Using machine learning models to estimate Escherichia coli concentration in an irrigation pond from water quality and drone-based RGB imagery data.

The rapid and efficient quantification of Escherichia coli concentrations is crucial for monitoring water quality. Remote sensing techniques and machine learning algorithms have been used to detect E. coli in water and estimate its concentrations. The application of these approaches, however, is challenged by limited sample availability and unbalanced water quality datasets. In this study, we estimated the E. coli concentration in an irrigation pond in Maryland, USA, during the summer season using demosaiced natural color (red, green, and blue: RGB) imagery in the visible and infrared spectral ranges, and a set of 14 water quality parameters. We did this by deploying four machine learning models - Random Forest (RF), Gradient Boosting Machine (GBM), Extreme Gradient Boosting (XGB), and K-nearest Neighbor (KNN) - under three data utilization scenarios: water quality parameters only, combined water quality and small unmanned aircraft system (sUAS)-based RGB data, and RGB data only. To select the training and test datasets, we applied two data-splitting methods: ordinary and quantile data splitting. These methods provided a constant splitting ratio in each decile of the E. coli concentration distribution. Quantile data splitting resulted in better model performance metrics and smaller differences between the metrics for both the training and testing datasets. When trained with quantile data splitting after hyperparameter optimization, models RF, GBM, and XGB had R2 values above 0.847 for the training dataset and above 0.689 for the test dataset. The combination of water quality and RGB imagery data resulted in a higher R2 value (>0.896) for the test dataset. Shapley additive explanations (SHAP) of the relative importance of variables revealed that the visible blue spectrum intensity and water temperature were the most influential parameters in the RF model. Demosaiced RGB imagery served as a useful predictor of E. coli concentration in the studied irrigation pond.

Prediction of Soluble-Solid Content in Citrus Fruit Using Visible-Near-Infrared Hyperspectral Imaging Based on Effective-Wavelength Selection Algorithm.

Citrus fruits were sorted based on external qualities, such as size, weight, and color, and internal qualities, such as soluble solid content (SSC), acidity, and firmness. Visible and near-infrared (VNIR) hyperspectral imaging techniques were used as rapid and nondestructive techniques for determining the internal quality of fruits. The applicability of the VNIR hyperspectral imaging technique for predicting the SSC in citrus fruits was evaluated in this study. A VNIR hyperspectral imaging system with a wavelength range of 400-1000 nm and 100 W light source was used to acquire hyperspectral images from citrus fruits in two orientations (i.e., stem and calyx ends). The SSC prediction model was developed using partial least-squares regression (PLSR). Spectrum preprocessing, effective wavelength selection through competitive adaptive reweighted sampling (CARS), and outlier detection were used to improve the model performance. The performance of each model was evaluated using the coefficient of determination (R2) and root mean square error (RMSE). In the present study, the PLSR model was developed using only a citrus cultivar. The SSC prediction CARS-PLSR model with outliers removed exhibited R2 and RMSE values of approximatively 0.75 and 0.56 °Brix, respectively. The results of this study are expected to be useful in similar fields such as agricultural and food post-harvest management, as well as in the development of an online system for determining the SSC of citrus fruits.

Detection of aflatoxins in ground maize using a compact and automated Raman spectroscopy system with machine learning.

