Monitoring phytoplankton species composition and their abundance are routine tasks in marine ecological research and environmental monitoring. As phytoplankton populations are highly heterogeneous in terms of size, morphology, and most significantly, their abundance can change drastically in a very short time, it is extremely difficult to quantify and monitor them and there are demands on the instrumentation. Conventional optical microscopy and flow cytometry are the main tools to enumerate and identify phytoplankton, but they have a compromise between spatial information and acquisition speed. While imaging flow cytometry has the potential to integrate the benefit of high spatial resolution from optical microscopy and the advantage of high throughput from flow cytometry, two intrinsic blur sources, motion blur and out-of-focus blur, prevent imaging flow cytometers from obtaining high spatial resolution images with high throughput. To address these limitations, in this work, a novel light sheet based fluorescence imaging flow cytometer has been proposed, constructed, and tested for phytoplankton analysis. Both 2D and 3D imaging mode of the light sheet based fluorescence imaging flow cytometer have been investigated. In the 2D imaging mode, the instrument can screen untreated costal water samples at a volumetric throughput up to 1 ml/min. The instrument demonstrated shows a high immunity to motion blur, and all-in-focus fluorescence images are captured with a lateral resolution of 0.75 ± 0.06 µm for a wide size range ~ 1 µm to ~ 200 µm that includes pico-, nano-and microphytoplankton. This is made possible by suppressing the out-of-focus blur using thin light sheet illumination and image deconvolution, and by precluding the motion blur with a unique flow configuration. With these abilities, the instrument demonstrated has high potential as a practical field instrument for monitoring phytoplankton. In the 3D imaging mode, the instrument can scan a large number of phytoplankton cells in a short time with spatial resolution as achieved by light sheet microscopy. The lateral resolution is 0.81 ± 0.07 µm, and axial resolution in terms of FWHM of the axial scattering PSF is 1.42 ± 0.15 µm. The volumetric throughput of the instrument is 0.5 µl/min. This is benefitted from the improvement that 3D images can be acquired without the need of sample immobilization, in contrast to existing 3D imaging approaches, such as confocal fluorescence microscopy. Preliminary results from untreated coastal water samples and cultured samples show promising potentials of the instrument for phytoplankton monitoring and scientific research.

M.Tech.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

<p>During bio-technical processing it is important to monitorbiological parameters such as cell growth, viability andproduct formation. Many of the analyses traditionally used areslow to perform and provide only average data for thepopulation. Flow cytometry is a laser-based technique, whichmeasures physical properties of a cell in a flowing stream, ata rate of several thousand cells per second. It offers theprospect of an at-line, multi-parameter analysis of individualmicroorganisms in a population.</p><p>In this project several methods for at-line measurements ofbioprocesses were developed such as protocol's for measuringcell concentration, viability and product formation. Theprimary focus was on prokaryotic organisms (<i>E. coli</i>) but eukaryotic organisms (<i>P. pastoris</i>) were included.</p><p>The possibility to use volumetric cell counting to measurecell concentration (cell number) was evaluated. It was shownthat the method was applicable for high cell density processesof both<i>E. coli</i>and<i>P. pastoris</i>.</p><p>The combination of Bis- (1,3-dibutylbarbituric acid)trimethine oxonol (depolarised membranes) and propidium iodide(loss of membrane integrity) as fluorescent markers was usefulto measure viability at-line of cells in high cell densityprocesses. The protocol was shown to be reproducible for<i>E. coli</i>and<i>P. pastoris</i>.</p><p>The viability staining was used to study the kinetics ofweak organic acids (food preservatives). The protocol provideddata about cell functions such as membrane depolarisation andloss of membrane integrity caused by introducing weak organicacids to shake flask cultures of<i>E. coli.</i></p><p>Labeling inclusion bodies with fluorescent antibodiesprovided a method, which could specifically monitor theincreased accumulation of recombinant promegapoetin proteinwith process time. This technique was further developed forintracellular staining by application of a permeabilising stepbefore labeling with antibodies. Staining of inclusion bodiesdirectly inside permeabilised cells gave information about thedistribution of protein expression in the cell population.</p><p>In conclusion, flow cytometry provides an at-line, singlecell technique for measurement of several biological parametersin bioprocesses.</p><p><b>Key words</b>: flow cytometry, Partec PAS, propidium iodide(PI), bis- (1,3-dibutylbarbituric acid) trimethine oxonol(BOX), Alexa fluor 488, bioprocess,<i>E. coli</i>,<i>P. pastoris</i>, inclusion body, food preservatives,viability, membrane potential</p>

