Infrared Microspectroscopy Imaging Research Articles

Application of FTIR imaging to detect dietary induced biochemical changes in brown and white adipocytes

Fourier Transform Infrared microspectroscopy Imaging (FTIR imaging) has powerful advantages when used as a complementary technique to histopathological investigations. The common histopathological investigations use a number of chemical fixatives and deparaffinizing agents that alter lipid profiles of biological tissues, particularly in lipid-rich adipose tissues. An infrared image is made of a multitude of pixels. For each pixel, identified by its (x,y) coordinates, corresponds an FTIR spectrum that reveals the chemical composition at this specific location. As a result, it allows biochemical analysis in selective locations within samples. Moreover, functional group distribution maps can be used as a staining free technique for spatial characterization of different biomolecules. Though FTIR imaging has been reported as a powerful approach to characterize adipose tissues, the biochemical heterogeneity within adipose tissues has not been acknowledged. This study shows relative changes in brown and white adipose tissues of mice due to consumption of high-fat diet. In particular, we report cryosectioning of adipose tissues, image acquisition, and different image analysis methods to evaluate dietary induced changes in lipids stored in brown and white adipocytes. Since biochemical changes in adipocytes constitute an important component in obesity investigations, this study shows the potential use of FTIR imaging to compare relative biochemical changes associated with dietary interventions.

Molecular weight and organization of cellulose at different stages of cotton fiber development

There is a continuous change in cell wall composition and organization during cotton fiber development. Cotton fiber strength correlates to the molecular weight (MW) and molecular weight distribution (MWD), and organization of cellulose chains in the secondary cell wall. These parameters change drastically during fiber development. This study reports on the MW, MWD, and organization of cellulose in cotton fibers harvested from two cotton cultivars of Gossypium hirsutum L., (Texas Marker-1 and TX55) at different levels of maturity. Fiber dissolution is necessary to estimate the molecular properties of cellulose. Cellulose in mature cotton fibers is larger in MW and highly crystalline and, therefore, poorly dissolves in common solvent systems. To facilitate the dissolution, fibers were first pretreated with 23% sodium hydroxide and then dissolved in a dimethylacetamide/lithium chloride solvent system. Gel permeation chromatography of dissolved fibers indicated that cellulose in both cultivars reaches its maximum MW around 30 days post anthesis. Fourier transform infrared microspectroscopy imaging in the transmission mode indicates changes in cellulose distribution in cotton fibers with fiber development. The distributions of infrared vibrations of cellulose at 897 (β-linkage of cellulose), 1161 (anti-symmetrical C-O-C stretching of cellulose), and 1429 cm−1 (CH2 scissoring of cellulose) provided information on cellulose deposition in intact cotton fibers.

Analysis of Counterfeit Coated Tablets and Multi-Layer Packaging Materials Using Infrared Microspectroscopic Imaging.

An effective method for detecting and characterizing counterfeit finished dosage forms and packaging materials is described in this study. Using attenuated total internal reflection Fourier transform infrared spectroscopic imaging, suspect tablet coating and core formulations as well as multi-layered foil safety seals, bottle labels, and cigarette tear tapes were analyzed and compared directly with those of a stored authentic product. The approach was effective for obtaining molecular information from structures as small as 6 μm.

The Advantages of an Attenuated Total Internal Reflection Infrared Microspectroscopic Imaging Technique for the Analysis of Polymer Laminates.

Until recently, the analysis of polymer laminates using infrared microspectroscopy involved the painstaking separation of individual layers by dissection or by obtaining micrometer thin cross-sections. The latter usually requires the expertise of an individual trained in microtomy and even then, the very structure of the laminate could affect the outcome of the spectral results. The recent development of attenuated total internal reflection (ATR) infrared microspectroscopy imaging has provided a new avenue for the analysis of these multilayer structures. This report compares ATR infrared microspectroscopy imaging with conventional transmission infrared microspectroscopy imaging. The results demonstrate that the ATR method offers improved spatial resolution, eliminates a variety of competing optical processes, and requires minimal sample preparation relative to transmission measurements. These advantages were illustrated using a polymer laminate consisting of 11 different layers whose thickness ranged in size from 4-20 μm. The spatial resolution achieved by using an ATR-FTIR (Fourier transform infrared spectroscopy) imaging technique was diffraction limited. Contrast in the ATR images was enhanced by principal component analysis.