Consumption of aflatoxin-contaminated food can cause severe illness when consumed by humans or livestock. Because the mycotoxin frequently occurs in cereal grains and other agricultural crops, it is crucial to develop portable devices that can be used non-destructively and in real-time to identify aflatoxin-contaminated food materials during early stages of harvesting or processing. In this study, an aflatoxin detection method was developed using a compact Raman device that can be used in the field. Data were obtained using maize samples naturally contaminated with aflatoxin, and the data were analyzed using a machine learning method. Of the multiple classification models evaluated, such as linear discriminant analysis (LDA), linear support vector machines (LSVM), quadratic discriminant analysis (QDA), and quadratic support vector machines and spectral preprocessing methods, the best classification accuracy was achieved at 95.7% using LDA in combination with Savitzky-Golay 2nd derivative (SG2) preprocessing. Partial least squares regression (PLSR) models demonstrated a close-range accuracy within the scope of standard normal variate (SNV) and multiplicative scatter correction (MSC) preprocessing methods, with determination of coefficient values of R2C and R2V of 0.9998 and 0.8322 respectively for SNV, and 0.9916 and 0.8387 respectively for MSC. This study demonstrates the potential use of compact and automated Raman spectroscopy, coupled with chemometrics and machine learning methods, as a tool for rapidly screening food and feed for hazardous substances at on-site field processing locations.

Advancement of non-destructive spectral measurements for the quality of major tropical fruits and vegetables: a review.

The quality of tropical fruits and vegetables and the expanding global interest in eating healthy foods have resulted in the continual development of reliable, quick, and cost-effective quality assurance methods. The present review discusses the advancement of non-destructive spectral measurements for evaluating the quality of major tropical fruits and vegetables. Fourier transform infrared (FTIR), Near-infrared (NIR), Raman spectroscopy, and hyperspectral imaging (HSI) were used to monitor the external and internal parameters of papaya, pineapple, avocado, mango, and banana. The ability of HSI to detect both spectral and spatial dimensions proved its efficiency in measuring external qualities such as grading 516 bananas, and defects in 10 mangoes and 10 avocados with 98.45%, 97.95%, and 99.9%, respectively. All of the techniques effectively assessed internal characteristics such as total soluble solids (TSS), soluble solid content (SSC), and moisture content (MC), with the exception of NIR, which was found to have limited penetration depth for fruits and vegetables with thick rinds or skins, including avocado, pineapple, and banana. The appropriate selection of NIR optical geometry and wavelength range can help to improve the prediction accuracy of these crops. The advancement of spectral measurements combined with machine learning and deep learning technologies have increased the efficiency of estimating the six maturity stages of papaya fruit, from the unripe to the overripe stages, with F1 scores of up to 0.90 by feature concatenation of data developed by HSI and visible light. The presented findings in the technological advancements of non-destructive spectral measurements offer promising quality assurance for tropical fruits and vegetables.

Protein and lipid content estimation in soybeans using Raman hyperspectral imaging.

Unlike standard chemical analysis methods involving time-consuming, labor-intensive, and invasive pretreatment procedures, Raman hyperspectral imaging (HSI) can rapidly and non-destructively detect components without professional supervision. Generally, the Kjeldahl methods and Soxhlet extraction are used to chemically determine the protein and lipid content of soybeans. This study is aimed at developing a high-performance model for estimating soybean protein and lipid content using a non-destructive Raman HSI. Partial least squares regression (PLSR) techniques were used to develop the model using a calibration model based on 70% spectral data, and the remaining 30% of the data were used for validation. The results indicate that the Raman HSI, combined with PLSR, resulted in a protein and lipid model Rp2 of 0.90 and 0.82 with Root Mean Squared Error Prediction (RMSEP) 1.27 and 0.79, respectively. Additionally, this study successfully used the Raman HSI approach to create a prediction image showing the distribution of the targeted components, and could predict protein and lipid based on a single seeds.

A hyperspectral plant health monitoring system for space crop production.

