Severe radiation toxicity can continue years after the completion of radiotherapy for prostate cancer patients. Currently, it is impossible to predict before treatment which patients will experience these long-term side effects. New approaches based on vibrational spectroscopy have advantages over lymphocyte and genomic assays in terms of minimal sample preparation, speed and cost. A high throughput method has been developed to measure Raman spectra from liquid plasma in a cover glass bottomed 96 well plate. However, the Raman spectra can show contributions from glass and water. The current study aims to optimise pre-processing steps to improve classification performance.
The clinical potential for Raman microscopic systems is well established for early diagnosis via cytology. Although Raman systems offer a complementary diagnostic tool providing molecular information, it is not yet utilised substantially in clinics. A few challenges for the clinical implementation of Raman spectroscopy are system and user variability. In this study, we asked how much variability occurs due to different Raman systems or users. To address these questions, we measured the same set of cells using two different Raman microscopes and by two different users. And classification models were generated using multivariate partial least squares discriminant analysis (PLS-DA) and analysed for clinical implementation. Raman spectra were measured from single exfoliated cells (n=400) from ThinPrep samples with negative cytology (n=10) and high-grade cytology (n=10). Raman spectra were acquired from the same set of cells via two identical HORIBA Jobin Yvon XploRATM systems (Villeneuve d'Ascq, France), as well as two different users. The Raman data was subjected to PLS-DA and cross-validated via leave-one-patient out. The study's findings suggest that the data acquired from the two Raman systems are 99% identical. However, the observed classification accuracy for the data obtained by user-one was 92%, whereas by user-two was 99%.
The gold standard diagnosis of oral pre-cancer/cancer is an invasive biopsy followed by a histological examination, which may present psychological trauma and risk of infection to patients. Moreover, multiple biopsies are often required to monitor precancer lesions' progression. With the aim of reducing the need for multiple biopsies, this study was undertaken to identify pre-cancerous lesions using minimally invasive brush biopsy and saliva samples, analysed by Raman spectroscopy.
Oral cancer has a poor prognosis of only 50% even in the light of current technological advances. This poor prognosis may be attributed to the still unmet clinical need to diagnose oral pre-cancer and dysplasia. Raman spectroscopy, which can detect subtle biochemical changes, has been explored for the diagnosis of cancer. This study aims to address the clinical need by exploiting the high amplification factor of Surface Enhanced Raman Spectroscopy (SERS) to analyse the saliva samples of 10 healthy controls and 10 patients with oral dysplasia. Furthermore, this technique was compared to conventional Raman spectroscopy. The saliva samples were centrifuged at 14000g for 15 minutes and the supernatant was applied directly on the SERS substrate and dried. Simultaneously, the saliva samples were prepared in the same way on slides for conventional Raman analysis. A peak at 2108 cm-1, attributed to salivary thiocyanate was present in all samples from dysplasia subjects but absent in samples from healthy non-smoking subjects. Partial least squares – discriminant analysis models for classification of oral pre-cancer were developed for both Raman spectroscopy and SERS to discriminate between healthy, mild and moderate dysplasia cohorts.
There is an unmet need for methods to help in the early detection of cervical precancer. Optical spectroscopy-based techniques, such as Raman spectroscopy, have shown great potential for diagnosis of different cancers, including cervical cancer. However, relatively few studies have been carried out on liquid-based cytology (LBC) pap test specimens and confounding factors, such as blood contamination, have been identified. Previous work reported a method to remove blood contamination before Raman spectroscopy by pretreatment of the slides with hydrogen peroxide. The aim of the present study was to extend this work to excessively bloody samples to see if these could be rendered suitable for Raman spectroscopy. LBC ThinPrep specimens were treated by adding hydrogen peroxide directly to the vial before slide preparation. Good quality Raman spectra were recorded from negative and high grade (HG) cytology samples with no blood contamination and with heavy blood contamination. Good classification between negative and HG cytology could be achieved for samples with no blood contamination (sensitivity 92%, specificity 93%) and heavy blood contamination (sensitivity 89%, specificity 88%) with poorer classification when samples were combined (sensitivity 82%, specificity 87%). This study demonstrates for the first time the improved potential of Raman spectroscopy for analysis of ThinPrep specimens regardless of blood contamination.
