Imran I. Patel

Distinguishing cell types or populations based on the computational analysis of their infrared spectra.

Infrared (IR) spectroscopy of intact cells results in a fingerprint of their biochemistry in the form of an IR spectrum; this has given rise to the new field of biospectroscopy. This protocol describes sample preparation (a tissue section or cytology specimen), the application of IR spectroscopy tools, and computational analysis. Experimental considerations include optimization of specimen preparation, objective acquisition of a sufficient number of spectra, linking of the derived spectra with tissue architecture or cell type, and computational analysis. The preparation of multiple specimens (up to 50) takes 8 h; the interrogation of a tissue section can take up to 6 h (∼100 spectra); and cytology analysis (n = 50, 10 spectra per specimen) takes 14 h. IR spectroscopy generates complex data sets and analyses are best when initially based on a multivariate approach (principal component analysis with or without linear discriminant analysis). This results in the identification of class clustering as well as class-specific chemical entities.

Infrared spectroscopy with multivariate analysis to interrogate endometrial tissue: a novel and objective diagnostic approach

Background:Endometrial cancer is the most common gynaecological malignancy in the United Kingdom. Diagnosis currently involves subjective expert interpretation of highly processed tissue, primarily using microscopy. Previous work has shown that infrared (IR) spectroscopy can be used to distinguish between benign and malignant cells in a variety of tissue types.Methods:Tissue was obtained from 76 patients undergoing hysterectomy, 36 had endometrial cancer. Slivers of endometrial tissue (tumour and tumour-adjacent tissue if present) were dissected and placed in fixative solution. Before analysis, tissues were thinly sliced, washed, mounted on low-E slides and desiccated; 10 IR spectra were obtained per slice by attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. Derived data was subjected to principal component analysis followed by linear discriminant analysis. Post-spectroscopy analyses, tissue sections were haematoxylin and eosin-stained to provide histological verification.Results:Using this approach, it is possible to distinguish benign from malignant endometrial tissue, and various subtypes of both. Cluster vector plots of benign (verified post-spectroscopy to be free of identifiable pathology) vs malignant tissue indicate the importance of the lipid and secondary protein structure (Amide I and Amide II) regions of the spectrum.Conclusion:These findings point towards the possibility of a simple objective test for endometrial cancer using ATR-FTIR spectroscopy. This would facilitate earlier diagnosis and so reduce the morbidity and mortality associated with this disease.

Combining immunolabeling and surface-enhanced Raman spectroscopy on cell membranes

We applied surface-enhanced Raman spectroscopy (SERS) to immunolabeled endothelial cells to derive enhanced spectra of the biomolecular makeup of the cellular surface. A two-step immunolabeling protocol with gold-conjugated antibodies coupled with silver enhancement to attach silver nanoparticles to the cell surface was employed. This approach generated ∼50-fold SERS enhancement of spectral signals. The SERS spectra exhibited several SERS-enhanced peaks associated with cell membrane components. The SERS detection of silver nanoparticles proved more far more sensitive than conventional light microscopy techniques. The SERS enhancement allowed us to carry out spectral mapping using wavenumbers associated with membrane components that correlated directly with the distribution of silver nanoparticles. SERS has the potential to detect immunolabeling at lower levels than is possible using conventional immunolabeling methods while simultaneously providing unique, spatially defined, biochemical information.

Concentration-dependent effects of carbon nanoparticles in gram-negative bacteria determined by infrared spectroscopy with multivariate analysis

With increasing production of carbon nanoparticles (CNPs), environmental release of these entities becomes an ever-greater inevitability. However, many questions remain regarding their impact on soil microorganisms. This study examined the effects of long or short multiwalled carbon nanotubes (MWCNTs), C60 fullerene and fullerene soot in Gram-negative bacteria. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was applied to derive signature spectral fingerprints of effects. A concentration-dependent response in spectral alterations was observed for each nanoparticle type. Long or short MWCNTs and fullerene soot gave rise to similar alterations to lipids, Amide II and DNA. The extent of alteration varies with nanoparticle size, with smaller short MWCNTs resulting in greater toxicity than long MWCNTs. Fullerene soot was the least toxic. C60 results in the most distinct and largest overall alterations, notably in extensive protein alteration. This work demonstrates a novel approach for assaying and discriminating the effects of CNPs in target systems.

High contrast images of uterine tissue derived using Raman microspectroscopy with the empty modelling approach of multivariate curve resolution-alternating least squares.

Approaches that allow one to rapidly understand tissue structure and functionality in situ remain to be developed. Such techniques are required in many instances, including where there is a need to remove with a high degree of confidence positive tumour margins during surgical excision. As biological tissue has little contrast, gold standard confirmation of surgical margins is conventionally undertaken by histopathological diagnosis of tissue architecture via optical microscopy. Vibrational spectroscopy techniques, when coupled to sophisticated computational analyses, are capable of constructing bio-molecular contrast images of unstained tissue. To assess the relative applicability of a range of candidate algorithms to distinguish the in situ bio-molecular structures of a complex tissue, the empty modelling approach of multivariate curve resolution-alternating least squares (MCR-ALS) was compared to hierarchical cluster analysis (HCA) or principal component analysis (PCA). Such chemometric analyses were applied to Raman images of benign (tumour-adjacent) endometrium, stage I and stage II endometrioid cancer. Re-constructed images from the in situ bio-molecular tissue architectures highlighted features associated with glandular epithelium, stroma, glandular lumen and myometrium. Of the tested chemometric analyses, MCR-ALS provided the best bio-molecular contrast images, superior to those derived following HCA or PCA, with clear and defined margins of histological features. Iteratively-resolved spectra identified wavenumbers responsible for the contrast image. Wavenumbers 1234 cm(-1) (Amide III), 1390 cm(-1) (CH(3) bend), 1675 cm(-1) (Amide I/lipid), 1275 cm(-1) (Amide III), 918 cm(-1) (proline) and 936 cm(-1) (proline, valine and proteins) were responsible for generating the majority of the contrast within MCR-ALS-generated images. Applications of sophisticated computational analyses coupled with vibrational spectroscopy techniques have the potential to lend novel functionality insights into bio-molecular structures in vivo.