The main goal in biopharmaceutical production is achieving high volumetric productivity while maintaining product quality (i.e. glycosylation). The objectives of this project were to explore the use of dielectric analysis in the early detection of cell demise and to analyze the impact of nucleotide / nucleotide sugar precursor feedings in biopharmaceutical production and glycosylation. Measurements of changes in the polarizability of individual cells can be performed in a dielectrophoretic (DEP) cytometer designed at the University of Manitoba. In this instrument the trajectory of individual cells was tracked according to their polarizability and recorded as a force index (FI). The identified sub-populations from a batch bioreactor and apoptosis-induced cultures were correlated with the fluorescent markers of apoptosis analyzed in a flow cytometer. Discrete cell sub-populations were identified as cells passed through the various stages of apoptosis. In the batch and the starvation culture the early changes in the measured FI of cells correlated with the Annexin V fluorescent assay associated with early phase apoptosis. For the oligomycin and staurosporine cultures changes in the FI could be correlated to modifications in the mitochondrial metabolism linked with early apoptosis for both inducers. In fed-batch experiments 10 mM galactose alone or 20 mM galactose in combination with 1 mM uridine or 1 mM uridine + 8 μM MnCl2 was added to the basal and feed medium for two CHO cell lines to determine their impact on the biopharmaceutical production and the glycosylation process. The results showed that the addition of all three precursors combined increased UDP-Gal, which increased and maintained the galactosylation index during the bioprocess for CHO-EG2 and CHO-DP12 cultures by 25.4% and 37.9%, respectively, compared to the non-supplemented fed-batch culture. In both cell lines saturation was reached when a further increase in the UDP-Gal concentration did not increase the galactosylation. A negative impact on cell growth was observed with the uridine addition in the CHO-EG2 culture, which was linked to the CHO-EG2 cell line being DHFR-/-. This work presents a dielectric detection method to monitor early changes in the cell metabolism and information for shifting and maintaining galactosylation during biopharmaceutical production. / February 2016

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2011 / BACKGROUND: Human Immunodeficiency Virus (HIV) is a global epidemic with growing numbers of people on highly active anti‐retroviral therapy (HAART) programmes. Effectiveness of treatment needs to be monitored to ensure the uncompromised well being of patients. This is currently done using both Viral Load (VL) and CD4 cell counts for HAART initiation and follow‐up. Although VL is the best predictor of disease progression it is often too expensive for monitoring patients in resource‐limited settings. There is thus a need for a cheaper, more accessible alternative to monitor long term patient response to therapy. METHODS: This study evaluated the use of a recently described flow cytometric assay of CD38 expression (previously developed at the Johannesburg Flow Cytometry Reference Laboratory) in a cohort of HIV+ patients failing 1st line therapy, who were subsequently enrolled onto 2nd line HAART. CD38 and CD8 were “piggy ‐backed” onto the PLG/CD4 protocol and mean fluorescence intensity (MFI) of the CD8/38 expression was monitored longitudinally. Patterns of CD38 expression were compared to 1st line treatment observations to establish equivalence in the predictive power of CD38 expression of fluctuation in viral load on 2nd line treatment patients. In addition, the effect of sample age on assay accuracy was tested before implementation of the CD38 assay at a secondary testing site. RESULTS: The patterns observed in the cohort of 2nd line therapy patients mirrored patterns previously seen in 1st line therapy with 55% of patients showing a continuous decline in CD38 MFI that mimicked changes in VL. The remaining 33% of patients had non‐specific increases in CD38 MFI without concurrent increases in VL and one patient showed irregular VL and CD38 MFI (non‐responder). The CD38 assay showed acceptable accuracy and reproducibility up to 48 hours after venesection (%CV<5%). Implementation at the secondary testing site was successful with 98% similarity (%CV<5%) compared to the reference laboratory. CONCLUSION: CD38 monitoring of 2nd line therapy patients showed comparable patterns to observations in 1st line therapy patients. The assay proved stable over time and easy to implement at another PLG/CD4 testing facility. As such, the CD38 assay offers a cost‐effective, reliable real time supplementary test to long‐term VL monitoring of HIV infected patients on the national ART programme.

