Milda Pucetaite

Molecular signatures of fossil leaves provide unexpected new evidence for extinct plant relationships

Gene sequences form the primary basis for understanding the relationships among extant plant groups, but genetic data are unavailable from fossils to evaluate the affinities of extinct taxa. Here we show that geothermally resistant fossil cuticles of seed-bearing plants, analysed with Fourier transform infrared (FTIR) spectroscopy and hierarchical cluster analysis (HCA), retain biomolecular suites that consistently distinguish major taxa even after experiencing different diagenetic histories. Our results reveal that similarities between the cuticular biochemical signatures of major plant groups (extant and fossil) are mostly consistent with recent phylogenetic hypotheses based on molecular and morphological data. Our novel chemotaxonomic data also support the hypothesis that the extinct Nilssoniales and Bennettitales are closely allied, but only distantly related to Cycadales. The chemical signature of the cuticle of Czekanowskia (Leptostrobales) is strongly similar to that of Ginkgo leaves and supports a close evolutionary relationship between these groups. Finally, our results also reveal that the extinct putative araucariacean, Allocladus, when analysed through HCA, is grouped closer to Ginkgoales than to conifers. Thus, in the absence of modern relatives yielding molecular information, FTIR spectroscopy provides valuable proxy biochemical data complementing morphological characters to distinguish fossil taxa and to help elucidate extinct plant relationships.A new palaeobotanical method analyses molecular signatures of fossil leaf cuticles to reveal hitherto obscure phylogenetic relationships, for example between Nilssoniales and Bennettitales; Ginkgoales and Leptostrobales.

Microspectroscopic infrared specular reflection chemical imaging of multi-component urinary stones: MIR vs. FIR

AbstractSpecular reflection infrared microspectroscopy was used for chemical imaging of cross-sectioned urinary stones to determine their chemical composition and morphology simultaneously. Absorption spectral bands were recovered from reflection spectra by Kramers-Kronig transform. FUse of far-infrared radiation provides high-contrast images and allows more precise constituent distribution determinations than mid-infrared because band asymmetry after the transform caused by diffuse reflection is less in the far-infrared.

Identification of kidney tumor tissue by infrared spectroscopy of extracellular matrix

Fourier transform infrared (FT-IR) spectroscopy was applied to characterize the extracellular matrix (ECM) of kidney tumor tissue and normal kidney tissue. Freshly resected tissue samples from 31 patients were pressed on a CaF₂ substrate. FT-IR spectra obtained from ECM of tumor tissue exhibit stronger absorption bands in the spectral region from 1000 to 1200 cm⁻¹ and around 1750 cm⁻¹ than those obtained from normal tissue. It is likely that the spectra of ECM of kidney tumor tissue with large increases in the intensities of these bands represent a higher concentration of fatty acids and glycerol. Amide I and amide II bands are stronger in the spectra of ECM from normal tissue, indicating a higher level of proteins. Our results suggest that FT-IR spectroscopy of the ECM is an innovative emerging technology for real-time intraoperative tumor diagnosis, which may improve margin clearance in renal cancer surgery.

Infrared spectroscopic imaging of kidney tumor tissue

Infrared spectroscopic imaging of cancerous kidney tissue was performed by means of FTIR microscopy. The spectra of thin tissue cryosections were collected with 64x64 MCT FPA detector and imaging area was increased up to 5.4×5.4 mm by mapping by means of PC controlled x,y stage. Chemical images of the samples were constructed using statistical treatment of the raw spectra. Several unsupervised and supervised statistical methods were used. The imaging results are compared with results of the standard histopathological analysis. It was concluded that application of method of cluster analysis ensures the best contrast of the images. It was found that border between cancerous and normal tissues visible in the infrared spectroscopic image corresponds with the border visible in histopathological image. Closer examination of the infrared spectroscopic image reveals that small domains of cancerous cells are found beyond the border in areas distant from the border up to 3 mm. Such domains are not visible in the histopathological images. The smallest domains found in the infrared images are approx. 60 μm.

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.

In situ detection of cancerous kidney tissue by means of fiber ATR-FTIR spectroscopy

The crucial goal of kidney-sparing surgical resection of a malignant tumor is complete removal of the cancerous tissue. The exact border between the cancerous and normal tissues is not always possible to identify by naked eye, therefore, a supplementary intraoperative diagnosis is needed. Unfortunately, intraoperative pathology methods used nowadays are time consuming and of inadequate quality rendering not definitive diagnosis. It has recently been shown that ATR-FTIR spectroscopy can be used for fast discrimination between cancerous and normal kidney tissues by analyzing the collected spectra of the tissue touch imprint smears. Most prominent differences are obtained in the wavenumber region from 950 cm-1 to 1250 cm-1, where the spectral bands due to the molecular vibrations of glycogen arise in the spectra of cancerous tissue smears. Such method of detection of cancerous tissue is limited by requirement to transfer the suspected tissue from the body to the FTIR instrument and stamp it on an ATR crystal of the spectrometer. We propose a spectroscopic tool which exploits the same principle of detection of cancerous cells as mentioned above, but does not require the tissue to be transferred from the body to the spectrometer. The portable spectrometer used in this design is equipped with fiber ATR probe and a sensitive liquid nitrogen cooled MCT detector. The design of the fiber probe allows the ATR tip to be changed easily in order to use only new sterilized tips for each measurement point of the tissue. It also enables sampling multiple areas of the suspected tissue with high lateral resolution which, in turn, increases accuracy with which the marginal regions between normal and cancerous tissues can be identified. Due to the loss of optical signal in the fiber probe the spectra have lower signal-to-noise ratio than in the case of standard ATR sampling setup. However, software for the spectral analysis used with the fiber probe design is still able to distinguish between cancerous and normal tissues with high accuracy.

