Characterisation and correction of signal fluctuations in successive acquisitions of microarray images

<p>Abstract</p> <p>Background</p> <p>There are many sources of variation in dual labelled microarray experiments, including data acquisition and image processing. The final interpretation of experiments strongly relies on the accuracy of the measurement of the signal intensity. For low intensity spots in particular, accurately estimating gene expression variations remains a challenge as signal measurement is, in this case, highly subject to fluctuations.</p> <p>Results</p> <p>To evaluate the fluctuations in the fluorescence intensities of spots, we used series of successive scans, at the same settings, of whole genome arrays. We measured the decrease in fluorescence and we evaluated the influence of different parameters (PMT gain, resolution and chemistry of the slide) on the signal variability, at the level of the array as a whole and by intensity interval. Moreover, we assessed the effect of averaging scans on the fluctuations. We found that the extent of photo-bleaching was low and we established that 1) the fluorescence fluctuation is linked to the resolution e.g. it depends on the number of pixels in the spot 2) the fluorescence fluctuation increases as the scanner voltage increases and, moreover, is higher for the red as opposed to the green fluorescence which can introduce bias in the analysis 3) the signal variability is linked to the intensity level, it is higher for low intensities 4) the heterogeneity of the spots and the variability of the signal and the intensity ratios decrease when two or three scans are averaged.</p> <p>Conclusion</p> <p>Protocols consisting of two scans, one at low and one at high PMT gains, or multiple scans (ten scans) can introduce bias or be difficult to implement. We found that averaging two, or at most three, acquisitions of microarrays scanned at moderate photomultiplier settings (PMT gain) is sufficient to significantly improve the accuracy (quality) of the data and particularly those for spots having low intensities and we propose this as a general approach. For averaging and precise image alignment at sub-pixel levels we have made a program freely available on our web-site <url>http://bioinfome.cgm.cnrs-gif.fr</url> to facilitate implementation of this approach.</p

Three-dimensional spectral phasor analysis characterises nuclear wide chromatin dynamics in living cells

Our understanding of the dynamic nature of chromatin and the models that describe it are becoming increasingly complex. This presents a need for live cell analysis techniques that characterise chromatin density in living cells. Since the discovery of the double helical structure of DNA in 1953 by Franklin, Watson and Crick, our understanding of DNA structure and that structure’s regulation of cell behaviour has grown increasingly complex. We now realise that this first order structure does not explain the dynamics and functionality of chromatin in the nuclear space. The nucleus is functionally compartmentalised where regions sharing macromolecular activity create spatiotemporal domains that regulate transcription events,5 the diffusion of molecules6, and ultimately, cell behaviour. This nuclear architecture is regulated by scaffold proteins, the post translational modification of histones, cationic interactions with histone proteins and the DNA backbone itself. These regulatory mechanisms suggest that epigenetic control of nuclear architecture is density dependant, where the expression of a particular loci is ultimately determined by the accessibility of that loci to the complex of proteins needed for its transcription. Several living cell applicable, in situ and in vitro techniques have been developed to characterise the density and conformation of chromatin. One such technique utilises a DNA density dependant spectral shift in the non-intercalating chromatin binding dye, Hoechst 33342, (H342). Modern confocal technologies such as spectral and hyperspectral analysis are now able to assemble three dimensional, 3D, replications of fluorescently stained cells by unmixing dyes using mapped spectral characteristics. This thesis utilises the spectral phasor approach to develop a living cell applicable, three dimensional, 3D, DNA density characterisation technique. Density dependant spectral shift in H342 was first confirmed and characterised. The phasor approach was then optimised for 3D analyses and subsequently applied in 3D to living L6 myoblasts to characterise relative DNA density throughout the cell cycle. This research effort demonstrates that the phasor approach to spectral analysis can characterise nuclear wide spectral shift in H342 and that this spectral shift is predictive of chromatin condensation and DNA density increases. Furthermore, spectral phasor analysis can be utilised to isolate discrete spectra in 3D and can be optimised for live cell acquisitions. Spectral phasor analysis is a promising new technology that may further elucidate the dynamic and complex architecture of the nucleus

Modular Instrumentation for Controlling and Monitoring In-Vitro Cultivation Environment and Image-based Functionality Measurements of Human Stem Cells