Compact and automated sensing systems are needed to monitor plant health for NASA's controlled-environment space crop production. A new hyperspectral system was designed for early detection of plant stresses using both reflectance and fluorescence imaging in visible and near-infrared (VNIR) wavelength range (400-1000 nm). The prototype system mainly includes two LED line lights providing VNIR broadband and UV-A (365 nm) light for reflectance and fluorescence measurement, respectively, a line-scan hyperspectral camera, and a linear motorized stage with a travel range of 80 cm. In an overhead sensor-to-sample arrangement, the stage translates the lights and camera over the plants to acquire reflectance and fluorescence images in sequence during one cycle of line-scan imaging. System software was developed using LabVIEW to realize hardware parameterization, data transfer, and automated imaging functions. The imaging unit was installed in a plant growth chamber at NASA Kennedy Space Center for health monitoring studies for pick-and-eat salad crops. A preliminary experiment was conducted to detect plant drought stress for twelve Dragoon lettuce samples, of which half were well-watered and half were under-watered while growing. A machine learning method using an optimized discriminant classifier based on VNIR reflectance spectra generated classification accuracies over 90% for the first four days of the stress treatment, showing great potential for early detection of the drought stress on lettuce leaves before any visible symptoms and size differences were evident. The system is promising to provide useful information for optimization of growth environment and early mitigation of stresses in space crop production.

Rapid Detection of Single- and Co-Contaminant Aflatoxins and Fumonisins in Ground Maize Using Hyperspectral Imaging Techniques.

Aflatoxins and fumonisins, commonly found in maize and maize-derived products, frequently co-occur and can cause dangerous illness in humans and animals if ingested in large amounts. Efforts are being made to develop suitable analytical methods for screening that can rapidly detect mycotoxins in order to prevent illness through early detection. A method for classifying contaminated maize by applying hyperspectral imaging techniques including reflectance in the visible and near-infrared (VNIR) and short-wave infrared (SWIR) regions, and fluorescence was investigated. Machine learning classification models in combination with different preprocessing methods were applied to screen ground maize samples for naturally occurring aflatoxin and fumonisin as single contaminants and as co-contaminants. Partial least squares-discriminant analysis (PLS-DA) and support vector machine (SVM) with the radial basis function (RBF) kernel were employed as classification models using cut-off values of each mycotoxin. The classification performance of the SVM was better than that of PLS-DA, and the highest classification accuracies for fluorescence, VNIR, and SWIR were 89.1%, 71.7%, and 95.7%, respectively. SWIR imaging with the SVM model resulted in higher classification accuracies compared to the fluorescence and VNIR models, suggesting that as an alternative to conventional wet chemical methods, the hyperspectral SWIR imaging detection model may be the more effective and efficient analytical tool for mycotoxin analysis compared to fluorescence or VNIR imaging models. These methods represent a food safety screening tool capable of rapidly detecting mycotoxins in maize or other food ingredients consumed by animals or humans.

Evaluating performance of SORS-based subsurface signal separation methods using statistical replication Monte Carlo simulation.

Spatially offset Raman spectroscopy (SORS) is a depth-profiling technique with deep information enhancement. However, the interference of the surface layer cannot be eliminated without prior information. The signal separation method is an effective candidate for reconstructing pure subsurface Raman spectra, and there is still a lack of evaluation means for the signal separation method. Therefore, a method based on line-scan SORS combined with improved statistical replication Monte Carlo (SRMC) simulation was proposed to evaluate the effectiveness of food subsurface signal separation method. Firstly, SRMC simulates the photon flux in the sample, generates a corresponding number of Raman photons at each voxel of interest, and collects them by external map scanning. Then, 5625 groups of mixed signals with different optical characteristic parameters were convoluted with spectra of public database and application measurement and introduced into signal separation methods. The effectiveness and application range of the method were evaluated by the similarity between the separated signals and the source Raman spectra. Finally, the simulation results were verified by three packaged foods. FastICA method can effectively separate Raman signals from subsurface layer of food and thus promote deep quality evaluation of food.

Hyperspectral reflectance imaging for nondestructive evaluation of root rot in Korean ginseng (Panax ginseng Meyer).