It is widely accepted that cervical screening has significantly reduced the incidence of cervical cancer worldwide. The primary screening test for cervical cancer is the Papanicolaou (Pap) test, which has extremely variable specificity and sensitivity. There is an unmet clinical need for methods to aid clinicians in the early detection of cervical precancer. Raman spectroscopy is a label-free objective method that can provide a biochemical fingerprint of a given sample. Compared with studies on infrared spectroscopy, relatively few Raman spectroscopy studies have been carried out to date on cervical cytology. The aim of this study was to define the Raman spectral signatures of cervical exfoliated cells present in liquid-based cytology Pap test specimens and to compare the signature of high-grade dysplastic cells to each of the normal cell types. Raman spectra were recorded from single exfoliated cells and subjected to multivariate statistical analysis. The study demonstrated that Raman spectroscopy can identify biochemical signatures associated with the most common cell types seen in liquid-based cytology samples; superficial, intermediate, and parabasal cells. In addition, biochemical changes associated with high-grade dysplasia could be identified suggesting that Raman spectroscopy could be used to aid current cervical screening tests.
Raman spectroscopy can provide a molecular-level signature of the biochemical composition and structure of cells with excellent spatial resolution and could be useful to monitor changes in composition for early stage and non-invasive cancer diagnosis, both ex-vivo and in vivo. In particular, the fingerprint spectral region (400–1,800 cm-1) has been shown to be very promising for optical biopsy purposes. However, limitations to discrimination of dysplastic and inflammatory processes based on the fingerprint region still persist. In addition, the Raman spectral signal of dysplastic cells is one important source of misdiagnosis of normal versus pathological tissues. The high wavenumber region (2,800–3,600 cm-1) provides more specific information based on N-H, O-H and C-H vibrations and can be used to identify the subtle changes which could be important for discrimination of samples. In this study, we demonstrate the potential of the highwavenumber spectral region by collecting Raman spectra of nucleoli, nucleus and cytoplasm from oral epithelial cancer (SCC-4) and dysplastic (DOK) cell lines and from normal oral epithelial primary cells, in vitro, which were then analyzed by area under the curve as a method to discriminate the spectra. In this region, we will show the discriminatory potential of the CH vibrational modes of nucleic acids, proteins and lipids. This technique demonstrated more efficient discrimination than the fingerprint region when we compared the cell cultures.
Raman spectroscopy coupled with K-means clustering analysis (KMCA) is employed to elucidate the biochemical structure of human skin tissue sections and the effects of tissue processing. Both hand and thigh sections of human cadavers were analyzed in their unprocessed and formalin-fixed, paraffin-processed (FFPP), and subsequently dewaxed forms. In unprocessed sections, KMCA reveals clear differentiation of the stratum corneum (SC), intermediate underlying epithelium, and dermal layers for sections from both anatomical sites. The SC is seen to be relatively rich in lipidic content; the spectrum of the subjacent layers is strongly influenced by the presence of melanin, while that of the dermis is dominated by the characteristics of collagen. For a given anatomical site, little difference in layer structure and biochemistry is observed between samples from different cadavers. However, the hand and thigh sections are consistently differentiated for all cadavers, largely based on lipidic profiles. In dewaxed FFPP samples, while the SC, intermediate, and dermal layers are clearly differentiated by KMCA of Raman maps of tissue sections, the lipidic contributions to the spectra are significantly reduced, with the result that respective skin layers from different anatomical sites become indistinguishable. While efficient at removing the fixing wax, the tissue processing also efficiently removes the structurally similar lipidic components of the skin layers. In studies of dermatological processes in which lipids play an important role, such as wound healing, dewaxed samples are therefore not appropriate. Removal of the lipids does however accentuate the spectral features of the cellular and protein components, which may be more appropriate for retrospective analysis of disease progression and biochemical analysis using tissue banks.
