Infrared attenuated total reflection spectroscopic surface analysis of bovine‐tail intervertebral discs after UV‐light‐activated riboflavin‐induced collagen cross‐linking

The tensile strength of the intervertebral disc (IVD) is mainly maintained by collagen cross‐links. Loss of collagen cross‐linking combined with other age‐related degenerative processes contributes to tissue weakening, biomechanical failure, disc herniation and pain. Exogenous collagen cross‐linking has been identified as an effective therapeutic approach for restoring IVD tensile strength. The current state‐of‐the‐art method to assess the extent of collagen cross‐linking in tissues requires destructive procedures and high‐performance liquid chromatography. In this study, we investigated the utility of infrared attenuated total reflection (IR‐ATR) spectroscopy as a nondestructive analytical strategy to rapidly evaluate the extent of UV‐light‐activated riboflavin (B2)‐induced collagen cross‐linking in bovine IVD samples. Thirty‐five fresh bovine‐tail IVD samples were equally divided into five treatment groups: (a) untreated, (b) cell culture medium Dulbecco's Modified Eagle's Medium only, (c) B2 only, (d) UV‐light only and (e) UV‐light‐B2. A total of 674 measurements have been acquired, and were analyzed via partial least squares discriminant analysis. This classification scheme unambiguously identified individual classes with a sensitivity >91% and specificity >92%. The obtained results demonstrate that IR‐ATR spectroscopy reliably differentiates between different treatment categories, and promises an excellent tool for potential in vivo, nondestructive and real‐time assessment of exogenous IVD cross‐linking.     

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.     

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.      

Translation of an esophagus histopathological FT‐IR imaging model to a fast quantum cascade laser modality

The technical progress in fast quantum cascade laser (QCL) microscopy offers a platform where chemical imaging becomes feasible for clinical diagnostics. QCL systems allow the integration of previously developed FT‐IR‐based pathology recognition models in a faster workflow. The translation of such models requires a systematic approach, focusing only on the spectral frequencies that carry crucial information for discrimination of pathologic features. In this study, we optimize an FT‐IR‐based histopathological method for esophageal cancer detection to work with a QCL system. We explore whether the classifier's performance is affected by paraffin presence from tissue blocks compared to removing it chemically. Working with paraffin‐embedded samples reduces preprocessing time in the lab and allows samples to be archived after analysis. Moreover, we test, whether the creation of a QCL model requires a preestablished FTIR model or can be optimized using solely QCL measurements.     

Segregation of ovarian cancer stage exploiting spectral biomarkers derived from blood plasma or serum analysis: ATR‐FTIR spectroscopy coupled with variable selection methods

Ovarian cancer is a solid tumor and a leading cause of mortality. Diagnostic tools for the detection of early stage (stage I) ovarian cancer are urgently needed. For this purpose, attenuated total reflection Fourier‐transform infrared spectroscopy (ATR‐FTIR) coupled with variable selection methods, successive projection algorithm or genetic algorithm (GA) combined with linear discriminant analysis (LDA), were employed to identify spectral biomarkers in blood plasma or serum samples for accurate diagnosis of different stages of ovarian cancer, histological type and segregation based on age. Three spectral datasets (stage I vs. stage II–IV; serous vs. non‐serous carcinoma; and, ≤60 years vs. >60 years) were processed: sensitivity and specificity required for real‐world diagnosis of ovarian cancer was achieved. Toward segregating stage I vs. stage II–IV, sensitivity and specificity (plasma blood) of 100% was achieved using a GA‐LDA model with 33 wavenumbers. For serous vs. non‐serous category (plasma blood), the sensitivity and specificity levels, using 29 wavenumbers by GA‐LDA, were remarkable (up to 94%). For ≤60 years and >60 years categories (plasma blood), the sensitivity and specificity, using 42 wavenumbers by GA‐LDA, gave complete accuracy (100%). For serum samples, sensitivity and specificity results gave relatively high accuracy (up to 91.6% stage I vs. stage II–IV; up to 93.0% serous vs. non‐serous; and, up to 96.0% ≤60 years vs. >60 years) using several wavenumbers. These findings justify a prospective population‐based assessment of biomarkers signatures using ATR‐FTIR spectroscopy as a screening tool for stage of ovarian cancer. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:832–839, 2015     