Root rot of Panax ginseng caused by Cylindrocarpon destructans, a soil-borne fungus is typically diagnosed by frequently checking the ginseng plants or by evaluating soil pathogens in a farm, which is a time- and cost-intensive process. Because this disease causes huge economic losses to ginseng farmers, it is important to develop reliable and non-destructive techniques for early disease detection. In this study, we developed a non-destructive method for the early detection of root rot. For this, we used crop phenotyping and analyzed biochemical information collected using the HSI technique. Soil infected with root rot was divided into sterilized and infected groups and seeded with 1-year-old ginseng plants. HSI data were collected four times during weeks 7-10 after sowing. The spectral data were analyzed and the main wavelengths were extracted using partial least squares discriminant analysis. The average model accuracy was 84% in the visible/near-infrared region (29 main wavelengths) and 95% in the short-wave infrared (19 main wavelengths). These results indicated that root rot caused a decrease in nutrient absorption, leading to a decline in photosynthetic activity and the levels of carotenoids, starch, and sucrose. Wavelengths related to phenolic compounds can also be utilized for the early prediction of root rot. The technique presented in this study can be used for the early and timely detection of root rot in ginseng in a non-destructive manner.

Determination of spectral resolutions for multispectral detection of apple bruises using visible/near-infrared hyperspectral reflectance imaging.

This study demonstrates a method to select wavelength-specific spectral resolutions to optimize a line-scan hyperspectral imaging method for its intended use, which in this case was visible/near-infrared imaging-based multiple-waveband detection of apple bruises. Many earlier studies have explored important aspects of developing apple bruise detection systems, such as key wavelengths and image processing algorithms. Despite the endeavors of many, development of a real-time bruise detection system is not yet a simple task. To overcome these problems, this study investigated selection of optimal wavelength-specific spectral resolutions for detecting bruises on apples by using hyperspectral line-scan imaging with the Random Track function for non-contiguous partial readout, with two experimental parts. The first part identified key-wavelengths and the optimal number of key-wavelengths to use for detecting low-, medium-, and high-impact bruises on apples. These parameters were determined by principal component analysis (PCA) and sequential forward selection (SFS) with four classification methods. The second part determined the optimal spectral resolution for each of the key-wavelengths by selecting and evaluating 21 combinations of exposure time and key-wavelength bandwidths, and then selecting the best combination based on the bruise detection accuracies achieved by each classification method. Each of the four classification methods was found to have a different optimized resolution for high accuracy bruise detection, and the optimized resolutions also allowed for use of shorter exposure times. The results of this work can be used to help develop multispectral imaging systems that provide rapid, cost-effective post-harvest processing to identify bruised apples on commercial processing lines.

Thermosensitive textiles made from silver nanoparticle-filled brown cotton fibers.

Filling fibers with nanomaterials can create new functions or modify the existing properties. However, as nanocomposite formation for natural cellulosic fibers has been challenging, little information is available on how the embedded nanomaterials alter the properties of cellulosic fibers. Here we filled brown cotton fibers with silver nanoparticles (Ag NPs) to examine their thermosensitive properties. Using naturally present tannins in brown cotton fibers as a reducing agent, Ag NP-filled brown cotton fibers (nanoparticle diameter of about 28 nm, weight fraction of 12 500 mg kg-1) were produced through a one-step process without using any external agents. The in situ formation of Ag NPs was uniform across the nonwoven cotton fabric and was concentrated in the lumen of the fibers. The insertion of Ag NPs into the fibers shifted the thermal decomposition of cellulose to lower temperatures with increased activation energy and promoted heat release during combustion. Ag NPs lowered the thermal effusivity of the fabric, causing the fabric to feel warmer than the control brown cotton. Ag NP-filled brown cotton was more effectively heated to higher temperatures than control brown cotton under the same heating treatments.

Comparative Determination of Phenolic Compounds in Arabidopsis thaliana Leaf Powder under Distinct Stress Conditions Using Fourier-Transform Infrared (FT-IR) and Near-Infrared (FT-NIR) Spectroscopy.

