An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging

AbstractAlthough live cell imaging is desirable, it is not always feasible and in many situations cells are fixed in order to provide a ‘snapshot’ of the nature and distribution of molecules within a cell while minimising changes from cell movement, sample degradation etc. There is a wide range of fixation methods available that act via different mechanisms, and on different cell components. Each method has advantages and disadvantages and a choice of what fixation method to choose for a particular experiment needs to take these factors into consideration. Here we used Raman spectroscopic imaging of live cells, and compared with cells preserved with aldehyde, or organic solvent-based fixation methods to assess the chemical changes induced by each fixative, and their impact on the quality of images that can be obtained from fixed cells. Overall, aldehyde fixation methods performed significantly better than organic solvents with less severe loss of biochemical information. Aldehyde based fixatives show an altered biochemical content of the cells, attributed to adduct formation, but this can be minimised by optimising fixation temperature, or through removal of adduct formation by detergent-based permeabilization treatments as a second step (at the cost of the loss of other biochemical information). The results showed that organic solvents, on the other hand, lead to a severe loss of cell content, attributed to the loss of membrane integrity after the removal of lipids. Additionally, fixation with aldehydes prior to permeabilization with organic solvents does not provide adequate protection of cytoplasmic content. The use of Raman imaging is ideal for comparing groups of cells in terms of their molecular content, and the results show that aldehyde fixations methods are preferable for studies where the overall molecular content of the samples is important. Although there is no universal fixation method for every application, the results here allow us to provide a few general principles: where spectral similarity to live cells is important, fixation with paraformaldehyde at room temperature is preferable, at the cost of some blebbing and vacuole formation. Where preservation of cellular structure or biomolecular distribution is important, a mix of paraformaldehyde and glutaraldehyde would be more appropriate, but at the cost of some changes to spectral profile, particularly in DNA-related bands

Correcting an acoustic wavefield for elastic effects

Finite-difference simulations are an important tool for studying elastic and acoustic wave propagation, but remain computationally challenging for elastic waves in three dimensions. Computations for acoustic waves are significantly simpler as they require less memory and operations per grid cell, and more significantly can be performed with coarser grids, both in space and time. In this paper, we present a procedure for correcting acoustic simulations for some of the effects of elasticity, at a cost considerably less than full elastic simulations. Two models are considered: the full elastic model and an equivalent acoustic model with the same P velocity and density. In this paper, although the basic theory is presented for anisotropic elasticity, the specific examples are for an isotropic model. The simulations are performed using the finite-difference method, but the basic method could be applied to other numerical techniques. A simulation in the acoustic model is performed and treated as an approximate solution of the wave propagation in the elastic model. As the acoustic solution is known, the error to the elastic wave equations can be calculated. If extra sources equal to this error were introduced into the elastic model, then the acoustic solution would be an exact solution of the elastic wave equations. Instead, the negative of these sources is introduced into a second acoustic simulation that is used to correct the first acoustic simulation. The corrected acoustic simulation contains some of the effects of elasticity without the full cost of an elastic simulation. It does not contain any shear waves, but amplitudes of reflected P waves are approximately corrected. We expect the corrected acoustic solution to be useful in regions of space and time around a P-wave source, but to deteriorate in some regions, for example, wider angles, and later in time, or after propagation through many interfaces. In this paper, we outline the theory of the correction method, and present results for simulations in a 2-D model with a plane interface. Reflections from a plane interface are simple enough that an analytic analysis is possible, and for plane waves, we give the correction to the acoustic reflection and transmission coefficients. Finally, finite-difference calculations for plane waves are used to confirm the analytic results. Results for wave propagation in more complicated, realistic models will be presented elsewher

Illness perceptions and work participation: a systematic review

Self-regulatory processes play an important role in mediating between the disease and the health outcomes, and potentially also work outcomes. This systematic review aims to explore the relationship between illness perceptions and work participation in patients with somatic diseases and complaints. The bibliographic databases Medline, PsycINFO and Embase were searched from inception to March 2008. Included were cross-sectional or longitudinal studies, patients with somatic diseases or complaints, illness perceptions based on at least four dimensions of the common sense model of self-regulation, and work participation. Two longitudinal and two cross-sectional studies selected for this review report statistically significant findings for one or more illness perception dimensions in patients with various complaints and illnesses, although some dimensions are significant in one study but not in another. Overall, non-working patients perceived more serious consequences, expected their illness to last a longer time, and reported more symptoms and more emotional responses as a result of their illness. Alternatively, working patients had a stronger belief in the controllability of their condition and a better understanding of their disease. The limited number of studies in this review suggests that illness perceptions play a role in the work participation of patients with somatic diseases or complaints, although it is not clear how strong this relationship is and which illness perception dimensions are most useful. Identifying individuals with maladaptive illness perceptions and targeting interventions toward changing these perceptions are promising developments in improving work participatio

