Cellular Discrimination Using In Vitro Raman Micro Spectroscopy: The Role Of The Nucleolus

Raman micro spectroscopy has attracted considerable attention over the last few years to explore its possible clinical applications as a non-invasive powerful label-free in vitro screening tool in cancer diagnosis and monitoring, subcellular analysis of biochemical processes, drug uptake, mode of action and mechanisms of interaction as well as toxicity of, for example, chemotherapeutic agents. However, in order to evaluate accurately the potential of Raman micro spectroscopy for such applications it is essential to optimise measurement and data processing protocols associated with subcellular analysis. To this end, in vitro differentiation of cell lines is a basic proof of concept for the potential of the technique, and although many studies have indicated successful differentiation based on Raman micro spectroscopy, it is important, as the measurement and processing techniques are improved, to establish the biochemical and subcellular basis of that discrimination. In this study, Raman micro spectroscopy is used to compare and differentiate normal and cancer cells from human lung origin, A549 adenocarcinoma cell line, Calu-1 epidermoid non-small-cell and BEAS-2B normal immortalized bronchial epithelium cell line. Spectra were taken from the three subcellular compartments, the cytoplasm, the nucleus and the nucleolus and Principal Components Analysis was used to compare the spectral profiles between the cell lines and, coupled to Linear Discriminant Analysis, to explore the optimum sensitivity and specificity of discrimination. To support the analysis, Raman micro spectroscopy was coupled with Flow Cytometry, Confocal Laser Scanning Microscopy and Atomic Force Microscopy. While all subcellular regions can be employed to differentiate the normal and cancer cell lines, optimum discrimination sensitivity and specificity is achieved using the spectra from the nucleolar regions alone. Notably, only the nucleolar spectral profiles differentiate the two cancer cell lines. The results point to the importance of the nucleolar regions in diagnostic applications of Raman microscopy as well as further applications in subcellular analysis of cytological processes

Generation of Intracellular Reactive Oxygen Species and Genotoxicity Effect to Exposure of Nanosized Polyamidoamine (PAMAM) Dendrimers in PLHC-1 Cells in Vitro

Polyamidoamine (PAMAM) dendrimers elicit systematically variable cyto- and eco-toxic responses, promising the basis for structure activity relationships governing nanotoxicological responses. Increased production of intracellular reactive oxygen species (ROS), genotoxicity and apoptosis due to in-vitro exposure of hepatocellular carcinoma cells to dendrimer generations G-4, G-5 and G-6 is demonstrated. A generation dependent increase in ROS and genotoxicity was observed, consistent with our previous studies. The responses correlate well with the number of surface amino groups per generation. Although ROS production initially increases approximately linearly, it saturates at higher doses. Notably, normalised to molar dose of surface amino groups, the dose-dependent ROS production for different generations overlap exactly, indicating that the response is due to these functional units. The percentage tail DNA formation is related to the generation and dose dependent production of intracellular ROS, at low levels. At the higher ROS levels, increased DNA damage is associated with the onset of necrosis

Optical Diagnostics – Spectropathology for the Next Generation

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Generation of Intracellular Reactive Oxygen Species and Genotixity Effect to Exposure of Nanosized Polyamidoamine (PAMAM) Dendrimers in PLHC-1 Cells in Vitro

Polyamidoamine (PAMAM) dendrimers elicit systematically variable cyto- and eco-toxic responses, promising the basis for structure activity relationships governing nanotoxicological responses. Increased production of intracellular reactive oxygen species (ROS), genotoxicity and apoptosis due to in-vitro exposure of hepatocellular carcinoma cells to dendrimer generations G-4, G-5 and G-6 is demonstrated. A generation dependent increase in ROS and genotoxicity was observed, consistent with our previous studies. The responses correlate well with the number of surface amino groups per generation. Although ROS production initially increases approximately linearly, it saturates at higher doses. Notably, normalised to molar dose of surface amino groups, the dose-dependent ROS production for different generations overlap exactly, indicating that the response is due to these functional units. The percentage tail DNA formation is related to the generation and dose dependent production of intracellular ROS, at low levels. At the higher ROS levels, increased DNA damage is associated with the onset of necrosis

Understanding the Molecular Information Contained in Principal Component Analysis of Vibrational Spectra of Biological Systems

K-means clustering followed by Principal Component Analysis (PCA) is employed to analyse Raman spectroscopic maps of single biological cells. K-means clustering successfully identifies regions of cellular cytoplasm, nucleus and nucleoli, but the mean spectra do not differentiate their biochemical composition. The loadings of the principal components identified by PCA shed further light on the spectral basis for differentiation but they are complex and, as the number of spectra per cluster is imbalanced, particularly in the case of the nucleoli, the loadings under-represent the basis for differentiation of some cellular regions. Analysis of pure bio-molecules, both structurally and spectrally distinct, in the case of histone, ceramide and RNA, and similar in the case of the proteins albumin, collagen and histone, show the relative strong representation of spectrally sharp features in the spectral loadings, and the systematic variation of the loadings as one cluster becomes reduced in number. The more complex cellular environment is simulated by weighted sums of spectra, illustrating that although the loading become increasingly complex; their origin in a weighted sum of the constituent molecular components is still evident. Returning to the cellular analysis, the number of spectra per cluster is artificially balanced by increasing the weighting of the spectra of smaller number clusters. While it renders the PCA loading more complex for the three-way analysis, a pair wise analysis illustrates clear differences between the identified subcellular regions, and notably the molecular differences between nuclear and nucleoli regions are elucidated. Overall, the study demonstrates how appropriate consideration of the data available can improve the understanding of the information delivered by PCA

