SignificancePancreatic surgery is a highly demanding and routinely applied procedure for the treatment of several pancreatic lesions. The outcome of patients with malignant entities crucially depends on the margin resection status of the tumor. Frozen section analysis for intraoperative evaluation of tissue is still time consuming and laborious.AimWe describe the application of fiber-based attenuated total reflection infrared (ATR IR) spectroscopy for label-free discrimination of normal pancreatic, tumorous, and pancreatitis tissue. A pilot study for the intraoperative application was performed.ApproachThe method was applied for unprocessed freshly resected tissue samples of 58 patients, and a classification model for differentiating between the distinct tissue classes was established.ResultsThe developed three-class classification model for tissue spectra allows for the delineation of tumors from normal and pancreatitis tissues using a probability score for class assignment. Subsequently, the method was translated into intraoperative application. Fiber optic ATR IR spectra were obtained from freshly resected pancreatic tissue directly in the operating room.ConclusionOur study shows the possibility of applying fiber-based ATR IR spectroscopy in combination with a supervised classification model for rapid pancreatic tissue identification with a high potential for transfer into intraoperative surgical diagnostics.
Pancreatic surgery is a highly demanding and routinely applied procedure for the treatment of several pancreatic lesions. The outcome of patients with malignant entities crucially depends on the margin resection status of the tumor. In this study we describe the application of fiber-based attenuated total reflection infrared (ATR IR) spectroscopy for label-free discrimination of normal pancreatic, tumorous and pancreatitis tissue. The method was applied for the unprocessed freshly resected tissue samples of 40 patients, and a classification model for differentiating between the distinct tissue classes was established. The developed three-class classification model for tissue spectra allows the delineation of tumors from normal and pancreatitis tissues. The classification algorithm provides probability values for each sample to be assigned to normal, tumor or pancreatitis classes. The established probability values were transferred to a Red-Green-Blue (RGB) color plot. Subsequently, the method was translated into intraoperative application. Fiber optic ATR IR spectra were obtained from freshly resected pancreatic tissue directly in the operating room. The spectroscopic findings could subsequently be confirmed by the histology gold standard. This study shows the possibility of applying fiber-based ATR IR spectroscopy in combination with a supervised classification model for rapid pancreatic tissue identification with a high potential for transfer into intraoperative surgical diagnostics.
The goal of this study is development of ultra-sensitive and reproducible SERS platform based on novel magnetoplasmonic nanoparticles produced by laser ablation. The magnetic part of hybrid nanoparticles ensures manipulation of the nanoparticles by magnetic field by arranging them at biological surfaces in a special geometry resulting in high and reproducible SERS. Magneto-plasmonic Au-Fe nanoparticles in colloidal suspension were prepared by picosecond laser ablation of evaporated iron and gold films on glass. The nanoparticles were characterized by UV-visible extinction, high resolution electronic microscopy, and Raman spectroscopy. EDX analysis revealed that the shell of nanoparticles (2−20 nm) consist of iron and the core is composed mostly of gold. The plasmonic behavior of nanoparticles was accessed by analysis of SERS spectra from adsorbed adenine as probe ligand. The fabrication of hybrid nanoparticles by laser ablation offers a new possibility for construction of SERS substrates with tunable optical and magnetic properties for biomedical sensing.
Vibrational spectroscopy is proved to be a reliable technique for biological tissue analysis and screening of pathological differences between healthy and cancerous tissues. Due to the ubiquity and inherent complexity of cancerous diseases together with existing challenges in diagnostic methods, the search for reliable separation of cancerous and healthy tissues is of great importance. In our previous works, we have shown that spectral differences between cancerous and healthy tissues of kidney can be found using FTIR–ATR and surface enhanced Raman scattering (SERS) spectroscopy. SERS allows identification and detection of molecules at low concentration and oversteps sensitivity limits of ordinary spectroscopic methods, thus is available for identification of cancerous tissues in cases where FTIR – ATR technique is not sensitive enough. For remote diagnostic applications (for example during surgeries) experimental configurations employing optical fibers are indispensable. In this work, we investigated the technical aspects of using optical fibers to accomplish SERS of biological tissues. For this purpose, we used commonly available optical fiber types: fused silica and acrylic glass (PMMA). One end of the fiber was coated with Ag nanoparticles. Different coating techniques were applied, such as formation of a self-assembled monolayer of the nanoparticles or drying of colloidal nanoparticle suspensions. Fiber-based SERS spectra of cancerous and healthy kidney tissues were analyzed. We found that reasonable quality fiberbased SERS spectra of normal and cancerous kidney tissue can be obtained through silica fiber with SERS activated tip. Furthermore, a distinction between cancerous and healthy kidney tissues from SERS spectra is found as cancerous kidney tissues contain spectral bands of glycogen which can be used as spectral markers of cancerous cells.