Artificial animal cell culture was successfully developed by Ross Harrison in 1907. But it was not until the 1940’s and 1950’s that several developments occurred, which expedited the cell culturing in-vitro (C-Vitro) to be a consistent and reproducible technique to study isolated living-cells in a controlled environment. Currently, CVitro is one of the major tools in cellular and molecular biology both in the academia and industry. They are extensively utilised to study the cellular physiology/biochemistry, to screen drugs/therapeutic compounds, to understand the effects of drugs/toxic compounds and also to identify the pathways of carcinogenesis/mutagenesis. It is also used in large scale manufacturing of vaccines and therapeutic proteins. In any experimental setup, it is important that the C-Vitro model should represent the physiological phenomena of interest with reasonable accuracy so that all experimental results are statistically consistent and reproducible. In this direction, sensors and measurement systems play important roles in in-situ detection and/or control/manipulation of cells/tissues/environment. This thesis aimed to develop new technology for tailored cell culturing and integrated measurements. Firstly, design and assembly of a portable Invert-upright microscope interchangeable modular cell culturing platform (iuCMP) was envisioned. In contrast to conventional methods, micro-scaled systems mimic the cells' natural microenvironment more precisely, facilitating accurate and tractable models. The iuCMP integrates modular measurement schemes with a mini culture chamber using biocompatible cell-friendly materials, automated environment-control (temperature and gas concentrations), oxygen sensing and simultaneous functional measurements (electrophysiological and image-based). Time lapse microscopy is very useful in cell biology, but integration of advanced >i>in-vitro/device based biological systems (e.g. lab/organ/body-on-chips, or mini-bioreactors/microfluidic systems) into conventional microscopes can be challenging in several circumstances due to multiple reasons. But in iuCMP the main advantage is, the microscope can be switched either as an inverted or as an upright system and therefore can accommodate virtually any in-vitro device. It can capture images from regions that are otherwise inaccessible by conventional microscopes, for example, cells cultured on physical or biochemical sensor systems. The modular design also allows accommodating more sensor or measurement systems quite freely. We have demonstrated the system for video-based beating analysis of cardiomyocytes, cell orientation analysis on nanocellulose, and simultaneous long-term in-situ microscopy with oxygen and temperature sensing in hypoxia. In an example application, the system was utilised for long-term temperature stressing and simultaneous mechanobiological analysis of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). For this the iuCMP together with a temperature sensor plate (TSP) and a novel non-invasive beating analysis software (CMaN—cardiomyocyte function analysis tool, scripted as a subpart of this thesis), was applied for automated temperature response studies in hiPSC-CM cultures. In-situ temperature sensing is usually challenging with bulky external sensors, but TSPs solved this issue. In the temperature response study, we showed that the relationship between hiPSC-CM beating frequency and temperature is non-linear and measured the Q10 temperature coefficients. Moreover, we observed the hiPSC-CM contractile networking, including propagation of the action potential signal between dissociated clusters and their non-invasive measurements. It was the first case where these events were reported in hiPSC-CM clusters and their noninvasive measurements by image processing. The software CMaN comes with a user-friendly interface and, is equipped with features for batch processing, movement centre detection and cluster finding. It can extract six different signals of the contractile motion of cardiomyocytes (clusters or single cells) per processing. This ensures a minimum of one useful beating signal even in the cases of complex beating videos. On the processing end, compared to similar tools, CMaN is faster, more sensitive, and computationally less expensive and allows ROI based processing. In the case of healthy cells, the waveform of the signal from the CMaN resembles an ECG signal with positive and negative segments, allowing the computation of contraction and relaxation features separately. In addition to iuCMP, a Modular optical pH measurement system (MO-pH) for 24/7 non-contact cell culture measurements was also developed. The MO-pH incorporates modular sterilisable optical parts and is used in phenol-red medium cell cultures. The modular assembly of MO-pH cassettes is unique and reusable. Measurements are carried out in a closed flow system without wasting any culture medium and requires no special manual attention or recalibrations during culture. Furthermore, a new absorption correction model was put forward that minimised errors caused e.g. by biolayers in spectrometric pH measurement, which improved the pH measurement accuracy. MO-pH has been applied in long-term human adipose stem cells (hASC) expansion cultures in CO2 dependent and independent media. Additionally, the MO-pH was also utilised to comprehend the behaviour of pH, temperature and humidity in water jacked incubators as well as to record the pH response as a function of temperature in the presence and absence of CO2 in the context of stem cell cultures. The resulting plots clearly showed the interplay between measured parameters indicating a few stress sources present all through the culture. Additionally, it provided an overall picture of behaviour of critical control parameters in an incubator and pointed out the need for bioprocess systems with automatic process monitoring and smart control for maximum yield, optimal growth and maintenance of the cells. Besides, we also integrated MO-pH into flasks with reclosable lids (RL-F) and tested its applicability in stem cell cultures. A standalone system around an RL-F flask was built by combining the cell culture, medium perfusion and optical measurements. The developed RL-F system has been successfully tested in ASC-differentiation cultures. Finally, a few trial experiments for image-based pH estimation aimed for iuCMP have also been carried out. This includes tests with LCD illumination, optical projection tomography, and webcam systems. In reality, the pH is not distributed uniformly in tissues, and has shown a gradient of up to 1.0 pH unit within 1 cm distance. Therefore, producing reliable pH maps also in in-vitro can be important in understanding various common pathologies and location of lesions. A reliable and adequately developed long-term pH mapping method will be an important addition into the iuCMP

