Tendinopathy diagnosis and treatment monitoring using attenuated total reflectance‐Fourier transform infrared spectroscopy

Tendinopathy, an important sports injury afflicting athletes and general public, is associated with huge economic losses. The currently used diagnostic tests are subjective, show moderate sensitivity and specificity; while treatment failures persist despite advances in therapy. This highlights the need for tendinopathy diagnostic and treatment monitoring tools. This study investigates tendon injury, natural healing and effect of treatment using ATR‐FTIR complemented with histopathology. Control (C), injured (I) and treated (T) rat tendons were extracted 3, 7, 14 and 28 days post‐injury/treatment, representing phases of healing; and subjected to hematoxylin & eosin staining as well as spectroscopy. While C showed no change, I‐ and T‐related histological changes could be clearly observed in stained sections. ATR‐FTIR spectra highlighted the biochemical changes within groups. Multivariate analysis could classify C, I and T with 75%; different days between groups with 84%; and different days within group with 65% efficiency. Results suggest that such analysis can not only identify C, I or T but also different phases of healing. Difference between I and T at different time points also suggest change in rate of healing. Further studies may help develop this technique for clinical diagnosis and treatment monitoring in future.      

Studies on photosynthetic oxygen-evolving complex by means of Fourier transform infrared spectroscopy: calcium and chloride cofactors

Structural roles of functional Ca²⁺ and Cl⁻ ions in photosynthetic oxygen-evolving complexes (OEC) were studied using low- (640–350 cm⁻¹) and mid- (1800–1200 cm⁻¹) frequency S₂/S₁ Fourier transform infrared (FTIR) difference spectroscopy. Studies using highly active Photosystem (PS) II core particles from spinach enabled the detection of subtle spectral changes. Ca²⁺-depleted and Ca²⁺-reconstituted particles produced very similar mid- and low-frequency spectra. The mid-frequency spectrum was not affected by reconstitution with ⁴⁴Ca isotope. In contrast, Sr²⁺-substituted particles showed unique spectral changes in the low-frequency Mn–O–Mn mode at 606 cm⁻¹ as well as in the mid-frequency carboxylate stretching modes. The mid-frequency spectrum of Cl⁻-depleted OEC exhibited marked changes in the carboxylate stretching modes and the suppression of protein modes compared with that of Cl⁻-reconstituted OEC. However, Cl⁻-depletion did not exert significant effects on the low-frequency spectrum.     

Temperature-pressure stability of green fluorescent protein: a Fourier transform infrared spectroscopy study

Green fluorescent protein (GFP) is widely used as a marker in molecular and cell biology. For its use in high-pressure microbiology experiments, its fluorescence under pressure was recently investigated. Changes in fluorescence with pressure were found. To find out whether these are related to structural changes, we investigated the pressure stability of wild-type GFP (wtGFP) and three of its red shift mutants (AFP, GFP(mut1), and GFP(mut2)) using Fourier transform infrared spectroscopy. For the wt GFP, GFP(mut1), and GFP(mut2) we found that up to 13-14 kbar the secondary structure remains intact, whereas AFP starts unfolding around 10 kbar. The 3-D structure is held responsible for this high-pressure stability. Previously observed changes in fluorescence at low pressure are rationalized in terms of the pressure-induced elastic effect. Above 6 kbar, loss of fluorescence is due to aggregation. Revisiting the temperature stability of GFP, we found that an intermediate state is populated along the unfolding pathway of wtGFP. At higher temperatures, the unfolding resulted in the formation of aggregates of wtGFP and its mutants.     

Minimally invasive screening for colitis using attenuated total internal reflectance fourier transform infrared spectroscopy

This article describes a rapid, simple and cost‐effective technique that could lead to a screening method for colitis without the need for biopsies or in vivo measurements. This screening technique includes the testing of serum using Attenuated Total Reflectance Fourier Transform Infrared (ATR‐FTIR) spectroscopy for the colitis‐induced increased presence of mannose. Chronic (Interleukin 10 knockout) and acute (Dextran Sodium Sulphate‐induced) models for colitis are tested using the ATR‐FTIR technique. Arthritis (Collagen Antibody Induced Arthritis) and metabolic syndrome (Toll like receptor 5 knockout) models are also tested as controls. The marker identified as mannose uniquely screens and distinguishes the colitic from the non‐colitic samples and the controls. The reference or the baseline spectrum could be the pooled and averaged spectra of non‐colitic samples or the subject's previous sample spectrum. This shows the potential of having individualized route maps of disease status, leading to personalized diagnosis and drug management.