Nowadays, with various available choices of non-prescription medication it is quite easy when under illness instead advising with the specialist to self-medicate and try different types of pharmaceuticals at once in order to save time or money. Such unguided or incorrect self-diagnosis of one’s health often results in overdose and other serious health risks. In these cases when such life threatening events occur it is of the most importance to apply early medical treatment. However, specific treatment can be only chosen when the species of pharmaceutical is known. Thus, in order to prevent serious outcomes it is necessary to have a tool which would allow to diagnose the type of drug that was used as soon as possible. The fastest way to gather the required information is to analyze chemically biofluids like blood, saliva or etc. since the pharmaceuticals or their metabolites are contained and transported via biofluids. But the concentration of the molecules to detect is often very low thus the technique not only should be fast but also very sensitive. Of course, there are several clinically available drug screening methods which have already been used for decades and can identify chemical constituents with high sensitivity. Even though such conventional clinically used drug screening methods like liquid chromatography and mass spectroscopy, are sensitive enough, the sample preparation procedure and analysis is not easy and take time. Faster methods would be beneficial and could help avoid serious complications of unguided selfmedication. Vibrational spectroscopy is known to be a reliable tool for chemical composition and structure analysis. Be that as it may, the sensitivity of the conventional vibrational spectroscopy techniques is not sufficient for the detection of trace amounts of molecules. Surface enhanced Raman scattering (SERS) spectroscopy - one of the unconventional methods of vibrational spectroscopy is known to be very sensitive and chemically specific thus suitable for detection of low concentration substances in bodily fluids. This work presents the possible approach of label-free SERS and EC-SERS spectroscopies for application as a faster method for drug screening from biological fluids.
The misuse or abuse of over-the-counter (OTC) drugs has a significant impact on patient health and if used without caution can lead to serious health issues. The prevalence of OTC drugs due to a well-developed market and the lack of patient knowledge about the drug safety has led to the increase in the number of hospitalizations related to the drug missuses. In order to choose a correct treatment procedure a fast and precise identification of the misused drug is necessary. Various accurate drug-screening methods are clinically available, however, they are rather slow or too complicated and more efficient method would be beneficial. In this work we present the first results of application of SERS spectroscopy for a fast screening of salicylic acid (metabolite of aspirin) and paracetamol directly in human blood. Conventional Raman spectroscopy is known to be a sensitive technique for identification and quantification of molecular substances in various molecular mixtures without the need of a large amount of the sample. However, when dealing with low concentrations of molecules under study in some biological fluids (like drugs or their metabolites in blood) unconventional sensitive spectral methods like surface enhanced Raman scattering (SERS) spectroscopy must be used. The extremely high sensitivity of the SERS allows identification of the blood metabolites in micromole or even in lower concentrations by analyzing the spectra of thin films of dried blood samples formed on the metal colloidal film. Additionally, the possibility to tailor the metal nanoparticles applied in colloidal SERS can be utilized to increase selectivity and sensitivity of the method. The SERS methodology proposed in this work allows fast and accurate identification of the aspirin with the detection limit – standard 500 mg one and half pills, while usage of up to 2 pills of paracetamol at once cannot be detected due to extremely low concentration of this drug in the blood.
Optical spectroscopy offers unique opportunities for a label-free investigation of tissues at the molecular level to identify the variety of diseases. To transfer spectroscopic analysis from the scientific laboratories to clinical environment, fiber optic probes are required as optical bridges between the equipment and tissue.