Due to its high lateral resolution, Raman microspectrsocopy is rapidly becoming an accepted technique for
the subcellular imaging of single cells. Although the potential of the technique has frequently been
demonstrated, many improvements have still to be realised to enhance the relevancy of the data collected.
Although often employed, chemical fixation of cells can cause modifications to the molecular composition
and therefore influence the observations made. However, the weak contribution of water to Raman spectra
offers the potential to study live cells cultured in vitro using an immersion lens, giving the possibility to
record highly specific spectra from cells in their original state. Unfortunately, in common 2-D culture
models, the contribution of the substrates to the spectra recorded requires significant data pre-processing
causing difficulties in developing automated methods for the correction of the spectra. Moreover, the 2-D
in vitro cell model is not ideal and dissimilarities between different optical substrates within in vitro cell
cultures results in morphological and functional changes to the cells. The interaction between the cells and
their microenvironment is crucial to their behavior but also their response to different external agents such
as radiation or anticancer drugs. In order to create an experimental model closer to the real conditions
encountered by the cell in vivo, 3-D collagen gels have been evaluated as a substrate for the spectroscopic
study of live cells. It is demonstrated that neither the medium used for cell culture nor the collagen gels
themselves contribute to the spectra collected. Thus the background contributions are reduced to that of the
water. Spectral measurements can be made in full cell culture medium, allowing prolonged measurement
times. Optimizations made in the use of collagen gels for live cells analysis by Raman spectroscopy are
encouraging and studying live cells within a collagenous microenvironment seems perfectly accessible.
Raman spectroscopy, as an evaluation of the products of ionising radiation exposure in biological systems, has been utilised mainly in the evaluation of the impact of exposure in tissue, cellular constituents and live animals. It has also been recently demonstrated that Raman spectroscopy can demonstrate key spectroscopic changes in the live cell associated with significant apoptotic and necrotic chemical damage. The present preliminary work utilises Raman spectroscopy at 514.5 nm to evaluate the results of exposure to γ-rays in HaCaT cells from a Co-60 therapy source, in tandem with other biological assays. The results demonstrate that Raman spectral changes may be correlated with changes in the cell also identified in parallel biochemical assays.
The application of vibrational spectroscopy to disease diagnosis is a relatively new, rapidly evolving scientific field. Techniques such as Raman and infrared spectroscopy have shown great promise in this regard over the past number of years. This study directly compared Raman spectroscopy and synchrotron infrared (SR-IR) spectroscopy on parallel cervical cancer samples. Both frozen and dewaxed formalin fixed paraffin preserved tissue sections were examined. Both tissue types produced good quality Raman and SR-IR spectra, although the lesser processed, frozen tissue sections displayed the most detailed spectra. Spectroscopy was shown capable of discriminating between different cell types in normal cervical tissue. Spectra recorded from invasive carcinoma showed a marked difference from those recorded from normal cervical epithelial cells. Spectral differences identified with the onset of carcinogenesis include increased nucleic acid contributions and decreased glycogen levels. These investigations pave the way for an enlarged study into this exciting new diagnostic field.