La cytométrie en flux en association avec la coloration et le marquage d'anticorps présente l'un des outils les plus précieux en biotechnologie actuelle fournissant des informations sur l'hétérogénéité des populations cellulaires, la taille et le volume des cellules, ainsi que l'expression de certaines molécules de surface et intracellulaires. L'augmentation du coût et la difficulté fondamentale de ces méthodes, cependant, sont attribués à l'exigence des molécules de marquage de surface. Diélectrophorèse (DEP) a été identifiée comme alternative sans marquage prometteuse. Cette thèse porte sur l'amélioration des technologies basée sur les DEP actuelles, et le développement d'une nouvelle méthode pour aborder les questions de cytometrie diélectrophorétique (DEP) permettant la mesure probabiliste des signatures diélectriques (DE) de cellules au niveau d’une population, ainsi que de permettre l'identification de biomarqueurs fiables pour les changements cellulaires.Tout d'abord, les améliorations de la cytométrie DEP sur la translation de cellules latérales induites par DEP sont explorées par fabrication. Un système de tri cellulaire benchmark microfluidique est présenté, et l'effet des désalignements des microcanaux sur les topologies des électrodes des cellules DEP vivantes est discuté. Un modèle de S. cerevisiae est présenté et validé expérimentalement dans des dispositifs microfluidiques fabriqués. Un nouveau procédé de fabrication permettant le prototypage rapide de dispositifs microfluidiques avec des électrodes intégrées bien alignées est présenté. Des dispositifs identiques ont été fabriqués avec des procédés standards de lithographie douce PDMS. Selon l'étude benchmark, la procédure standard PDMS est tombée bien en dessous de la gamme nécessaire pour le tri des cellules par DEP. Le temps de fabrication et les coûts de la méthode proposée se sont révélés être à peu près les mêmes.Deuxièmement, une nouvelle méthode appelée cytométrie DEP distribuée (2DEP cytométrie) a été développée. Elle utilise une translation verticale de cellules induite par effet de DEP en liaison avec la vélocimétrie par image de particules (PIV) afin de mesurer la répartition probabiliste de forces DEP sur une population cellulaire entière. La méthode a été intégrée dans un dispositif microfluidique avec des électrodes intégrées. Les cellules passant à travers le micro-canal sont sollicitées par des forces de sédimentation, tandis que les forces DEP soit s’opposent à la sédimentation, prennent en charge la sédimentation, ou aucun des deux, en fonction des signatures DE des cellules. Les hauteurs à laquelle les cellules se stabilisent correspondent à leur signature DE et sont mesurées indirectement en utilisant PIV.Les données expérimentales quantifient la signature DE d'une population de S. cerevisiae et la lignée cellulaire human immortalise leucemie myeloide K562. Tout d'abord, l'effet de la surexpression de certaines protéines membranaires a été étudié dans des cellules S. cerevisiae. La répartition mesurée des forces DEP a été comparée à la population de cellules exprimant une protéine cytoplasmique au même taux. Deuxièmement, 2DEP cytométrie a été appliquée à la lignée cellulaire K562. Les effets de la réponse à un stress provoqué par divers inducteurs sur la signature DE de la population cellulaire ont été analysées.Enfin, l'analyse statistique des données définies estimation par noyau ajustées pour surmonter la nature finie des données mesurées. En combinaison avec des spectres en distance de Wasserstein, notés signatures Wasserstein, ont été quantifiés et liée à certains changements cellulaires. Ces signatures peuvent être utilisées comme marqueurs biologiques fiables pour certains changements cellulaires.En conclusion, 2DEP cytométrie a montré être suffisamment sensible pour identifier certains changements d’états cellulaires. Le nouveau dispositif 2DEP cytométrie est donc une alternative prometteuse à la cytométrie en flux classique / Flow cytometry in combination with staining and antibody labelling presents one of the most valuable tools in current biotechnology providing information about cell population heterogeneity, cell size and volume, as well as expression of certain surface and intracellular molecules. The increased cost and the fundamental difficulty of these methods, however, are attributed to the requirement of the surface marker molecules. Attractive alternatives to flow cytometry are label-free methods, such as micro-filtration, dielectric spectroscopy, and electro-kinetic methods. Out of these methods, dielectrophoresis (DEP) was selected as the most promising approach. This thesis focuses on