Utilising daily diaries to examine oral health experiences associated with dentine hypersensitivity

Background: The current investigation examined the determinants of oral health experiences associated with dentine hypersensitivity using prospective diary methodology. Methods: Staff and students from a large UK university who had self-diagnosed dentine hypersensitivity completed an online daily diary and text survey for two weeks recording their mood, oral health-related coping behaviours, coping and pain appraisals, pain experiences and functional limitations. Cross sectional and lagged path analyses were employed to examine relationships. Results: 101 participants took part in the diary study. Participants had a mean age of 26.3 years (range=18-63) and most were female (N=69). Individuals who used more oral health-related coping behaviours predicted and experienced greater levels of pain on subsequent days. Negative mood also predicted worse pain outcomes. The daily diary method provided a useful avenue for investigating variations in oral health experiences and relationships between variables that can fluctuate daily. Conclusions: Psychological variables such as coping and mood play an important role in the pain experiences of people with dentine hypersensitivity. The study highlights the benefits of using prospective methods to elucidate the experiences of people with oral condition

Application of chemometric analysis to infrared spectroscopy for the identification of wood origin

Chemical characteristics of wood are used in this study for plant taxonomy classification based on the current Angiosperm Phylogeny Group classification (APG III System) for the division, class and subclass of woody plants. Infrared spectra contain information about the molecular structure and intermolecular interactions among the components in wood but the understanding of this information requires multivariate techniques for the analysis of highly dense datasets. This article is written with the purposes of specifying the chemical differences among taxonomic groups, and predicting the taxa of unknown samples with a mathematical model. Principal component analysis, t-test, stepwise discriminant analysis and linear discriminant analysis, were some of the chosen multivariate techniques. A procedure to determine the division, class, subclass and order of unknown samples was built with promising implications for future applications of Fourier Transform Infrared spectroscopy in wood taxonomy classification

Subcellular peptide localization in single identified neurons by capillary microsampling mass spectrometry

Single cell mass spectrometry (MS) is uniquely positioned for the sequencing and identification of peptides in rare cells. Small peptides can take on different roles in subcellular compartments. Whereas some peptides serve as neurotransmitters in the cytoplasm, they can also function as transcription factors in the nucleus. Thus, there is a need to analyze the subcellular peptide compositions in identified single cells. Here, we apply capillary microsampling MS with ion mobility separation for the sequencing of peptides in single neurons of the mollusk Lymnaea stagnalis, and the analysis of peptide distributions between the cytoplasm and nucleus of identified single neurons that are known to express cardioactive Phe-Met-Arg-Phe amide-like (FMRFamide-like) neuropeptides. Nuclei and cytoplasm of Type 1 and Type 2 F group (Fgp) neurons were analyzed for neuropeptides cleaved from the protein precursors encoded by alternative splicing products of the FMRFamide gene. Relative abundances of nine neuropeptides were determined in the cytoplasm. The nuclei contained six of these peptides at different abundances. Enabled by its relative enrichment in Fgp neurons, a new 28-residue neuropeptide was sequenced by tandem MS

Evaluation of a novel magneto-optical method for the detection of malaria parasites

Improving the efficiency of malaria diagnosis is one of the main goals of current malaria research. We have recently developed a magneto-optical (MO) method which allows high-sensitivity detection of malaria pigment (hemozoin crystals) in blood via the magnetically induced rotational motion of the hemozoin crystals. Here, we evaluate this MO technique for the detection of Plasmodium falciparum in infected erythrocytes using in-vitro parasite cultures covering the entire intraerythrocytic life cycle. Our novel method detected parasite densities as low as approximately 40 parasites per microliter of blood (0.0008% parasitemia) at the ring stage and less than 10 parasites/microL (0.0002% parasitemia) in the case of the later stages. These limits of detection, corresponding to approximately 20 pg/microL of hemozoin produced by the parasites, exceed that of rapid diagnostic tests and compete with the threshold achievable by light microscopic observation of blood smears. The MO diagnosis requires no special training of the operator or specific reagents for parasite detection, except for an inexpensive lysis solution to release intracellular hemozoin. The devices can be designed to a portable format for clinical and in-field tests. Besides testing its diagnostic performance, we also applied the MO technique to investigate the change in hemozoin concentration during parasite maturation. Our preliminary data indicate that this method may offer an efficient tool to determine the amount of hemozoin produced by the different parasite stages in synchronized cultures. Hence, it could eventually be used for testing the susceptibility of parasites to antimalarial drugs