Multivariate Analysis of Phenol in Freeze-Dried and Spray-Dried Insulin Formulations by NIR and FTIR

Dehydration is a commonly used method to stabilise protein formulations. Upon dehydration, there is a significant risk the composition of the formulation will change especially if the protein formulation contains volatile compounds. Phenol is often used as excipient in insulin formulations, stabilising the insulin hexamer by changing the secondary structure. We have previously shown that it is possible to maintain this structural change after drying. The aim of this study was to evaluate the residual phenol content in spray-dried and freeze-dried insulin formulations by Fourier transform infrared (FTIR) spectroscopy and near infrared (NIR) spectroscopy using multivariate data analysis. A principal component analysis (PCA) and partial least squares (PLS) projections were used to analyse spectral data. After drying, there was a difference between the two drying methods in the phenol/insulin ratio and the water content of the dried samples. The spray-dried samples contained more water and less phenol compared with the freeze-dried samples. For the FTIR spectra, the best model used one PLS component to describe the phenol/insulin ratio in the powders, and was based on the second derivative pre-treated spectra in the 850–650 cm⁻¹ region. The best PLS model based on the NIR spectra utilised three PLS components to describe the phenol/insulin ratio and was based on the standard normal variate transformed spectra in the 6,200–5,800 cm⁻¹ region. The root mean square error of cross validation was 0.69% and 0.60% (w/w) for the models based on the FTIR and NIR spectra, respectively. In general, both methods were suitable for phenol quantification in dried phenol/insulin samples.     

Infrared spectroscopy with multivariate analysis potentially facilitates the segregation of different types of prostate cell

The prostate gland is conventionally divided into zones or regions. This morphology is of clinical significance as prostate cancer (CaP) occurs mainly in the peripheral zone (PZ). We obtained tissue sets consisting of paraffin-embedded blocks of cancer-free transition zone (TZ) and PZ and adjacent CaP from patients (n = 6) who had undergone radical retropubic prostatectomy; a seventh tissue set of snap-frozen PZ and TZ was obtained from a CaP-free gland removed after radical cystoprostatectomy. Paraffin-embedded tissue slices were sectioned (10-mum thick) and mounted on suitable windows to facilitate infrared (IR) spectra acquisition before being dewaxed and air dried; cryosections were dessicated on BaF(2) windows. Spectra were collected employing synchrotron Fourier-transform infrared (FTIR) microspectroscopy in transmission mode or attenuated total reflection-FTIR (ATR) spectroscopy. Epithelial cell and stromal IR spectra were subjected to principal component analysis to determine whether wavenumber-absorbance relationships expressed as single points in "hyperspace" might on the basis of multivariate distance reveal biophysical differences between cells in situ in different tissue regions. After spectroscopic analysis, plotted clusters and their loadings curves highlighted marked variation in the spectral region containing DNA/RNA bands ( approximately 1490-1000 cm(-1)). By interrogating the intrinsic dimensionality of IR spectra in this small cohort sample, we found that TZ epithelial cells appeared to align more closely with those of CaP while exhibiting marked structural differences compared to PZ epithelium. IR spectra of PZ stroma also suggested that these cells are structurally more different to CaP than those located in the TZ. Because the PZ exhibits a higher occurrence of CaP, other factors (e.g., hormone exposure) may modulate the growth kinetics of initiated epithelial cells in this region. The results of this pilot study surprisingly indicate that TZ epithelial cells are more likely to exhibit what may be a susceptibility-to-adenocarcinoma spectral signature. Thus, IR spectroscopy on its own may not be sufficient to identify premalignant prostate epithelial cells most likely to progress to CaP.     

Use of random forest in FTIR analysis of LDL cholesterol and tri‐glycerides for hyperlipidemia

A quantitative determination method for the diagnosis of hyperlipidemia was developed using Fourier transform infrared (FTIR) spectroscopy. Random forest (RF) was demonstrated as a potential multivariate algorithm for the FTIR analysis of low‐density lipoprotein cholesterol (LDL‐C) and tri‐glycerides (TG) in human serum samples. The informative wavebands for LDL‐C and TG were selected based on the Gini importance. The selected wavebands were mainly within the fingerprint region. The RF modeling results were better than those derived using PLS in validation process, because the chance for over‐fitting was possibly eliminated in RF algorithm. ARF also demonstrated favorable results in the test process. The prospective model exhibited a higher than 90% true prediction in negative/positive properties for male and female samples. These clinical statistical results indicated the optimization of RF algorithm performed accurately in the FTIR determination of LDL‐C and TG. RF is evaluated as a promising tool for diagnosing and controlling hyperlipidemia in populations. The parameter optimization methodology is useful in the improving model accuracy using FTIR spectroscopic technology. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1693–1702, 2015     