Raman Spectroscopy in Nanomedicine: Current Status and Future Perspectives

Raman spectroscopy is a branch of vibration spectroscopy which is capable of probing the chemical composition of materials. Recent advances in Raman microscopy have added significantly to the range of applications which now extend from medical diagnostics to exploring interfaces between biological organisms and nanomaterials. In this review, Raman is introduced in a general context, highlighting some of the areas in which the technique has found success in the past, as well as some of the potential benefits it offers over other analytical modalities. The subset of Raman techniques which specifically probe the nanoscale, namely Surface Enhanced and Tip Enhanced Raman Spectroscopy, will be described and specific applications relevant to nanomedical applications will be reviewed. Progress in the use of traditional label-free Raman applied to investigation of nanoscale interactions will be described, and recent developments in Coherent Anti-Stokes Raman Scattering will be explored, particularly applications to biomedical and nanomedical fields

Structure-Property Relationships for Electron-Vibrational Coupling in Conjugated Organic Oligomeric Systems

A series of ð-conjugated oligomers containing one to six monomer units were studied by absorption and photoluminescence spectroscopy. As is common for these systems, a linear relationship between the positioning of the lowest-energy absorption and the highest-energy photoluminescence maxima plotted versus inverse conjugation length is observed, in good agreement with a simple nearly free electron model, one of the earliest descriptions of the properties of one-dimensional organic molecules. It was observed that the Stokes shift and therefore Huang-Rhys factor also exhibit a well-defined relationship with increasing conjugation length, implying a correlation between the electron-vibrational coupling and chain length. This correlation is further examined using Raman spectroscopy, whereby the integrated relative Raman scattering is seen to behave superlinearly with chain length. The Stokes shift and the Raman activity are also well-correlated in these systems. There is a clear indication that the vibrational activity and thus nonradiative decay processes are controllable through molecular structure

Extraoral maxillofacial prosthesis implant retention systems: a critical review.

Objectives: The purpose of this literature review is to provide information on the different available techniques for implant-supported prosthetic retention, which are bar-clip, o-ring or magnets. Through presenting the practitioner preferences reported in literature, although limited from strict comparison due to the heterogeneity of methodologies and studied individuals, this review aims to identify the choices for maxillofacial prosthesis implant retention systems, regarding patient comfort and good aesthetic outcome, as an aid to surgical and prosthetic planning for implant-supported extraoral maxillofacial prosthetics. With proper knowledge of each implant retention system, a practitioner can design a treatment plan which allows for a more natural and comfortable prosthetic. Methods and Materials: Papers were searched through the PubMed and Scopus databases. The literature search was restricted to papers published from 2001-2013 although patient studies may have been conducted prior to 2001. MeSH terms for the searches were “Maxillofacial Prosthesis” and “Craniofacial Prosthesis OR Craniofacial Prostheses”. Overall, 2630 papers were returned. After eliminating duplicates, titles and abstracts were analyzed, 25 papers were filtered and reviewed. Of these, 12 papers were excluded, because they were case reports or non-systematic literature reviews. Of the remaining 13, 10 papers presented group analysis and were deemed appropriate to access practitioner’s choices, as cited in the abstract. These papers refer to 1611 prosthesis. Three papers do not mention the type of prosthetic connection chosen, so they were not counted for this purpose. Results: The most popular choices of retention system for different patient conditions were analysed, even though the sites and corresponding retention systems were not specified in all of the 10 papers based on group analysis. The bar-clip system was the most used in auricular (6 papers out of 10) and nasal prosthesis (4 papers out of 10). For the orbital region, 6 out of the 10 favored magnets. Conclusions and relevance: Non-osseointegrated mechanical or adhesive retention techniques are the least expensive and have no contraindication. When osseointegrated implants are possible, there is a more commonly used system for each facial region. The choice of implant retention system is mostly determined by two factors: standard practice and maxillofacial surgeon and maxillofacial prosthetist abilities

Polyamidoamine Dendrimer Nanoparticle Cytotoxicity, Oxidative Stress, Caspase Activation and Infammatory Response: Experimental Observation and Numerical Simulation

Mechanisms underlying the in vitro cytotoxicity of Polyamidoamine nano-dendrimers in human keratinocytes are explored. Previous studies demonstrated a systematic, dendrimer-generation-dependent cytotoxicity, oxidative stress, and genotoxicity. The emerging picture is of dendrimer endocytosis, endosomal rupture and subsequent mitochondrial attack and cell death. To understand the underlying mechanisms, the evolution of reactive oxygen species, intracellular glutathione, caspase activation, mitochondrial membrane potential decay, and inflammatory responses have been examined. Early-stage responses are associated with endosomal encapsulation, later-stage with mitochondrial attack. In all cases, the magnitude and evolution of responses depend on dendrimer generation and dose. The early-stage response is modelled using a rate equation approach, qualitatively reproducing the time, dose and generation dependences, using only two variable parameters. The dependence of the response on the nanoparticle physicochemical properties can thus be separated from internal cellular parameters, and responses can be quantified in terms of rate constants rather than commonly employed effective concentrations