improvements of current DEP-based technologies, and development and establishment of a new method to address the issues of dielectrophoretic (DEP) cytometry enabling label-free non-invasive probabilistic measurement of cell dielectric (DE) signatures on population level, as well as enabling identification of reliable biomarkers for cell changes.First, improvements of DEP cytometry based on DEP-induced lateral cell translation through fabrication are explored. A benchmark microfluidic live cell sorting system is presented, and the effect of microchannel misalignment above electrode topologies on live cell DEP is discussed in detail. Simplified model of budding S. cerevisiae cell is presented and validated experimentally in fabricated microfluidic devices. A novel fabrication process enabling rapid prototyping of microfluidic devices with well-aligned integrated electrodes is presented and the process flow is described. Identical devices were produced with standard PDMS soft lithography processes. The presented fabrication process significantly improved the alignment of the microstructures. According to the benchmark study, the standard PDMS procedure fell well outside the range required for reasonable cell sorting efficiency. The fabrication time and costs of the proposed methodology were found to be roughly the same.Second, a method called distributed dielectrophoretic cytometry (2DEP cytometry) was developed. It uses a DEP-induced vertical translation of live cells in conjunction with PIV in order to measure probabilistic distribution of live cell DE signatures on an entire cell population. The method was integrated in a microfluidic device with integrated electrodes. Cells passing through the microchannel are acted on by sedimentation forces, while DEP forces either oppose sedimentation, support sedimentation, or neither, depending on the DE signatures of the cells. The heights at which cells stabilize correspond to their DE signature and are measured indirectly using particle image velocimetry (PIV).Experimental data quantify the DE signature of a S. cerevisiae population and Human immortalised myelogenous leukaemia cell line K562. First, the effect of over-expression of certain membrane protein was studied in S. cerevisiae cells. Measured distribution of DEP forces was compared to cell population expressing a cytoplasmic protein at the same rate. Second, 2DEP cytometry was applied to K562 cell line. Effects of stress response triggered by various inducers on the DE signature of the cell population were analysed.Finally, statistical data analysis defined adjusted kernel density estimation to overcome the finite nature of the measured data. In combination with Wasserstein pseudometrics from sampled data, the Wasserstein distance spectra, denoted as Wasserstein signatures, were quantified and linked to certain cell changes. These signatures may be used as reliable biomarkers for cell changes.In conclusion, 2DEP cytometry showed it is sensitive enough to identify certain changes in cell states. The novel 2DEP cytometry device is therefore a promising alternative to conventional flow cytometry

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on December 13, 2007) Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 153-161).

Platelets play a pivotal role in hemostasis and thrombosis. It induces vascular retraction and clot formation through platelets activation and signal transmission, which promote ligands expressing on the surface of platelets, such as glycoproteins and P-selectin. Some surface glycoproteins gatherd to form complexes after activation. It bound to extracellular receptors such as collagen and thrombin to induce aggregation, which could also be induced by releasing agonists, such as arachidonic acid, adenosine diphosphate, epinephrine, serotonin and fibrinogen, to active the nearby platelets. The standard process of plateletapheresis in the Blood Center was to hold the platelets in still for one hour before stored on a vibrator. The process of holding platelets still for one hour before storage was omitted in some hospitals. It was not clear whether to omit the process has any effect on the quality of platelets. The expressions of P-selectin and vWF receptor, CD42b (Gp Ib£\) on platelets were analyzed by flow cytometry in this study. No significant differences (p= 0.77 and p= 0.62, respectively) were found. Similar results were obtained when functions of platelets were evaluated by agonists. It was concluded that leaving the platelets in room temperature for one hour to recover before keeping it on a vibrator would not enhance the functions of platelets aggregation significantly.