We developed single and combined fiber optic probes for the following set of spectroscopy methods: Mid IR-absorption, Raman scattering, Diffuse NIR-reflection, and auto-fluorescence. We benchmarked these methods and selected the optimal one (or their combination), that differentiate between healthy and malignant tissue, based on optical spectra. We tested cancer-normal tissue pairs of human body such as colon, kidney, brain as well as cartilages with and without injuries. Equines cartilage samples with and without osteoarthritis were tested as well. Obtained spectral data were evaluated by multivariate discrimination analysis to enable clear separation of malignant and normal tissues. Data fusion was revealed a synergic effect resulted in increasing of sensitivity, specificity and accuracy (up to 98% for kidney cancer).
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.
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.
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.
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.
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.
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.
In this work the infrared absorption spectra of intercellular fluid of normal and tumor kidney tissue were recorded and
analyzed. The samples were prepared by stamping freshly resected tissue onto a CaF2 substrate. FT-IR spectra obtained
from intracellular fluid of tumor tissue exhibit stronger absorption bands in the spectral region from 1000-1200 cm-1 and
around 1750 cm-1 than those obtained from normal tissue. It is likely the spectra of extracellular matrix 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 spectra of normal tissue indicating a higher level of proteins. The results
demonstrate that FT-IR spectroscopy of intercellular fluids is a novel approach for a quick diagnosis during surgical
resection, which can improve the therapy of kidney tumors.
Surface-enhanced Raman scattering (SERS) spectroscopy can be a useful tool in regard to disease diagnosis and prevention. Advantage of SERS over conventional Raman spectroscopy is its significantly increased signal (up to factor of 106-108) which allows detection of trace amounts of substances in the sample. So far, this technique is successfully used for analysis of food, pieces of art and various biochemical/biomedical samples. In this work, we survey the possibility of applying SERS spectroscopy for detection of trace components in urinary deposits. Early discovery together with the identification of the exact chemical composition of urinary sediments could be crucial for taking appropriate preventive measures that inhibit kidney stone formation or growth processes. In this initial study, SERS spectra (excitation wavelength - 1064 nm) of main components of urinary deposits (calcium oxalate, uric acid, cystine, etc.) were recorded by using silver (Ag) colloid. Spectra of 10-3-10-5 M solutions were obtained. While no/small Raman signal was detected without the Ag colloid, characteristic peaks of the substances could be clearly separated in the SERS spectra. This suggests that even small amounts of the components could be detected and taken into account while determining the type of kidney stone forming in the urinary system. We found for the first time that trace amounts of components constituting urinary deposits could be detected by SERS spectroscopy. In the future study, the analysis of centrifuged urine samples will be carried out.
We used infrared reflection microspectroscopy for chemical imaging of urinary
calculi and showed that contribution of diffuse reflection, influencing the imaging results, can be
suppressed by decreasing surface roughness and (or) increasing wavelength of infrared radiation
applied for the imaging.
We have used infrared microspectroscopy for chemical analysis of urinary sediments. We showed that Mie scattering from urinary sediments as small as 10-100 μm is influencing the spectra and the influence can be
suppressed and quality of the spectra can be improved by applying RMieS-EMSC procedure.
Results of the structural analysis of urinary sediments by means of infrared spectral microscopy are presented. The results are in good agreement with the results of standard optical microscopy in the case of single-component and crystalline urinary sediments. It is found that for noncrystalline or multicomponent sediments, the suggested spectroscopic method is superior to optical microscopy. The chemical structure of sediments of any molecular origin can be elucidated by this spectroscopic method. The method is sensitive enough to identify solid particles of drugs present in urine. Sulfamethoxazole and traces of other medicines are revealed in this study among the other sediments. We also show that a rather good correlation exists between the type of urinary sediments and the renal stones removed from the same patient. Spectroscopic studies of urinary stones and corresponding sediments from 76 patients suffering from renal stone disease reveal that in 73% of cases such correlation exists. This finding is a strong argument for the use of infrared spectral microscopy to prevent kidney stone disease because stones can be found in an early stage of formation by using the nonintrusive spectroscopic investigation of urinary sediments. Some medical recommendations concerning the overdosing of certain pharmaceuticals can also be derived from the spectroscopic studies of urinary sediments.