Near‐infrared spectroscopic evaluation of lyophilized viral vaccine formulations

This article examines the applicability of near‐infrared spectroscopy (NIRS) to evaluate the virus state in a freeze‐dried live, attenuated vaccine formulation. Therefore, this formulation was freeze‐dried using different virus volumes and after applying different pre‐freeze‐drying virus treatments (resulting in different virus states): (i) as used in the commercial formulation; (ii) without antigen (placebo); (iii) concentrated via a centrifugal filter device; and (iv) stressed by 96 h exposure to room temperature. Each freeze‐dried product was measured directly after freeze‐drying with NIR spectroscopy and the spectra were analyzed using principal component analysis (PCA). Herewith, two NIR spectral regions were evaluated: (i) the 7300–4000 cm^?1 region containing the amide A/II band which might reflect information on the coated proteins of freeze‐dried live, attenuated viruses; and (ii) the C–H vibration overtone regions (10,000–7500 and 6340–5500 cm^?1) which might supply information on the lipid layer surrounding the freeze‐dried live, attenuated viruses. The different pre‐freeze‐drying treated live, attenuated virus formulations (different virus states and virus volumes) resulted in different clusters in the scores plots resulting from the PCA of the collected NIR spectra. Secondly, partial least squares discriminant analysis models (PLS‐DA) were developed and evaluated, allowing classification of the freeze‐dried formulations according to virus pretreatment. The results of this study suggest the applicability of NIR spectroscopy for evaluating live, attenuated vaccine formulations with respect to their virus pretreatment and virus volume. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1573–1586, 2013     

Fiber attenuated total reflection infrared spectroscopy of kidney tissue during live surgery

More than 90% of solid kidney tumors are cancerous and have to be treated by surgical resection where surgical outcomes and patient prognosis are dependent on the tumor discrimination. The development of alternative approaches based on a new generation of fiber attenuated total reflection (ATR) probes could aid tumor identification even under intrasurgical conditions. Herein, fiber ATR IR spectroscopy is employed to distinguish normal and cancerous kidney tissues. Freshly resected tissue samples from 34 patients are investigated under nearly native conditions. Spectral marker bands that allow a reliable discrimination between tumor and normal tissue are identified by a supervised classification algorithm. The absorbance values of the bands at 1025, 1155 and 1240 cm^?1 assigned to glycogen and fructose 1,6‐bisphosphatase are used as the clearest markers for the tissue discrimination. Absorbance threshold values for tumor and normal tissue are determined by discriminant analysis. This new approach allows the surgeon to make a clinical diagnosis.     

FTIR spectroscopy for the detection and evaluation of live attenuated viruses in freeze dried vaccine formulations

This article examines the applicability of Fourier Transform Infrared (FTIR) spectroscopy to detect the applied virus medium volume (i.e., during sample filling), to evaluate the virus state and to distinguish between different vaccine doses in a freeze dried live, attenuated vaccine formulation. Therefore, different formulations were freeze dried after preparing them with different virus medium volumes (i.e., 30, 100, and 400 µl) or after applying different pre‐freeze‐drying sample treatments (resulting in different virus states); i.e., (i) as done for the commercial formulation; (ii) samples without virus medium (placebo); (iii) samples with virus medium but free from antigen; (iv) concentrated samples obtained via a centrifugal filter device; and (v) samples stressed by 96h exposure to room temperature; or by using different doses (placebo, 25‐dose vials, 50‐dose‐vials and 125‐dose vials). Each freeze‐dried product was measured directly after freeze‐drying with FTIR spectroscopy. The collected spectra were analyzed using principal component analysis (PCA) and evaluated at three spectral regions, which might provide information on the coated proteins of freeze dried live, attenuated viruses: (i) 1700–1600 cm^?1 (amide I band), 1600–1500 cm^?1 (amide II band) and 1200–1350 cm^?1 (amide III band). The latter spectral band does not overlap with water signals and is hence not influenced by residual moisture in the samples. It was proven that FTIR could distinguish between the freeze‐dried samples prepared using different virus medium volumes, containing different doses and using different pre‐freeze‐drying sample treatments in the amide III region. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1107–1118, 2015     

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)‐FTIR and HT‐Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.     