The increasing interest in plant phenolic compounds in the past few years has become necessary because of their several important physicochemical properties. Thus, their identification through non-destructive methods has become crucial. This study carried out comparative non-destructive measurements of Arabidopsis thaliana leaf powder sample phenolic compounds using Fourier-transform infrared and near-infrared spectroscopic techniques under six distinct stress conditions. The prediction analysis of 600 leaf powder samples under different stress conditions (LED lights and drought) was performed using PLSR, PCR, and NAS-based HLA/GO regression analysis methods. The results obtained through FT-NIR spectroscopy yielded the highest correlation coefficient (Rp2) value of 0.999, with a minimum error (RMSEP) value of 0.003 mg/g, based on the PLSR model using the MSC preprocessing method, which was slightly better than the correlation coefficient (Rp2) value of 0.980 with an error (RMSEP) value of 0.055 mg/g for FT-IR spectroscopy. Additionally, beta coefficient plots present spectral differences and the identification of important spectral signatures sensitive to the phenolic compounds in the measured powdered samples. Thus, the obtained results demonstrated that FT-NIR spectroscopy combined with partial least squares regression (PLSR) and suitable preprocessing method has a solid potential for non-destructively predicting phenolic compounds in Arabidopsis thaliana leaf powder samples.

Multispectral Wavebands Selection for the Detection of Potential Foreign Materials in Fresh-Cut Vegetables.

Ensuring the quality of fresh-cut vegetables is the greatest challenge for the food industry and is equally as important to consumers (and their health). Several investigations have proven the necessity of advanced technology for detecting foreign materials (FMs) in fresh-cut vegetables. In this study, the possibility of using near infrared spectral analysis as a potential technique was investigated to identify various types of FMs in seven common fresh-cut vegetables by selecting important wavebands. Various waveband selection methods, such as the weighted regression coefficient (WRC), variable importance in projection (VIP), sequential feature selection (SFS), successive projection algorithm (SPA), and interval PLS (iPLS), were used to investigate the optimal multispectral wavebands to classify the FMs and vegetables. The application of selected wavebands was further tested using NIR imaging, and the results showed good potentiality by identifying 99 out of 107 FMs. The results indicate the high applicability of the multispectral NIR imaging technique to detect FMs in fresh-cut vegetables for industrial application.

Estimation of Cold Stress, Plant Age, and Number of Leaves in Watermelon Plants Using Image Analysis.

Watermelon (Citrullus lanatus) is a widely consumed, nutritious fruit, rich in water and sugars. In most crops, abiotic stresses caused by changes in temperature, moisture, etc., are a significant challenge during production. Due to the temperature sensitivity of watermelon plants, temperatures must be closely monitored and controlled when the crop is cultivated in controlled environments. Studies have found direct responses to these stresses include reductions in leaf size, number of leaves, and plant size. Stress diagnosis based on plant morphological features (e.g., shape, color, and texture) is important for phenomics studies. The purpose of this study is to classify watermelon plants exposed to low-temperature stress conditions from the normal ones using features extracted using image analysis. In addition, an attempt was made to develop a model for estimating the number of leaves and plant age (in weeks) using the extracted features. A model was developed that can classify normal and low-temperature stress watermelon plants with 100% accuracy. The R2, RMSE, and mean absolute difference (MAD) of the predictive model for the number of leaves were 0.94, 0.87, and 0.88, respectively, and the R2 and RMSE of the model for estimating the plant age were 0.92 and 0.29 weeks, respectively. The models developed in this study can be utilized in high-throughput phenotyping systems for growth monitoring and analysis of phenotypic traits during watermelon cultivation.

A packaged food internal Raman signal separation method based on spatially offset Raman spectroscopy combined with FastICA.