Abstract 4292: Fast and automated assessment of tumor response: Infrared imaging

Abstract The rabbit Vx2 tumor is a fast-growing carcinoma model, which is commonly used to study different aspects of tumor behaviour under cancer treatments. The reduction of tumor viability and the degree of induced necrosis are the most common criteria to evaluate the efficacy of cancer treatments. The most recent developments in infrared microspectroscopy (IRMS) imaging aimed at automating the procedure of tissue recognition and quantification by using statistical methods and prediction algorithm. We used IRMS for the automatic characterization and quantification of the Vx2 liver tumor viability after a chemoembolization treatment. Twenty-eight rabbits with Vx2 liver tumor were included in this study: 20 rabbits were subjected to a Doxorubicin eluting beads (DEB) treatment and compared to a control (CTRL) group of 8 rabbits. The tumor bearing livers were resected, fixed in formalin and embedded in paraffin. Two adjacent sections were cut from each sample using a microtome. The first section was mounted on a calcium fluoride window suitable for IRMS imaging. The second section was put on a standard glass slide and stained with HES to serve as a control for IRMS imaging. On a first series of 14 different tumor sections (CTRL: 7 sections, DEB: 7 sections), we developed and validated a prediction algorithm. The protocol consisted of K-means (KM) clustering followed by linear discriminant analysis (LDA). The KM clustering was used to classify the spectra from the infrared images and to build a data base containing a large number of reference spectra (397.289 spectra) characteristics for tumor necrosis, viable tumor, fibrosis, liver parenchyma and liver parenchyma necrosis. Once the model validated, we empirically attributed a color for each type of tissue. Then, the predictive model was applied to infrared images of 52 new test tumor sections (CTRL: 9 sections, DEB: 43 sections). The result of the LDA model analysis is a false color image where each color corresponds to a type of tissue. The percentage of pixels corresponding to each color is automatically recorded by the LDA model. This advantage was used to calculate the mean percentage of surface occupied by each type of tissue and to determine the tumor viability after the DEB treatment. The LDA false color images reproduced the histological structures of Vx2 liver tumors. The sensitivity and specificity of the LDA model were high to 86.7% and 96.7% for the 5 tissue types respectively. For DEB group, the LDA model determined that the surface of necrotic tissue represented 77.68±23 % (CTRL group: 16.89±9 %, Mann Whitney: P<0.0001), the viable tumor 14.29±23 % (CTRL group: 74.74±7 %, MW: P< 0.0001) and fibrosis 3.89±6 % of the tumor (CTRL group: 1.1±2 %, MW: P= 0.6262). The remaining percentage corresponded to unclassified spectra (DEB group: 3.94±7 %, CTRL group: 6.40±5 %, MW: P=0.0135). Our results show that IR imaging coupled with LDA model analysis could be a helpful to easily assess tumor response. Citation Format: Hadrien D'inca, Florentina Pascale, Saida Homayra Ghegediban, Michel Wassef, Cyril Gobinet, Julien Namur, Alexandre Laurent, Michel Manfait. Fast and automated assessment of tumor response: Infrared imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4292. doi:10.1158/1538-7445.AM2014-4292

Direct observation of bulk and surface chemical morphologies of Ginkgo biloba leaves by Fourier transform mid- and near-infrared microspectroscopic imaging