Use of intensity - and spatial-based image descriptors to characterise and quantify neoplastic lesions in positron emission tomography

Intra-tumour biological heterogeneity is a characteristic shared by all cancers and is thought to contribute to treatment failure. Within-lesion spatial heterogeneity can be qualitatively visualised in Positron Emission Tomography (PET) imaging. Quantifying the variability of the biological processes and the complexity of the signal being measured in PET oncology is essential. The aim of this thesis was to develop and validate intensity- and spatial-based metrics to quantitatively account for the complexity of radiotracer uptake and to annotate intra-tumour PET heterogeneity. Texture analysis was employed to characterise the in vivo tumour heterogeneity of cell proliferation in breast tumours using 18F-fluorothymidine (18F-FLT) PET. The repeatability of the feature measurements was assessed in patients who had two PET scans prior to therapy. Associations between features at baseline and clinical response measured after three cycles of chemotherapy were explored. Associations between feature changes at one week after the start of chemotherapy and clinical response were also explored. Furthermore, the influence of analysis parameters and imaging protocols were studied. A subset of textural features produced reliable measurements and were associated with treatment response. A technique based on multifractal analysis was also developed for characterising the space-filling properties of an object of interest in PET imaging. The derived spatial index was further combined with intensity metrics and the technique was shown to correct for partial volume effects. The method was illustrated on mathematical objects, validated on test-retest 18F-FLT PET clinical data and applied to realistic PET simulations. This work contributes to the demonstration that intensity- and spatial-based image analysis methods can supplement existing methods in PET quantification studies. These techniques provide some improvements on existing methods to derive classical quantitative PET indices and permit extraction of additional information to further characterise patient populations in the clinical setting and in relation to therapy.Open Acces

Tools for single cell proteomics

Despite recent advances that offer control of single cells, in terms of manipulation and sorting and the ability to measure gene expression, the need to measure protein copy number remains unmet. Measuring protein copy number in single cells and related quantities such as levels of phosphorylation and protein-protein interaction is the basis of single cell proteomics. A technology platform to undertake the analysis of protein copy number from single cells has been developed. The approach described is ‘all-optical’ whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by mechanical shearing caused by laser-induced microcavitation; and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. This demonstrates the ability count protein copy number from single cells in a manner which could be applied in principle to any set of proteins and for any cell type without the need for genetic engineering. Metabolism can undergo alteration in diseases such as cancer and heart failure and also as cells differentiate during development. In order to assess how it may inform a proteomic measurement, multidimensional two-photon fluorescence metabolic imaging is conducted on a cultured cancer cell line, primary adult rat cardiomyocytes and human embryonic stem cells. By measuring the parameters of fluorescence such as intensity and lifetime of the autofluorescent metabolic co-factors NADH and FAD, it was found to be possible to contrast cells under various conditions and metabolic stimuli. In particular, human embryonic stem cells were able to be contrasted at 3 stages of development as they underwent differentiation into embryonic stem cell derived cardiomyocytes. Metabolic imaging provides a non-destructive method to monitor cellular metabolic activity with high resolution. This is complimentary to the single cell proteomic platform and the convergence of both techniques holds promise in future investigations into how metabolism influences cell function and the proteome in development and disease