Fourier transform infrared (FTIR) spectroscopic imaging has been used to probe the biochemical composition of human renal tumor tissue and adjacent normal tissue. Freshly resected renal tumor tissue from surgery was prepared as a thin cryosection and examined by FTIR spectroscopic imaging. Tissue types could be discriminated by utilizing a combination of fuzzy k-means cluster analysis and a supervised classification algorithm based on a linear discriminant analysis. The spectral classification is compared and contrasted with the histological stained image. It is further shown that renal tumor cells have spread in adjacent normal tissue. This study demonstrates that FTIR spectroscopic imaging can potentially serve as a fast and objective approach for discrimination of renal tumor tissue from normal tissue and even in the detection of tumor infiltration in adjacent 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.
Polarization Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS) is a very sensitive imaging
technique for the characterization of molecular films. In order to achieve a spatial resolution close to the diffraction limit
a very small pinhole which acts as a point-source has to be used. However, such a small pinhole, the typical diameter
would be app. 100 μm, may reduces dramatically the intensity of the infrared beam. Using a common FTIR spectrometer
the spatial resolution is mainly limited by the brilliance of the globar infrared source. Therefore, an improvement in
lateral resolution requires a more brilliant light source.
The free electron laser (FEL) is such a high brilliant infrared source. The combination of the FEL with the PM-IRRAS
imaging system is a new approach to capture spectroscopic images with an excellent spatial resolution close to the
diffraction limit. PM-IRRAS images of a self assembly monolayer of phosphonic acid molecules onto a microstructures
gold / aluminum oxide surface where characterized. The spectroscopic image exhibits a spatial resolution of app. 5 μm.
An evaluation of characteristic absorbance bands of the phosphate group reveals that phosphonic acid molecules bound
with a high degree of orientation but differently at the gold and aluminum oxide surfaces. However, the spectroscopic
image reveals also several domains of disordering across the surface. Such domains have a dimension of only few
micrometers and can be identified in a high resolved PM-IRRAS image.
Amorphous carbon films were formed on Si (111) wafers from argon-acetylene gas mixture at atmospheric pressure by
direct current (DC) plasma torch discharge. The Ar/C2H2 gas volume ratio varied from 12 to 100, and the distance
between plasma torch nozzle exit and the samples was 0.005, 0.01 and 0.02 m. SEM revealed carbon coatings thickness
in the range of 20-270 &mgr;m, and variation of the growth rate from 0.067 &mgr;m/s to 1.5 &mgr;m/s. Growth rate of the coatings
increases decreasing Ar/C2H2 gas ratio and the distance. The Raman spectra of carbon films indicate the upward shift of
the D (~1360 cm-1) and G (~1600 cm-1) peaks, compared to typical diamond-like carbon (DLC). a-C:H coatings
deposited at higher Ar/C2H2 gas ratio (60 and 100) and distance d greater than or equal to 0.01 m contain high sp3 bond fraction and are
attributed to DLC films. However Raman spectra shape and ID/IG ratio demonstrate existence of diamond phase mixed
with glassy carbon phase. Films produced at lower Ar/C2H2 ratios are graphite-like carbon (GLC). The Fourier transform
infrared (FTIR) spectroscopy has shown that film transparency increases decreasing acetylene gas content. Reflectance
of the films depends on Ar/C2H2 gas ratio and distance, and varies from 60% up to 90%. The IR spectra showed clear
evidence of C=C and C=O bonds in GLC films and presence of sp3 CH2 symmetric (2850 cm-1) and antisymmetric (2920
cm-1) modes in DLC coatings.
Functionality of biosensor arrays based on SPR imaging is mainly defined by quality of patterned SAM, which is used for sensing of biological substances of interest. The monolayers are commonly not uniform in the micrometer scale. In the current work we describe experimental setups for PM-IRRAS mapping and SPR imaging of self
assembly monolayer (SAM) formed by octadecyl phosphonic acid (CH3(CH2)17PO(OH)2) on a patterned Au/Al2O3 surface. By combining highly resolved surface plasmon resonance (SPR) images of the surface with the corresponding PM-IRRAS maps we achieved enhancement of the resolution. In this work we show, that changes of molecular structure in the thin films also induce changes in the SPR signal. Contrary, changes in SPR have their origin in molecular structure within the film.
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