Raman spectroscopy attempts to reflect food quality by characterizing molecular vibration and rotation. However, the blocking of optical signals by packaging materials and the interference of the optical signal generated by the packaging itself make the detection of internal food quality without destroying packaging highly difficult. In this regard, this paper proposes a novel packaged food internal signal separation based on spatially offset Raman spectroscopy (SORS) coupled with improved fast independent component analysis (FastICA). Firstly, the Raman scattering image of the packaged food with offset laser incident point was obtained. Then, the movable quadratic mean of information entropy was used to select the observation feature region of the image. Thirdly, the main independents decomposed by the optimized FastICA method were identified by spectral attenuation characteristics of the SORS peak signal. Finally, the non-negativity of the separated signal was ensured by baseline recognition and correction. The effectiveness of this method was verified by refactoring the similarity between the signal and the reference signal by testing three different packaging and four internal materials under standard experimental conditions. The applicability of the method was proved by the internal signal separation of three packaged foods on sale. The experimental results indicate that the proposed method can separate the Raman signal of packaged food and can be used as a pretreatment method and auxiliary analysis means for the detection of packaged food.

Combining deep learning and fluorescence imaging to automatically identify fecal contamination on meat carcasses.

Food safety and foodborne diseases are significant global public health concerns. Meat and poultry carcasses can be contaminated by pathogens like E. coli and salmonella, by contact with animal fecal matter and ingesta during slaughter and processing. Since fecal matter and ingesta can host these pathogens, detection, and excision of contaminated regions on meat surfaces is crucial. Fluorescence imaging has proven its potential for the detection of fecal residue but requires expertise to interpret. In order to be used by meat cutters without special training, automated detection is needed. This study used fluorescence imaging and deep learning algorithms to automatically detect and segment areas of fecal matter in carcass images using EfficientNet-B0 to determine which meat surface images showed fecal contamination and then U-Net to precisely segment the areas of contamination. The EfficientNet-B0 model achieved a 97.32% accuracy (precision 97.66%, recall 97.06%, specificity 97.59%, F-score 97.35%) for discriminating clean and contaminated areas on carcasses. U-Net segmented areas with fecal residue with an intersection over union (IoU) score of 89.34% (precision 92.95%, recall 95.84%, specificity 99.79%, F-score 94.37%, and AUC 99.54%). These results demonstrate that the combination of deep learning and fluorescence imaging techniques can improve food safety assurance by allowing the industry to use CSI-D fluorescence imaging to train employees in trimming carcasses as part of their Hazard Analysis Critical Control Point zero-tolerance plan.

Nondestructive Prediction of Isoflavones and Oligosaccharides in Intact Soybean Seed Using Fourier Transform Near-Infrared (FT-NIR) and Fourier Transform Infrared (FT-IR) Spectroscopic Techniques.

The demand for rapid and nondestructive methods to determine chemical components in food and agricultural products is proliferating due to being beneficial for screening food quality. This research investigates the feasibility of Fourier transform near-infrared (FT-NIR) and Fourier transform infrared spectroscopy (FT-IR) to predict total as well as an individual type of isoflavones and oligosaccharides using intact soybean samples. A partial least square regression method was performed to develop models based on the spectral data of 310 soybean samples, which were synchronized to the reference values evaluated using a conventional assay. Furthermore, the obtained models were tested using soybean varieties not initially involved in the model construction. As a result, the best prediction models of FT-NIR were allowed to predict total isoflavones and oligosaccharides using intact seeds with acceptable performance (R2p: 0.80 and 0.72), which were slightly better than the model obtained based on FT-IR data (R2p: 0.73 and 0.70). The results also demonstrate the possibility of using FT-NIR to predict individual types of evaluated components, denoted by acceptable performance values of prediction model (R2p) of over 0.70. In addition, the result of the testing model proved the model's performance by obtaining a similar R2 and error to the calibration model.

Handheld Multispectral Fluorescence Imaging System to Detect and Disinfect Surface Contamination.