Photoluminescence intensity PL measurements were taken for a range of PmPV concentrations, in which HiPco single walled carbon nanotubes (SWNTs) at 100%, 10%, 1%, 0.1%, 0.01% and 0% mass fractions were added. The PL intensity of the composite was shown to decrease for all mass fractions, relative to the polymer up to 1.56x10-3g/l of PmPV, above which there is an initial increase in the composite emission yield with respect to the polymer. This increase is associated with an interaction within the composite, which results in a decrease in polymer aggregate formation, which has been shown to quench intensity yields. Within the concentration range studied 5.9x10-8g/l to 2g/l the photoluminescence intensity yield for each system is highly non linear. Previously the ratio of the maximum PL intensity of the composite, which includes both, bound and unbound polymer chains, and the maximum PL intensity of the polymer, which includes only unbound polymer chains was plotted as a function of polymer concentration. From this the authors calculated the amount of free polymer within each composite and derived a model, which showed that as the polymer concentration is lowered the bundles break up until isolated SWNTs are stable at low concentrations. In particular for their 100% mass fraction polymer/HiPco SWNT it was shown that individual nanotubes are stable in solutions ~3x10-5kg/m3. Here we utilize this approach and results indicate that as the mass fraction of nanotubes in reduced, individual nanotubes are stable at higher polymer concentrations. In particular for our 100% mass fraction results indicate that below ~1.5x10-4g/l individual nanotubes are stable. This result indicates that the choice of polymer and or solvent has a significant effect on the debundling and aggregation within these systems.
Spectral characteristics based on in-vivo fluorescence measurements were taken of five laboratory uni-algal cultures: Isochrysis galbana, Nannochloris atomus, Tetraselmis chui, Tetraselmis pseuicia and Tetraselmis seucica. Two diffrent techniques for fluorescence measurements have been used to detection of fluorescence of marine algae. The techniqeus used were Confocal Microscope LSM 510 and LabRam 1B Spectroscopic Microscope. Both instruments were able to detect the in vivo fluroescence signal of all uni-algal cultures. Different excitation wavelengths have been used. The excitation wavelength of 488 nm produced the fluorescence signal with highest intensity. Confocal fluorescence microscopy shows that the samples are spectrally uniform from cell to cell. This result suggests that fluorescence measurements can be used for identification of algal species. Raman spectroscopy is employed to identify differences in molecular structure between the samples. Chlorophyll fluorescence of different species shows a change. As fluorescence is directly linked to the physiological state of the photosynthetic system and very sensitive to stress being experienced further research in the direction of fluorescence detection of toxic algae species will be of interest.
Raman spectroscopy was employed to spectroscopically fingerprint the range of animo acids, purines and pyramidines. Irradiation of these components by 10 Gray gamma rays shows that several of the amino acids and pyrimidines are particularly prone to molecular degradation. The spectroscopic signature of the degradation is utilized to identify the molecular origin of the degradation observed in the various biological macromolecules. Irradiation in solution compared to solid sate is employed in an effort to differentiate between primary and secondary ionization processes.
In this study Raman spectroscopy was employed distinguish between normal and abnormal human tissue. Raman spectra were obtained using a confocal Raman microspectrometer and a laser excitation of 514.532nm for a number of healthy tissues (Tonsil and Cervix) as well as for their diseased counterparts. Each tissue type was classified and its various spectral components were associated with vibrations, rotations etc. of the chemical bonds in the tissues' constituent components. Raman bands have been attributed to biological compounds such as proteins, lipids and DNA as well as to chemical bonds within these compounds such as, C=O stretch in Amide I (1645-1680 cm-1), NH bending in Amide III (1230-1310 cm-1), phenyl ring in Phenylalanine (1004 cm-1). Changes in the relative intensities, position, and width of these bands relate to the composition of the sample. Differences between healthy and diseased tissue with progression to malignancy have been identified and include; an increase in intensity of the phenylalanine ring breathing band (1004 cm-1), the C-C stretch of proteins (1082 cm-1), Amide I band (1655 cm-1) and the C-N stretching modes of proteins (1082cm-1). Decreases in relative intensities were also observed, and include a decrease in intensity of the CH2 deformation (1295 cm-1), CH2 bending of proteins and lipids (1445 cm-1), and the C=O stretching of proteins (1695 cm-1). The molecular origin of the observed spectral changes is discussed.
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