Fourier transform infrared microspectroscopy is a powerful tool to obtain knowledge about the spatial and/or temporal distributions of the chemical compositions of plants for better understanding of their biological properties. However, the chemical morphologies of plant leaves in the plane of the blade are barely studied, because sections in this plane for mid-infrared transmission measurements are difficult to obtain. Besides, native compositions may be changed by chemical reagents used when plant samples are microtomed. To improve methods for direct infrared microspectroscopic imaging of plant leaves in the plane of the blade, the bulk and surface chemical morphologies of nonmicrotomed Ginkgo biloba leaves were characterized by near-infrared transmission and mid-infrared attenuated total reflection microspectroscopic imaging. A new self-modeling curve resolution procedure was proposed to extract the spectral and concentration information of pure compounds. Primary and secondary metabolites of secretory cavities, veins, and mesophylls of Ginkgo biloba leaf blades were analyzed, and the distributions of cuticle, protein, calcium oxalate, cellulose, and ginkgolic acids on the adaxial surface were determined. By the integration of multiple infrared microspectroscopic imaging and chemometrics methods, it is possible to analyze nonmicrotomed leaves and other plant samples directly to understand their native chemical morphologies in detail.

A Comparison of Reflectance and Attenuated Total Internal Reflection Infrared Microspectroscopic Imaging Techniques for the Analysis of Kidney Stones

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Characterization of the structure of human skin substitutes by infrared microspectroscopy

The skin acts mainly as a protective barrier from the external environment, thanks to the stratum corneum which is the outermost layer of the skin. As in vitro tests on skin are essential to elaborate new drugs, the development of skin models closer to reality becomes essential. It is now possible to produce in vitro human skin substitutes through tissue engineering by using the self-assembly method developed by the Laboratoire d'Organogénèse Expérimentale. In the present work, infrared microspectroscopy imaging analyses were performed to get in-depth morpho-spectral characterization of the three characteristic layers of human skin substitutes and normal human skin, namely the stratum corneum, living epidermis, and dermis. An infrared spectral analysis of the skin is a powerful tool to gain information on the order and conformation of the lipid chains and the secondary structure of proteins. On one hand, the symmetric stretching mode of the lipid methylene groups (2,850cm(-1)) is sensitive to the acyl chain conformational order. The evolution profile of the frequency of this vibrational mode throughout the epidermis suggests that lipids in the stratum corneum are more ordered than those in the living epidermis. On the other hand, the frequencies of the infrared components underneath the envelop of the amide I band provide information about the overall protein conformation. The analysis of this mode establishes that the proteins essentially adopt an α-helix conformation in the epidermis, probably associated with the presence of keratin, while modifications of the protein content are observed in the dermis (extracellular matrix made of collagen). Finally, the lipid organization, as well as the protein composition in the different layers, is similar for human skin substitutes and normal human skin, confirming that the substitutes reproduce essential features of real skin and are appropriate biomimetics.

Hyperspectral Data Restoration in Synchrotron-based Infrared Microspectroscopic Imaging

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Discrimination between healthy and tumor tissues on formalin-fixed paraffin-embedded breast cancer samples using IR imaging

This work presents a pilot study to illustrate the potential of Fourier transform infrared (FT-IR) imaging in breast cancer research. Using this technique, we have acquired infrared (IR) microspectroscopic images from healthy and cancerous breast tissue section from one patient. First of all, a Student t-test was applied, showing DNA/RNA spectral region (1400–1000 cm−1) as the most discriminant for the differentiation between healthy and tumor samples. Afterwards, a supervised pattern recognition method, Partial Least Squares (PLS) was used to develop an automated classifier to discriminate the two classes of data. Infrared spectra of independent IR measurements were used to test the classifier. The class identity was correlated with information obtained by histopathologic gold standard. The results showed that more than 95% of the training and validation spectra were correctly identified. We demonstrate that combination between IR microspectroscopic imaging and multivariate data analysis can be used as a complement to present diagnostic tools for breast cancer.