Gold nanoparticles explore cells : cellular uptake and their use as intracellular probes

Understanding uptake of nanomaterials by cells and their use for intracellular sensing is important for studying their interaction and toxicology as well as for obtaining new biological insight. Here, we investigate cellular uptake and intracellular dynamics of gold nanoparticles and demonstrate their use in reporting chemical information from the endocytotic pathway and cytoplasm. The intracellular gold nanoparticles serve as probes for surface-enhanced Raman spectroscopy (SERS) allowing for biochemical characterisation of their local environment. In particular, in this work we compare intracellular SERS using non-functionalised and functionalised nanoparticles in their ability to segregate different but closely related cell phenotypes. The results indicate that functionalised gold nanoparticles are more efficient in distinguishing between different types of cells. Our studies pave the way for understanding the uptake of gold nanoparticles and their utilisation for SERS to give rise to a greater biochemical understanding in cell-based therapies.

Constitutive expression of bioactivating enzymes in normal human prostate suggests a capability to activate pro‐carcinogens to DNA‐damaging metabolites

The constitutive bioactivating capacity of human prostate may play a role in determining risk of adenocarcinoma developing in this tissue. Expression of candidate enzymes that convert exogenous and/or endogenous agents into reactive DNA‐damaging species would suggest the potential to generate initiating events in prostate cancer (CaP).

Coherent anti-Stokes Raman scattering for label-free biomedical imaging

Coherent anti-Stokes Raman scattering (CARS) has established itself as an imaging technique capable of providing video-rate imaging of biological specimens through vibrational coherence of endogenous molecules. Current techniques predominantly involve the application of costly, invasive and potentially non-specific dyes or labels for imaging biomolecules. CARS microscopy can however provide a high-resolution and non-invasive alternative for imaging biomolecules of interest without the need for exogenous labels. Here we provide an overview of CARS including the technique and common instrumentation as well as its applications in biomedical imaging. We discuss the major biomedical areas where CARS has been applied such as in evaluating liver disease, progression of atherosclerosis, tumour classification and tracking drug delivery, whilst also assessing the future challenges for clinical translation.

CARS based label-free assay for assessment of drugs by monitoring lipid droplets in tumour cells

Coherent anti-Stokes Raman scattering (CARS) is becoming an established tool for label-free multi-photon imaging based on molecule specific vibrations in the sample. The technique has proven to be particularly useful for imaging lipids, which are abundant in cells and tissues, including cytoplasmic lipid droplets (LD), which are recognized as dynamic organelles involved in many cellular functions. The increase in the number of lipid droplets in cells undergoing cell proliferation is a common feature in many neoplastic processes [1] and an increase in LD number also appears to be an early marker of drug-induced cell stress and subsequent apoptosis [3]. In this paper, a CARS-based label-free method is presented to monitor the increase in LD content in HCT116 colon tumour cells treated with the chemotherapeutic drugs Etoposide, Camptothecin and the protein kinase inhibitor Staurosporine. Using CARS, LDs can easily be distinguished from other cell components without the application of fluorescent dyes and provides a label-free non-invasive drug screening assay that could be used not only with cells and tissues ex vivo but potentially also in vivo.

MGMT promoter hypermethylation and K-RAS, PTEN and TP53 mutations in tamoxifen-exposed and non-exposed endometrial cancer cases.

Background:Tamoxifen has anti-oestrogenic and anti-tumour activity in the breast, but is oestrogenic and carcinogenic in the endometrium. It can induce experimental tumours by both hormonal and DNA-damaging mechanisms, but its carcinogenic mode of action in human endometrium remains unclear.Methods:We investigated whether an epigenetic mechanism, involving promoter hypermethylation of the gene for the DNA repair enzyme MGMT (O6-methylguanine DNA methyltransferase), was associated with K-RAS, TP53 and PTEN mutations in endometrial tumours from women treated with tamoxifen (TAM, n=30) or unexposed to the drug (EC, n=38).Results:There were significant (P<0.05) differences in tumour grade between the TAM and EC groups, with more favourable morphology in the latter. K-RAS mutations, predominantly G>A, occurred in small numbers in both groups. TP53 mutations were of mainly A>G, C>T and indel modifications in both groups, but more frequent in TAM cases. PTEN mutations dominated in EC tumours and were of the type that has large impact on protein function, such as indel or nonsense mutations. These observations alongside the mutational spectrum in PTEN suggest that the malignancies arise from different backgrounds, hence pointing to an effect of tamoxifen. Both groups displayed MGMT promoter hypermethylation. This coincided with mutations more frequently in the TAM (78%) than in the EC (50%) group, even though there were significantly (P<0.05) fewer mutations and methylations in TAM cases.Conclusions:Although the difference in coincidence did not reach significance with the current sample size, the findings suggest that epigenetic processes may play a role in the way tamoxifen induces endometrial cancer.