Assignment of vibrational spectral bands of kidney tissue by means of low temperature SERS spectroscopy

Surface enhanced Raman scattering (SERS) spectroscopy is a useful method for detection of trace amounts of molecules. It has already been successfully implemented for detection of explosives, food additives, biomarkers in blood or urine, etc. In the last decade, SERS spectroscopy was introduced into the field of health sciences and has been especially focused on early disease detection. In the recent years, application of SERS spectroscopy for detection of various types of human cancerous tissues emerged. Furthermore, SERS spectroscopy of extracellular fluid shows great potential for the differentiation of normal and cancerous tissues; however, due to high variety of molecules present in such biological samples, the experimental spectrum is a combination of many different overlapping vibrational spectral bands. Thus, precise assignment of these bands to the corresponding molecular vibrations is a difficult task. In most cases, researchers try to avoid this task satisfying just with tentative assignment. In this study, low temperature SERS measurements of extracellular fluid of cancerous and healthy kidney tissue samples were carried out in order to get a deeper understanding of the nature of vibrational spectral bands present in the experimental spectrum. The SERS spectra were measured in temperature range from 300 K down to 100 K. SERS method was implemented using silver nanoparticle colloidal solution. The results of the low temperature SERS experiment were analysed and compared with the results of theoretical calculations. The analysis showed that the SERS spectrum of extracellular fluid of kidney tissue is highly influenced by the vibrational bands of adenine and Lcystine molecules.

Intra-operative on-line discrimination of kidney cancer from normal tissue by IR ATR spectroscopy of extracellular fluid

Determination of cancerous and normal kidney tissues during partial, simple or radical nephrectomy surgery was performed by using differences in the IR absorption spectra of extracellular fluid taken from the corresponding tissue areas. The samples were prepared by stamping of the kidney tissue on ATR diamond crystal. The spectral measurements were performed directly in the OR during surgery for 58 patients. It was found that intensities of characteristic spectral bands of glycogen (880-1200 cm-1) in extracellular fluid are sensitive to the type of the tissue and can be used as spectral markers of tumours. Characteristic spectral band of lactic acid (1730 cm-1) - product of the anaerobic glycolysis, taking place in the cancer cells is not suitable for use as a spectral marker of cancerous tissue, since it overlaps with the band of carbonyl stretch in phospholipids and fatty acids. Results of hierarchical cluster analysis of the spectra show that the spectra of healthy and tumour tissue films can be reliably separated into two groups. On the other hand, possibility to differentiate between tumours of different types and grades remains in question. While the fluid from highly malignant G3 tumour tissue contains highly pronounced glycogen spectral bands and can be well separated from benign and G1 tumours by principal component analysis, the variations between spectra from sample to sample prevent from obtaining conclusive results about the grouping between different tumour types and grades. The proposed method is instant and can be used in situ and even in vivo.

Detection of cancerous biological tissue areas by means of infrared absorption and SERS spectroscopy of intercellular fluid

We present a novel approach to the detection of cancerous kidney tissue areas by measuring vibrational spectra (IR absorption or SERS) of intercellular fluid taken from the tissue. The method is based on spectral analysis of cancerous and normal tissue areas in order to find specific spectral markers. The samples were prepared by sliding the kidney tissue over a substrate - surface of diamond ATR crystal in case of IR absorption or calcium fluoride optical window in case of SERS. For producing the SERS signal the dried fluid film was covered by silver nanoparticle colloidal solution. In order to suppress fluorescence background the measurements were performed in the NIR spectral region with the excitation wavelength of 1064 nm. The most significant spectral differences – spectral markers - were found in the region between 400 and 1800 cm-1, where spectral bands related to various vibrations of fatty acids, glycolipids and carbohydrates are located. Spectral markers in the IR and SERS spectra are different and the methods can complement each other. Both of them have potential to be used directly during surgery. Additionally, IR absorption spectroscopy in ATR mode can be combined with waveguide probe what makes this method usable in vivo.

Study of single walled carbon nanotube functionalization by means of surface enhanced Raman spectroscopy

Raman spectroscopy is known to provide information about the quality of the single walled carbon nanotubes (SWCNT). The information is based on the intensity ratio of D and G spectral modes and the frequency of RBM modes. However due to resonance nature of Raman spectrum of the nanotubes this method is not suitable to detect functionalization of the nanotubes. Surface enhanced Raman spectroscopy (SERS) is known to enhance the Raman bands up to fourteen orders of magnitude. Preferable adsorption sites for small silver nanoparticles are expected to be the functional groups of SWCNT; therefore SERS technique allows detecting small amounts of functional groups despite strong resonance Raman from backbone of SWCNT. In this study functionalized nanotubes were dispersed in silver colloid and dried on the standard silver plate for Raman measurements. Spectra of SWCNT without colloid in the spectral range between 50 and 1800 cm-1 exhibit only four main spectral features: G, D, and RBM modes between 200 and 400 cm-1. Spectra of SWCNT with the colloid exhibit several additional spectral bands which do not belong to the colloid. These bands attributed to vibrations of C-O, C-C and O-H from the functional groups and the carbon atom of the SWCNT attached to the corresponding group. The bands associated with the vibrations involving O atom is an indication that silver nanoparticles interact with the functional group attached to SWCNT.