Contamination inspection is an ongoing concern for food distributors, restaurant owners, caterers, and others who handle food. Food contamination must be prevented, and zero tolerance legal requirements and damage to the reputation of institutions or restaurants can be very costly. This paper introduces a new handheld fluorescence-based imaging system that can rapidly detect, disinfect, and document invisible organic residues and biofilms which may host pathogens. The contamination, sanitization inspection, and disinfection (CSI-D) system uses light at two fluorescence excitation wavelengths, ultraviolet C (UVC) at 275 nm and violet at 405 nm, for the detection of organic residues, including saliva and respiratory droplets. The 275 nm light is also utilized to disinfect pathogens commonly found within the contaminated residues. Efficacy testing of the neutralizing effects of the ultraviolet light was conducted for Aspergillus fumigatus, Streptococcus pneumoniae, and the influenza A virus (a fungus, a bacterium, and a virus, respectively, each commonly found in saliva and respiratory droplets). After the exposure to UVC light from the CSI-D, all three pathogens experienced deactivation (> 99.99%) in under ten seconds. Up to five-log reductions have also been shown within 10 s of UVC irradiation from the CSI-D system.

High-Throughput Phenotyping Approach for the Evaluation of Heat Stress in Korean Ginseng (Panax ginseng Meyer) Using a Hyperspectral Reflectance Image.

Panax ginseng has been used as a traditional medicine to strengthen human health for centuries. Over the last decade, significant agronomical progress has been made in the development of elite ginseng cultivars, increasing their production and quality. However, as one of the significant environmental factors, heat stress remains a challenge and poses a significant threat to ginseng plants' growth and sustainable production. This study was conducted to investigate the phenotype of ginseng leaves under heat stress using hyperspectral imaging (HSI). A visible/near-infrared (Vis/NIR) and short-wave infrared (SWIR) HSI system were used to acquire hyperspectral images for normal and heat stress-exposed plants, showing their susceptibility (Chunpoong) and resistibility (Sunmyoung and Sunil). The acquired hyperspectral images were analyzed using the partial least squares-discriminant analysis (PLS-DA) technique, combining the variable importance in projection and successive projection algorithm methods. The correlation of each group was verified using linear discriminant analysis. The developed models showed 12 bands over 79.2% accuracy in Vis/NIR and 18 bands with over 98.9% accuracy at SWIR in validation data. The constructed beta-coefficient allowed the observation of the key wavebands and peaks linked to the chlorophyll, nitrogen, fatty acid, sugar and protein content regions, which differentiated normal and stressed plants. This result shows that the HSI with the PLS-DA technique significantly differentiated between the heat-stressed susceptibility and resistibility of ginseng plants with high accuracy.

Continuous gradient temperature Raman spectroscopy of 1-stearoyl- 2-docosahexaenoyl, 1-stearoyl- 2-arachidonoyl, and 1,2-stearoyl phosphocholines.

Mixed chain phospholipids containing a saturated fatty acid at sn1 and a polyunsaturated fatty acid in sn2 are common in the specialized biological membranes prevalent in neural, retinal and organ tissues. Particularly important are mixed lipids containing palmitic or stearic acid and arachidonic or docosahexaenoic acid. Gradient temperature Raman spectroscopy (GTRS) applies the temperature gradients utilized in differential scanning calorimetry to Raman spectroscopy, providing a straightforward technique to identify molecular rearrangements and phase transitions. Herein we utilize GTRS for 1-18:0, 2-20:4n-6 PC; 1-18:0 2-22:6n-3 PC; and 1-18:0, 2-18:0 PC from -80 to 50 °C temperatures. 20 Mb three-dimensional data arrays with 0.2 °C increments and first/second derivatives allowed detailed vibrational mode assignment and analysis. Samples were analyzed neat and with molecular hydration. Previously reported phase transitions for hydrated 18:0-20:4PC and 18:0-22:6PC and numerous spectral differences resulting from hydration and the double bond structure were clearly observed. Molecular models showed that the addition of minimal water molecules results in significant structural differences compared to the neat molecules; 18:0-22:6PC is strikingly compact with water when viewed from the hydrophilic end. This precise Raman data cannot be observed in typically utilized fully hydrated vesicle samples, however the improved GTRS will allow for more precise analysis in fully hydrated vesicles because the underlying modes in the unavoidably broadened spectra can be identified.