Microseismic Full Waveform Modeling in Anisotropic Media with Moment Tensor Implementation

Seismic anisotropy which is common in shale and fractured rocks will cause travel-time and amplitude discrepancy in different propagation directions. For microseismic monitoring which is often implemented in shale or fractured rocks, seismic anisotropy needs to be carefully accounted for in source location and mechanism determination. We have developed an efficient finite-difference full waveform modeling tool with an arbitrary moment tensor source. The modeling tool is suitable for simulating wave propagation in anisotropic media for microseismic monitoring. As both dislocation and non-double-couple source are often observed in microseismic monitoring, an arbitrary moment tensor source is implemented in our forward modeling tool. The increments of shear stress are equally distributed on the staggered grid to implement an accurate and symmetric moment tensor source. Our modeling tool provides an efficient way to obtain the Green’s function in anisotropic media, which is the key of anisotropic moment tensor inversion and source mechanism characterization in microseismic monitoring. In our research, wavefields in anisotropic media have been carefully simulated and analyzed in both surface array and downhole array. The variation characteristics of travel-time and amplitude of direct P- and S-wave in vertical transverse isotropic media and horizontal transverse isotropic media are distinct, thus providing a feasible way to distinguish and identify the anisotropic type of the subsurface. Analyzing the travel-times and amplitudes of the microseismic data is a feasible way to estimate the orientation and density of the induced cracks in hydraulic fracturing. Our anisotropic modeling tool can be used to generate and analyze microseismic full wavefield with full moment tensor source in anisotropic media, which can help promote the anisotropic interpretation and inversion of field data

Structural investigations of RNA through the application of Raman, Raman optical activity and surface enhanced spectroscopies

RNA molecules are involved in a wide range of cellular processes from genetic coding to catalytic activities, and determination of their secondary and tertiary structures is essential for the understanding of their functions. The work presented in this thesis shows the structural information that can currently be obtained using Raman and ROA spectroscopies for nucleic acid components and RNA molecules. Raman and ROA spectra can be used to identify particular building blocks, for example, with some marker bands characteristic for mono- and tri-nucleotides. In oligonucleotides and RNA sequences, Raman is particularly sensitive to the environment of the bases, while ROA is most sensitive to the conformation of the ribose-phosphate backbone. Therefore this complementary information can be used to identify single base changes and the introduction of a bulge sequence in the EMCV IRES Domain I, as well as allow tentative identification of structural features within the Adenovirus VA RNAr. Moreover, perturbation induced changes can also be monitored using Raman spectroscopy. Results presented here show these studies can be used to identify changes in specific parts of the molecule with particular temperatures, as well as identifying particular changes in secondary and/or tertiary structure with other perturbations, such as Mg2+ concentrations. The main limitation for ROA studies of RNA is the requirement for relatively high concentrations and long data collection times which can limit the range of RNA molecules that can be studied. Surface enhanced spectroscopic techniques can reduce the sample concentration and data collection requirements through the interaction of a molecule with surface plasmons associated with a nanoscale· roughened metal surface. The second half of this thesis concentrates on the implications of surface enhancement for Raman (SERS) and ROA (SEROA) studies of RNA. The application of SERS to RNA appears to be more complex than for other analyte species because the enhancement process is strongly time dependent. This, together with the spectral profile, can be influenced by a number of factors, including the nature of the aggregating agent and the concentration of the reagents involved. The effects of changes of these variables on the spectral profile and the time dependence of SERS enhancement are presented and discussed in the context of physical changes occurring during the experiment. Overall, Raman and ROA studies of RNA have provided information about the secondary and tertiary structures of RNA molecules despite their varying conformations and transitions, a matter that can be complicated for high-resolution techniques, such as X-ray crystallography. Surface enhanced Raman studies of RNA can, with careful preparation, provide meaningful results whilst reducing sample concentration requirements. However, the processes involved in generating surface enhanced ROA spectra appear to be significantly more complex and, for nucleic acid components, SEROA has not been successfully measured.EThOS - Electronic Theses Online ServiceGBUnited Kingdo