     

Study of tumor cell invasion by Fourier transform infrared microspectroscopy

Lung cancer is usually fatal once it becomes metastatic. However, in order to develop metastases, a tumor usually invades the basal membrane and enters the vascular or lymphatic system. In this study, a three-dimensional artificial membrane using collagen type I, one of the main components of basal membranes, was established in order to investigate tumor cell invasion. Lung cancer cell line CALU-1 was seeded on this artificial membrane and cell invasion was studied using the Fourier transform infrared (FTIR) imaging technique. This approach allowed identification of tumor cells invading the collagen type I membrane by means of their infrared spectra and images. The mapping images obtained with FTIR microspectroscopy were validated with standard histological section analysis. The FTIR image produced using a single wavenumber at 1080 cm(-1), corresponding to PO2- groups in DNA from cells, correlated well with the histological section, which clearly revealed a cell layer and invading cells within the membrane. Furthermore, the peaks corresponding to amide A, I, and II in the spectra of the invading cells shifted compared to the noninvading cells, which may relate to the changes in conformation and/or heterogeneity in the phenotype of the cells. The data presented in this study demonstrate that FTIR microspectroscopy can be a fast and reliable technique to assess tumor invasion in vitro.     

Rapid intra‐operative diagnosis of kidney cancer by attenuated total reflection infrared spectroscopy of tissue smears

Herein, a technique to analyze air‐dried kidney tissue impression smears by means of attenuated total reflection infrared (ATR‐IR) spectroscopy is presented. Spectral tumor markers—absorption bands of glycogen—are identified in the ATR‐IR spectra of the kidney tissue smear samples. Thin kidney tissue cryo‐sections currently used for IR spectroscopic analysis lack such spectral markers as the sample preparation causes irreversible molecular changes in the tissue. In particular, freeze‐thaw cycle results in degradation of the glycogen and reduction or complete dissolution of its content. Supervised spectral classification was applied to the recorded spectra of the smears and the test spectra were classified with a high accuracy of 92% for normal tissue and 94% for tumor tissue, respectively. For further development, we propose that combination of the method with optical fiber ATR probes could potentially be used for rapid real‐time intra‐operative tissue analysis without interfering with either the established protocols of pathological examination or the ordinary workflow of operating surgeon. Such approach could ensure easier transition of the method to clinical applications where it may complement the results of gold standard histopathology examination and aid in more precise resection of kidney tumors.      

Rapid intraoperative diagnosis of gynecological cancer by ATR‐FTIR spectroscopy of fresh tissue biopsy

A rapid and reliable intraoperative diagnostic technique to support clinical decisions was developed using Fourier‐transform infrared (FTIR) spectroscopy. Twenty‐six fresh tissue samples were collected intraoperatively from patients undergoing gynecological surgeries. Frozen section (FS) histopathology aimed to discriminate between malignant and benign tumors was performed, and attenuated total reflection (ATR) FTIR spectra were collected from these samples. Digital dehydration and principal component analysis and linear discriminant analysis (PCA‐LDA) models were developed to classify samples into malignant and benign groups. Two validation schemes were employed: k‐fold and “leave one out.” FTIR absorption spectrum of a fresh tissue sample was obtained in less than 5 minutes. The fingerprint spectral region of malignant tumors was consistently different from that of benign tumors. The PCA‐LDA discrimination model correctly classified the samples into malignant and benign groups with accuracies of 96% and 93% for the k‐fold and “leave one out” validation schemes, respectively. We showed that a simple tissue preparation followed by ATR‐FTIR spectroscopy provides accurate means for very rapid tumor classification into malignant and benign gynecological tumors. With further development, the proposed method has high potential to be used as an adjunct to the intraoperative FS histopathology technique.     

Use of Fourier transform infrared (FT-IR) spectroscopy as a tool for pollen identification

Airborne pollen are largely studied to obtain information about the atmospheric content of natural allergens. Aerobiological monitoring networks have been established to provide reliable data that facilitate the timely initiation of preventive actions aimed at minimizing allergic symptoms. Airborne pollen are usually identified and counted using an optical microscope, but as such procedures are extremely time-consuming, more expedient options are being explored. We have assessed the potential of Fourier transform infrared (FT-IR) spectroscopy as an alternative method for the rapid and reliable identification of allergenic pollen using six well-known allergenic pollen taxa and obtaining the respective FT-IR spectra. In doing this, a first IR spectral library has been created. The spectra of unknown pollen were compared to those of the reference library, and two pollen taxa of a mixed sample were identified.     