Discrimination between healthy and tumor tissues on formalin-fixed paraffin-embedded breast cancer samples using IR imaging

This work presents a pilot study to illustrate the potential of Fourier transform infrared (FT-IR) imaging in breast cancer research. Using this technique, we have acquired infrared (IR) microspectroscopic images from healthy and cancerous breast tissue section from one patient. First of all, a Studentt-test was applied, showing DNA/RNA spectral region (1400–1000 cm−1) as the most discriminant for the differentiation between healthy and tumor samples. Afterwards, a supervised pattern recognition method, Partial Least Squares (PLS) was used to develop an automated classifier to discriminate the two classes of data. Infrared spectra of independent IR measurements were used to test the classifier. The class identity was correlated with information obtained by histopathologic gold standard. The results showed that more than 95% of the training and validation spectra were correctly identified. We demonstrate that combination between IR microspectroscopic imaging and multivariate data analysis can be used as a complement to present diagnostic tools for breast cancer.

Analysis of Composition Distribution in High Impact Polypropylene Particles Using Synchrotron Infrared Microspectroscopy Imaging

Composition distributions in high impact polypropylene particles synthesized with Ziegler-Natta catalysts were investigated by using the SPring-8 infrared microscope apparatus. High impact polypropylene is prepared in two stages. At first the homopolymers are produced from propylene, and in the following stage copolymers are synthesized from propylene and ethylene. In most of the second stage, gas phase polymerization processes using heterogeneous Ziegler-Natta catalysts are applied. A particle of the heterogeneous catalyst is the aggregation of the first microparticles. In the polymerization reaction, gaseous monomers contact the microparticles to produce polymers around them, and the growing microparticles result in increasing the size of the second macroparticles. Several kinds of high impact polypropylene particles were cut into cross sections by means of single edge industrial blades. The composition distributions of the samples were measured by using infrared microspectroscopy imaging. It was found that the propylene-ethylene copolymer with high ethylene concentration spots was formed around the surface of the particles. In the case of the particle having voids inside, high ethylene concentration spots were formed not only around the surface but also near the interface of the voids. Mechanism of the formation of high ethylene concentration spots was considered.

The Advantages of an Attenuated Total Internal Reflection Infrared Microspectroscopic Imaging Approach for Kidney Biopsy Analysis

The benefits of an attenuated total reflection Fourier transform infrared (ATR-FTIR) imaging approach for kidney biopsy analysis are described. Biopsy sections collected from kidney-stone formers are analyzed at the initial stages of stone development to provide insights into stone growth and formation. The majority of tissue analysis currently conducted with IR microspectroscopy is performed with a transflection method. The research presented in this manuscript demonstrates that ATR overcomes many of the disadvantages of transflection or transmission measurements for tissue analysis including an elimination of spectral artifacts. When kidney biopsies with small mineral inclusions are analyzed with a transflection approach, specular reflection and the Christiansen effect (anomalous dispersion) can occur, leading to spectral artifacts. Another effect specific to the analysis of mineral inclusions present in kidney biopsies is known as the reststrahlen effect whereby the inclusions become strong reflectors near an absorption band. ATR eliminates these effects by immersing the sample in a high index medium. Additionally, the focused beam size for ATR is decreased by a factor of four when a germanium internal reflection element is used, allowing the acquisition of spectra from small mineral inclusions several micrometers in diameter. If quantitative analysis of small mineral inclusions is ultimately desired, ATR provides the photometrically accurate spectra necessary for quantification.