Fucoidan degradation by marine bacteria

The oceans are an important carbon sink that have sequestered about half of all anthropogenic CO2 emissions. Marine carbon cycling is driven by the deposition of photosynthetic micro- and macroalgae in ocean sediments, where carbon is stored over thousands of years. The algal polysaccharide fucoidan is considered to be recalcitrant to microbial degradation and may therefore facilitate long-term carbon storage. Yet, factors that render fucoidan recalcitrant against microbial degradation remain unidentified, hampering our understanding of fucoidans in the carbon cycle. Fucoidans originating from the cell wall of brown algae are often co-extracted with other cell wall components. In Chapter I, I develop a simple step-wise protocol to purify fucoidans from different brown algae. Using mass spectrometry and nuclear magnetic resonance analyses, I describe the highly diverse and branched structures of different fucoidans. In Chapter II, I examine how marine bacteria degrade those complex branched fucoidans. Using genomics, proteomics and biochemistry, I characterize the newly isolated Verrucomicrobium a Lentimonasa sp. CC4 and show that fucoidan degradation requires highly dedicated pathways of over 100 enzymes covering 20% of the a Lentimonasa sp. CC4 proteome. The complexity of these pathways implies that only highly specialized bacteria can effectively degrade fucoidans and gives a clue why it may be recalcitrant. The proteomic analysis of a Lentimonasa sp. CC4 in chapter II suggested that two protein families, S1 15 and GH29, are key in fucoidan degradation. In Chapter III, I biochemically and structurally characterize one S1 15 sulfatase and one GH29 fucosidase, revealing their exo-enzyme activity and a novel catalytic pair of two aspartate residues. This provides insights into the molecular mechanism of exo-enzymatic fucoidan degradation. In Chapter IV, I trace the dynamics of different polysaccharides during a diatom spring bloom in Helgoland. I found that the dominant bloom-forming diatom Chaetoceros socialis secretes fucoidan in dissolved form, which aggregates and accumulates in particles at the end of the bloom. Known enzymes to degrade this polysaccharide are not expressed in the microbial community which indicates that fucoidans are not microbially degraded and act as vector for organic carbon drawdown. To summarize, fucoidans are diverse, highly branched polysaccharides whose degradation requires a large set of enzymes found in very few specialized marine bacteria. Their stability-enhancing properties lead to increased brown algal deposition in coastal sediments and in the open ocean they may acts as aggregation nuclei that enhance aggregation and settling of phytoplankton aggregates. Their abundance, recalcitrant nature and stickiness make fucoidans a likely key players in oceanic carbon sequestration

Nanoparticles in medicine: Automating the analysis process of high-throughput microscopy data.

Automated tracking of cells across timelapse microscopy image sequences typically employs complex segmentation routines and/or bio-staining of the tracking objective. Often accurate identification of a cell's morphology is not of interest and the accurate segmentation of cells in pursuit of non-morphological parameters is complex and time consuming. This thesis explores the potential of internalized quantum dot nanoparticles as alternative, bio- and photo-stable optical markers for tracking the motions of cells through time. CdTe/ZnS core-shell quantum dots act as nodes in moving light display networks within A549, epithelial, lung cancer cells over a 40 hour time period. These quantum dot fluorescence sources are identified and interpreted using simplistic algorithms to find consistent, non-subjective centroids that represent cell centre locations. The presented tracking protocols yield an approximate 91% success rate over 24 hours and 78% over the full 40 hours. The nanoparticle moving light displays also provide simultaneous collection of cell motility data, resolution of mitotic traversal dynamics and identification of familial relationships enabling the construction of multi-parameter lineage trees. This principle is then developed further through inclusion of 3 different coloured quantum dots to create cell specific colour barcodes and reduce the number of time points necessary to successfully track cells through time. The tracking software and identification of parameters without detailed morphological knowledge is also demonstrated through automated extraction of DOX accumulation profiles and Cobalt agglomeration accruement statistics from two separate toxicology assays without the need for cell segmentation

A proteomic investigation of the immune response of the South African abalone, Haliotis midae

Includes bibliographical referencesHaliotis midae is a commercially important abalone in South Africa, previously harvested from a stable, quota-managed fishery. However, the combined effects of overharvesting, increased illegal catches and negative environmental factors led to a collapse in wild populations in the mid-90s. Consequently, land-based aquaculture of H. midae has grown significantly in South Africa, to satisfy the global demand for abalone and alleviate pressure on wild stocks. Unfortunately, disease outbreaks have had a severe impact on the abalone aquaculture industry internationally and remain one of the single biggest factors contributing to economic loss. Understanding the effects of pathogen infection of abalone is therefore crucial to mitigating and controlling infection outbreaks on farms. Despite this, the molecular mechanisms underlying the immune response of H. midae remain obscure. High-throughput proteomics, a powerful tool to analyse global protein expression changes, can provide an integrated view of the immune system. Thus, this study aimed to elucidate the haemocyte proteome of H. midae and gain insight into regulatory molecular pathways underlying innate immunity. In this study, a comparative shotgun proteomics approach using isobaric tagging for relative and absolute quantification (iTRAQ) coupled with LC-MS/MS was employed to investigate H. midae proteome changes in response to Vibrio anguillarum challenge. A preliminary iTRAQ challenge trial was conducted which identified a putative early (1 and 2 hours post-injection) and late (48 hours post-injection) proteome response to bacterial-challenge. Using these time points, four independent challenge trials were conducted and analysed by iTRAQ and the results combined to produce a high-confidence dataset with good quantitative reproducibility for statistical analysis. A parallel set of experiments was conducted using mock-infected samples