Dehydrating phospholipid vesicles measured in real-time using ATR Fourier transform infrared spectroscopy

According to the water replacement hypothesis, trehalose stabilizes dry membranes by preventing the decrease in spacing between adjacent phopspholipid headgroups during dehydration. Alternatively, the water-entrapment hypothesis postulates that in the dried state sugars trap residual water at the biomolecule sugar interface. In this study, Fourier transform infrared spectroscopy with an attenuated total reflection accessory was used to investigate the influence of trehalose on the dehydration kinetics and residual water content of egg phosphatidylcholine liposomes in real time under controlled relative humidity conditions. In the absence of trehalose, the lipids displayed a transition to a more ordered gel phase upon drying. The membrane conformational disorder in the dried state was found to decrease with decreasing relative humidity. Even at a relative humidity as high as 94% the conformational disorder of the lipid acyl chains decreased after evaporation of the bulk water. The presence of trehalose affects the rate of water removal from the system and the lipid phase behavior. The rate of water removal is decreased and the residual water content is higher, as compared to drying in the absence of trehalose. During drying, the level of hydrogen bonding to the head groups remains constant. In addition, the conformational disorder of the lipid acyl chains in the dried state more closely resembles that of the lipids in the fully hydrated state. We conclude that water entrapment rather than water replacement explains the effect of trehalose on lipid phase behavior of phosphatidylcholine lipid bilayers during the initial phase of drying.     

Comparative analysis of the amino- and carboxy-terminal domains of calmodulin by Fourier transform infrared spectroscopy

Fourier transform infrared spectra were obtained for mammalian calmodulin and two of its fragments produced by limited proteolysis with trypsin TR₁C (1–77) and TR₂C (78–148). Experiments were done in H₂O, D₂O and D₂O/trifluoroethanol (TFE) mixtures. Information about secondary structure was obtained from analysis of the amide I and II bands; while characteristic absorbances for tyrosine, phenylalanine and carboxylate groups were analyzed for changes in tertiary structure. Our data indicate that the secondary and tertiary structure is preserved in the two half molecules of CaM, both in the apo- and Ca²⁺-saturated state. Addition of the structure-inducing solvent TFE causes marked changes only in the apo-TR₁C domain. The maximum wavenumber for the amide I band of the two domains of CaM in D20 was markedly different (1642 cm^–1 for TR₁C versus 1646/1648 cm^–1 for Ca ²⁺ and apo-TR₂C). This renders the amide I band for the intact protein very broad in comparison to that in other proteins and is indicative of a distribution of -helices with slightly different hydrogen bonding patterns.     

Evaluation of the biological stability of waste during landfill stabilization by thermogravimetric analysis and Fourier transform infrared spectroscopy

This study seeks to assess the biological stability of landfilled municipal solid waste (MSW) based on the changes in organic matter, as revealed by thermogravimetric analysis and Fourier transform infrared (FTIR) spectroscopy. Derivate thermogravimetry profiles (DTG) showed a reduction in peak intensity at 200-350 °C (DTG2), while an increase in peak intensity and a shift towards higher temperature at 400-600 °C (DTG3). The decrease in the peak intensity of the aliphatic methylene at 2920 and 2850 cm(-1), and the increase of aromatic substances and polysaccharide at 1640 cm(-1) in the FTIR spectra also confirm the changes. Well-fitted correlations of the peak intensity ratio (2920/1640) and peak area ratio (DTG2/DTG3) to C/N ratio were also established, confirming that the 2920/1640 and the DTG2/DTG3 ratios can be considered as reliable parameters for tracking the biological stability of MSW during landfill stabilization.     

Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy

Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of freezing-induced membrane dehydration was found to be dependent on the ice nucleation temperature, and showed Arrhenius behavior. The presence of glycerol did not prevent the freezing-induced membrane phase transition, but membrane dehydration occurred more gradual and over a wider temperature range. We describe a method to determine membrane hydraulic permeability parameters (E_Lp, Lpg) at subzero temperatures from membrane phase behavior data. In order to do this, it was assumed that the measured freezing-induced shift in wavenumber position of the symmetric CH₂ stretching band arising from the lipid acyl chains is proportional to cellular dehydration. Membrane permeability parameters were also determined by analyzing the H₂O-bending and -libration combination band, which yielded higher values for both E_Lp and Lpg as compared to lipid band analysis. These differences likely reflect differences between transport of free and membrane-bound water. FTIR allows for direct assessment of membrane properties at subzero temperatures in intact cells. The derived biophysical membrane parameters are dependent on intrinsic cell properties as well as freezing extender composition.     