Delimitation of squamous cell cervical carcinoma using infrared microspectroscopic imaging

Infrared (IR) spectroscopic imaging coupled with microscopy has been used to investigate thin sections of cervix uteri encompassing normal tissue, precancerous structures, and squamous cell carcinoma. Methods for unsupervised distinction of tissue types based on IR spectroscopy were developed. One-hundred and twenty-two images of cervical tissue were recorded by an FTIR spectrometer with a 64x64 focal plane array detector. The 499,712 IR spectra obtained were grouped by an approach which used fuzzy C-means clustering followed by hierarchical cluster analysis. The resulting false color maps were correlated with the morphological characteristics of an adjacent section of hematoxylin and eosin-stained tissue. In the first step, cervical stroma, epithelium, inflammation, blood vessels, and mucus could be distinguished in IR images by analysis of the spectral fingerprint region (950-1480 cm(-1)). In the second step, analysis in the spectral window 1420-1480 cm(-1) enables, for the first time, IR spectroscopic distinction between the basal layer, dysplastic lesions and squamous cell carcinoma within a particular sample. The joint application of IR microspectroscopic imaging and multivariate spectral processing combines diffraction-limited lateral optical resolution on the single cell level with highly specific and sensitive spectral classification on the molecular level. Compared with previous reports our approach constitutes a significant progress in the development of optical molecular spectroscopic techniques toward an additional diagnostic tool for the early histopathological characterization of cervical cancer.

Hepatobiliary and pancreatic: Liver histopathology: Fourier transform infrared microspectroscopic imaging for objective and quantifiable assessment of liver biopsies

Histopathological changes in the liver are conventionally assessed using histochemical stains and examination by an experienced histopathologist. Sampling error and interobserver variation were addressed by Bedossa et al. who called for objective measurements by imaging technologies. We are investigating the applicability of FourierTransform Infrared Microspectroscopic Imaging (FTIRMI) to the analysis of liver biopsies. One previous study has been reported. Moreover, microimaging could find additional applications, including investigating lesions not amenable to histochemical study such as drug accumulations in liver cells. The applications of infrared spectroscopy range from analysis of interstellar dust particles to detection of cancer cells in cervical biopsies. The infrared spectrum of any compound is in effect a unique molecular fingerprint. The positions of observed peaks depend on the masses of the atoms, the strengths of the bonds between them, and the interaction with the surrounding molecular environment. Furthermore, the absorbance is directly proportional to analyte concentration. Lewis et al. revolutionized infrared imaging analysis by combining a focal plane array detector with a FTIR microscope, allowing insight into the molecular structure of tissues. The diagnostic and monitoring capacity of FTIRMI has the premise that a chemical change must accompany morphological expression. Thus, morphological changes are readily detected by FTIRMI as changes in the concentration and conformational orientation of functional groups associated with proteins, lipids, nucleic acids and carbohydrates. Techniques such as Unsupervised Hierarchical Cluster Analysis (UHCA) can be used to analyze the ‘spectral data hypercubes’ that constitute the individual pixels in the focal plane array. Our images exemplify the technique applied to a cirrhotic liver. Figure 1 is a Masson stain (original magnification ¥ 100) while Figure 2 is an image derived by performing UHCA on spectral data obtained with the DigiLab ‘Stingray’ FPA system. The images show regions both with measurably high levels of collagen (fibrosis) and glycogen (liver parenchyma).

Effective time averaging of multiplexed measurements: a critical analysis.

Multiplexing and time averaging of signal are effective noise reduction protocols applied in many analytical measurement systems. The efficacy of these protocols may be reduced by random occurrences of high-magnitude noise that do not conform to the statistical distribution of noise for all other measurements in the data set. This high-magnitude noise, which may have an insignificant probability of occurrence for a single measurement, almost certainly affects data collected in a multichannel, multiplexed modality, such as Fourier transform infrared (FT-IR) spectroscopic imaging employing focal plane array detectors. To recover time-averaging advantages in these cases, we present a general coaddition method that uses two statistical measures, the mean and median of the ensemble of measurements of a signal, to obtain a better estimate of the true signal than that estimated by time averaging alone. This method, termed median filtered time averaging, is shown to be an effective noise removal procedure for FT-IR imaging data. The effects of noise removal on time averaging and multiplexing are examined theoretically and are demonstrated for hyperspectral infrared microspectroscopic imaging data obtained from human skin biopsies by using a rapid data acquisition procedure.