative features in practice. Experimental results from 54 MSI sequences show that our spectral inconsistency measurement is potentially invaluable for MSI-based ocular disease diagnosis.

Method Development for Container Closure Integrity Evaluation via Headspace Gas Ingress by Using Frequency Modulation Spectroscopy.

USP <1207.1> Section 3.5 states that "A deterministic leak test method having the ability to detect leaks at the product's maximum allowable leakage limit is preferred when establishing the inherent integrity of a container-closure system." Ideally, container closure integrity of parenteral packaging would be evaluated by measuring a physical property that is sensitive to the presence of any package defect that breaches package integrity by increasing its leakage above its maximum allowable leakage limit. The primary goals of the work presented herein were to demonstrate the viability of the nondestructive, deterministic method known as laser-based gas headspace analysis for evaluating container closure integrity and to provide a physical model for predicting leak rates for a variety of container volumes, headspace conditions, and defect sizes. The results demonstrate that laser-based headspace analysis provides sensitive, accurate, and reproducible measurements of the gas ingress into glass vial-stopper package assemblies that are under either diffusive or effusive leak conditions. Two different types of positive controls were examined. First, laser-drilled micro-holes in thin metal disks that were crimped on top of 15R glass vials served as positive controls with a well-characterized defect geometry. For these, a strong correlation was observed between the measured ingress parameter and the size of the defect for both diffusive and effusive conditions. Second, laser-drilled holes in the wall of glass vials served as controls that more closely simulate real-world defects. Due to their complex defect geometries, their diffusive and effusive ingress parameters did not necessarily correlate; this is an important observation that has significant implications for standardizing the characterization of container defects. Regardless, laser-based headspace analysis could readily differentiate positive and negative controls for all leak conditions, and the results provide a guide for method development of container closure integrity tests.LAY ABSTRACT: The new USP 39 <1207>, "Package Integrity Evaluation-Sterile Products", states in section 3.4.1: "tracer gas tests performed using … laser-based gas headspace analysis [have] been shown to be sensitive enough to quantitatively analyze leakage through the smallest leak paths found to pose the smallest chance of liquid leakage or microbial ingress in rigid packaging." In addition, USP <1207> also states that "for such methods, the limit of detection can be mathematically predicted on the basis of gas flow kinetics." Using the above statements as a foundation, this paper presents a theoretical basis for predicting the gas ingress through well-defined defects in product vials sealed under a variety of headspace conditions. These calculated predictions were experimentally validated by comparing them to measurements of changes in the headspace oxygen content or total pressure for several different positive controls using laser-based headspace analysis. The results demonstrated that laser-based headspace analysis can, by readily differentiating between negative controls and positive controls with a range of defect sizes on the micron scale, be used to assess container closure integrity. The work also demontrated that caution must be used when attempting to correlate a leak rate to an idealized defect-size parameter.

A Prospective Clinical Trial Comparing Visible Light Spectroscopy to Handheld Doppler for Postoperative Free Tissue Transfer Monitoring.

BACKGROUND: Early detection of compromised free flap perfusion is critical. A common modality of thrombosis detection, physical examination augmented with hand-held Doppler, provides only intermittent data and is insensitive to venous compromise. Visible light spectroscopy provides continuous, noninvasive evaluation of tissue perfusion. The authors hypothesized that visible light spectroscopy is a more sensitive and specific monitoring method for early detection of postoperative flap compromise than intermittent hand-held Doppler and clinical examination. METHODS: The authors prospectively conducted a controlled study evaluating the sensitivity, specificity, and accuracy of the T-Stat Model 303 VLS oximeter versus that of intermittent Doppler and clinical examination. The authors prospectively collected and analyzed patient data, complications, reoperations, flap failures, and tissue oxygen saturation. RESULTS: Sixty-eight patients with 81 flaps completed the study. The majority of flaps (86.4 percent) were either transverse rectus abdominis musculocutaneous or deep inferior epigastric artery perforator flaps. The mean tissue oxygen saturation for all flaps in the study was 56.7 ± 7.1 percent (range, 39.4 to 72.1 percent) and did not differ significantly with patient comorbidity or flap type. During the course of the study, three flaps were returned to the operating room for exploration because of a perfusion abnormality, and the resulting salvage rate was 100 percent. The sensitivity, specificity, and accuracy of visible light spectroscopy were found to be greater than both intermittent Doppler and clinical examination. CONCLUSION: Visible light spectroscopy is a reliable, continuous adjunct to free tissue transfer monitoring with advantages over intermittent hand-held Doppler and clinical examination. CLINICAL QUESTION/LEVEL OF EVIDENCE: Diagnostic, II.

Development and validation of a noncontact spectroscopic device for hemoglobin estimation at point-of-care.

Anemia severely and adversely affects human health and socioeconomic development. Measuring hemoglobin with the minimal involvement of human and financial resources has always been challenging. We describe a translational spectroscopic technique for noncontact hemoglobin measurement at low-resource point-of-care settings in human subjects, independent of their skin color, age, and sex, by measuring the optical spectrum of the blood flowing in the vascular bed of the bulbar conjunctiva. We developed software on the LabVIEW platform for automatic data acquisition and interpretation by nonexperts. The device is calibrated by comparing the differential absorbance of light of wavelength 576 and 600 nm with the clinical hemoglobin level of the subject. Our proposed method is consistent with the results obtained using the current gold standard, the automated hematology analyzer. The proposed noncontact optical device for hemoglobin estimation is highly efficient, inexpensive, feasible, and extremely useful in low-resource point-of-care settings. The device output correlates with the different degrees of anemia with absolute and trending accuracy similar to those of widely used invasive methods. Moreover, the device can instantaneously transmit the generated report to a medical expert through e-mail, text messaging, or mobile apps.

Accurate and interpretable classification of microspectroscopy pixels using artificial neural networks.

This paper addresses the problem of classifying materials from microspectroscopy at a pixel level. The challenges lie in identifying discriminatory spectral features and obtaining accurate and interpretable models relating spectra and class labels. We approach the problem by designing a supervised classifier from a tandem of Artificial Neural Network (ANN) models that identify relevant features in raw spectra and achieve high classification accuracy. The tandem of ANN models is meshed with classification rule extraction methods to lower the model complexity and to achieve interpretability of the resulting model. The contribution of the work is in designing each ANN model based on the microspectroscopy hypothesis about a discriminatory feature of a certain target class being composed of a linear combination of spectra. The novelty lies in meshing ANN and decision rule models into a tandem configuration to achieve accurate and interpretable classification results. The proposed method was evaluated using a set of broadband coherent anti-Stokes Raman scattering (BCARS) microscopy cell images (600 000  pixel-level spectra) and a reference four-class rule-based model previously created by biochemical experts. The generated classification rule-based model was on average 85% accurate measured by the DICE pixel label similarity metric, and on average 96% similar to the reference rules measured by the vector cosine metric.