ATR microspectroscopy with multivariate analysis segregates grades of exfoliative cervical cytology

Although cervical cancer screening in the UK has led to reductions in the incidence of invasive disease, this programme remains flawed. We set out to examine the potential of infrared (IR) microspectroscopy to allow the profiling of cellular biochemical constituents associated with disease progression. Attenuated total reflection-Fourier Transform IR (ATR) microspectroscopy was employed to interrogate spectral differences between samples of exfoliative cervical cytology collected into liquid based cytology (LBC). These were histologically characterised as normal (n = 5), low-grade (n = 5), high-grade (n = 5) or severe dyskaryosis (? carcinoma) (n = 5). Examination of resultant spectra was coupled with principal component analysis (PCA) and subsequent linear discriminant analysis (LDA). The interrogation of LBC samples using ATR microspectroscopy with PCA-LDA facilitated the discrimination of different categories of exfoliative cytology and allowed the identification of potential biomarkers of abnormality; these occurred prominently in the IR spectral region 1200 cm(-1) - 950 cm(-1) consisting of carbohydrates, phosphate, and glycogen. Shifts in the centroids of amide I (approximately 1650 cm(-1)) and II (approximately 1530 cm(-1)) absorbance bands, indicating conformational changes to the secondary structure of intracellular proteins and associated with increasing disease progression, were also noted. This work demonstrates the potential of ATR microspectroscopy coupled with multivariate analysis to be an objective alternative to routine cytology.     

Application of ATR‐FTIR spectroscopy to the study of thermally induced changes in secondary structure of protein molecules in solid state

FTIR spectroscopy in combination with ATR sampling technique is the most accessible analytical technique to study secondary structure of proteins both in solid and aqueous solution. Although several studies have demonstrated the applications of ATR‐FTIR to study conformational changes of solid dried proteins due to dehydration, there are no reports that demonstrate the application of ATR‐FTIR in the study of thermally induced changes of secondary structure of biomolecules directly on the solid state. In this study, four biomolecules of pharmaceutical interest, lysozyme, myoglobine, chymotripsin and human growth hormone (hGH), were studied on the solid state before and after different thermal treatments in order to relate changes of secondary structure to partial or total thermal denaturation processes. The results obtained provide experimental evidence that protein thermal denaturation in the solid state can be detected by displacement of carbonyl bands which correspond to conformational transformations between α–helix to β‐sheet or intermolecular β‐sheet; the molecules studied undergo this transformation when exposed to a temperature close to their denaturation temperature which may become irreversible depending on the extent of the heating treatment. These findings demonstrate that ATR‐FTIR is an effective and time efficient technique that allows the monitoring of the protein thermal denaturation process of solid samples without further reconstitution or prior sample preparation. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 574–584, 2015.     

Bladder cancer diagnosis from bladder wash by Fourier transform infrared spectroscopy as a novel test for tumor recurrence

This study proposes Fourier Transform Infrared (FTIR) spectroscopy as a more sensitive, rapid, non‐destructive and operator‐independent analytical diagnostic method for bladder cancer recurrence from bladder wash than other routinely used urine cytology and cystoscopy methods. A total of 136 patients were recruited. FTIR spectroscopic experiments were carried out as a blind study, the classification results of which were then compared with those of cytology and cystoscopy. Firstly, 71 samples (n = 37; bladder cancer and n = 34; control) were studied with transmittance FTIR spectroscopy. After achieving successful differentiation of the groups, to develop a more rapid diagnostic tool and check the reproducibility of the results, the work was continued with different samples (n = 65 as n = 44; bladder cancer and n = 21; control), using the reflection mode (ATR) of FTIR spectroscopy by a different operator. The results revealed significant alterations in moleculer content in the cancer group. Based on the spectral differences, using transmittance FTIR spectroscopy coupled with chemometrics, the diseased group was successfully differentiated from the control. When only carcinoma group was taken into consideration a sensitivity value of 100% was achieved. Similar results were also obtained by ATR‐FTIR spectroscopy. This study shows the power of infrared spectroscopy in the diagnosis of bladder cancer.