Time-resolved rapid-scan Fourier transform infrared difference spectroscopy on a noncyclic photosystem: rhodopsin photointermediates from Lumi to Meta II

The visual pigment rhodopsin has been extensively studied for the kinetics of its photointermediates by various spectroscopic methods. Unlike such archaeal retinal proteins as bacteriorhodopsin, visual rhodopsin does not thermally recover its dark state after photoexcitation, which precludes repeated excitation of a single sample and thereby complicates time-resolved experiments. Kinetic data on the late rhodopsin photointermediates have so far been available mainly from time-resolved ultraviolet (UV)-visible spectroscopy, but not from Fourier transform infrared (FTIR) spectroscopy. The latter has the advantage of being informative of structural changes of both chromophore and protein, but does not allow the highly reproducible, automated sample exchange procedures available to UV-visible spectroscopy. Using rapid-scan FTIR difference spectroscopy, we obtained time-resolved data sets that were analyzed by a maximum entropy inverse Laplace-transform. Covering the time range from 8 ms to 15 s at temperatures of 0 and -7 degrees C, the transitions from the Lumi to the Meta I and from the Meta I to the Meta II photoproduct states could be resolved. In the transition from Meta I to Meta II, our data reveal a partial deprotonation of the retinal Schiff base preceding the conformational change of the receptor protein to Meta II. The technique and the results are discussed in regard to its advantages as well as its limitations.     

Fourier transform infrared spectroscopy as a new tool to determine rosmarinic acid in situ

A new procedure has been developed for the in situ FT-IR determination of rosmarinic acid (RA) in suspension cultures of Lavandula officinalis. The method involves sample preparation on ZnSe crystals or microplates from silicon, and measuring absorbance spectra between 4000 and 700 cm(-1). First derivative spectra were analysed after normalisation using partial least square (PLS) algorithm. The correlation between spectral analysis and HPLC measurements of cell extracts shows that the FT-IR procedure is suitable for qualitative and quantitative analyses of RA in cell suspension cultures.     

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.     

Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy

The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Up to now, the determination of kinetic data has been achieved by techniques such as pH-stat titration, ion-selective electrodes, and fluorogenic substrate analogs. We report a new assaying method using in situ Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR) for the real-time determination of reaction rates. The method employs changes in the P-O-R stretching vibration of DFP and nerve agent substrates when hydrolyzed to their corresponding phosphoric and phosphonic acids. It is shown that the Lambert-Beer law holds and that changes in absorbance can be directly related to changes in concentration. Compared with other methods, the use of in situ FTIR spectroscopy results in a substantially reduced reaction volume that adds extra work safety when handling highly toxic substrates. In addition, the new method allows the noninvasive measurement of buffered solutions with varying ionic strengths complementing existing methods. Because the assay is independent of the used enzyme, it should also be applicable to other phosphotriesterase enzymes such as organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA), and paraoxonase (PON).     

Rapid discrimination of commercial strawberry cultivars using Fourier transform infrared spectroscopy data combined by multivariate analysis

To determine whether pattern recognition based on metabolite fingerprinting for whole cell extracts can be used to discriminate cultivars metabolically, leaves and fruits of five commercial strawberry cultivars were subjected to Fourier transform infrared (FT-IR) spectroscopy. FT-IR spectral data from leaves were analyzed by principal component analysis (PCA) and Fisher’s linear discriminant function analysis. The dendrogram based on hierarchical clustering analysis of these spectral data separated the five commercial cultivars into two major groups with originality. The first group consisted of Korean cultivars including ‘Maehyang’, ‘Seolhyang’, and ‘Gumhyang’, whereas in the second group, ‘Ryukbo’ clustered with ‘Janghee’, both Japanese cultivars. The results from analysis of fruits were the same as of leaves. We therefore conclude that the hierarchical dendrogram based on PCA of FT-IR data from leaves represents the most probable chemotaxonomical relationship between cultivars, enabling discrimination of cultivars in a rapid and simple manner. The authors Suk Weon Kim and Sung Ran Min contributed equally to this work.     

Application of Fourier transform infrared spectroscopy for monitoring hydrolysis and synthesis reactions catalyzed by a recombinant amidase

This study demonstrates the use of Fourier transform infrared (FTIR) spectroscopy for monitoring both synthesis and hydrolysis reactions catalyzed by a recombinant amidase (EC 3.5.1.4) from Pseudomonas aeruginosa. The kinetics of hydrolysis of acetamide, propionamide, butyramide, acrylamide, benzamide, phenylalaninamide, alaninamide, glycinamide, and leucinamide were determined. This revealed that very short-chain substrates displayed higher amidase activity than did branched side-chain or aromatic substrates. In addition, on reducing the polarity and increasing the substrates' bulkiness, a reduction of the amidase affinity for the substrates took place. Using FTIR spectroscopy it was possible to monitor and quantify the synthesis of several hydroxamic acid derivatives and ester hydrolysis products. These products may occur simultaneously in a reaction catalyzed by the amidase. The substrates used for the study of such reactions were ethyl acetate and glycine ethyl ester. Hydroxylamine was the nucleophile substrate used for the synthesis of acetohydroxamate compounds. Results presented in this article demonstrate the usefulness of FTIR spectroscopy as an important tool for understanding the enzyme structure-activity relationship because it provides a simple and rapid real-time assay for the detection and quantification of amidase hydrolysis and synthesis reactions in situ.