     

Comparison between high definition FT‐IR,Raman and AFM‐IR for subcellular chemical imaging of cholesteryl esters in prostate cancer cells

The family of vibrational spectroscopic imaging techniques grows every few years and there is a need to compare and contrast new modalities with the better understood ones, especially in the case of demanding biological samples. Three vibrational spectroscopy techniques (high definition Fourier‐transform infrared [FT‐IR], Raman and atomic force microscopy infrared [AFM‐IR]) were applied for subcellular chemical imaging of cholesteryl esters in PC‐3 prostate cancer cells. The techniques were compared and contrasted in terms of image quality, spectral pattern and chemical information. All tested techniques were found to be useful in chemical imaging of cholesterol derivatives in cancer cells. The results obtained from FT‐IR and Raman imaging showed to be comparable, whereas those achieved from AFM‐IR study exhibited higher spectral heterogeneity. It confirms AFM‐IR method as a powerful tool in local chemical imaging of cells at the nanoscale level. Furthermore, due to polarization effect, p‐polarized AFM‐IR spectra showed strong enhancement of lipid bands when compared to FT‐IR.     

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.      

Discrimination of single‐porin Escherichia (E.) coli mutants by ATR and transmission mode FTIR spectroscopy

Vibrational spectroscopy has long been used in bacterial identification with different levels of taxonomic discrimination but its true potential for intra‐species differentiation remains poorly explored. Herein, both transmission Fourier‐transform infrared (FTIR) and attenuated total reflectance (ATR)‐FTIR spectroscopy are used to analyse E. coli strains that differ solely in their porin expression profile. In this previously unreported approach, the applicability of both FTIR‐spectroscopy techniques is compared with the same collection of unique strains. ATR‐FTIR spectroscopy proved to reliably distinguish between several E. coli porin mutants with an accuracy not replicated by FTIR in transmission mode (using previously optimized procedures). Further studies should allow the identification of the individual contribution of the single porin channel to the overall bacterial infrared spectrum and of molecular predictive patterns of porin alterations. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Real‐time optical spectroscopic monitoring of nonirrigated lesion progression within atrial and ventricular tissues

Despite considerable advances in guidance of radiofrequency ablation (RFA) therapy for the treatment of cardiac arrhythmias, success rates have been hampered by a lack of tools for precise intraoperative evaluation of lesion extent. Near‐infrared spectroscopic (NIRS) techniques are sensitive to tissue structural and biomolecular properties, characteristics that are directly altered by radiofrequency (RF) treatment. In this work, a combined NIRS‐RFA catheter is developed for real‐time monitoring of tissue reflectance during RF energy delivery. An algorithm is proposed for processing NIR spectra to approximate nonirrigated lesion depth in both atrial and ventricular tissues. The probe optical geometry was designed to bias measurement influence toward absorption enabling enhanced sensitivity to changes in tissue composition. A set of parameters termed “lesion optical indices” are defined encapsulating spectral differences between ablated and unablated tissue. Utilizing these features, a model for real‐time tissue spectra classification and lesion size estimation is presented. Experimental validation conducted within freshly excised porcine cardiac specimens showed strong concordance between algorithm estimates and post‐hoc tissue assessment.      

Detection of fungal infection in Lolium perenne by Fourier transform infrared spectroscopy

Aims The goal of the study was to apply Fourier transform infrared (FTIR) spectroscopy followed by chemometrical data treatment for the differentiation of fungi-infected perennial ryegrass (Lolium perenne) from uninfected grass.Methods FTIR was used to rapidly discriminate between leaves of perennial ryegrass (L. perenne) infected by a fungal endophyte (Epichlo?; asexual forms: Neotyphodium) and uninfected leaves. Besides drying and grinding of the sampled leaves, no other preparation steps were needed. FTIR measurements were performed in the attenuated total reflection (ATR) mode. Aliquots of powdered leaf samples were placed on a ZnSe crystal and the spectra were collected, followed by chemometrical analysis (multidimensional factor analysis, hierarchical cluster analysis).Important findings ATR-FTIR allowed a rapid detection of fungal infections in the plant material and proved to be a fast and reliable tool for the differentiation of plant biomass without the need of time-consuming sample preparation.