RiboBright reveals cell-type-specific differences in ribosome organization and movement.

Across species and environments, the ribosome content of cell populations correlates with population growth rate. The robustness and universality of this correlation have led to its classification as a "growth law." This law has fueled theories about how evolution selects for microbial organisms that maximize their growth rate based on nutrient availability, and it has informed models about how individual cells regulate their growth rates and ribosomal content. However, due to methodological limitations, this growth law has rarely been studied at the level of individual cells. While populations of fast-growing cells tend to have more ribosomes than populations of slow-growing cells, it is unclear whether individual cells tightly regulate their ribosome content to match their environment. Here, we employ recent groundbreaking single-cell RNA sequencing techniques to study this growth law at the single-cell level in two different microbes, S. cerevisiae (a single-celled yeast and eukaryote) and B. subtilis (a bacterium and prokaryote). In both species, we observe significant variation in the ribosomal content of single cells that is not predictive of growth rate. Fast-growing populations include cells exhibiting transcriptional signatures of slow growth and stress, as do cells with the highest ribosome content we survey. Broadening our focus to non-ribosomal transcripts reveals subpopulations of cells in unique transcriptional states suggestive that they have evolved to do things other than maximize their rate of growth. Overall, these results indicate that single-cell ribosome levels are not finely tuned to match population growth rates or nutrient availability and cannot be predicted by a Gaussian process model that assumes measurements are sampled from a normal distribution centered on the population average. This work encourages the expansion of growth law and other models that predict how growth rates are regulated or how they evolve to consider single-cell heterogeneity. To this end, we provide extensive data and analysis of ribosomal and transcriptomic variation across thousands of single cells from multiple conditions, replicates, and species.

Human embryonal carcinoma (EC) cell lines exhibit considerable heterogeneity in their levels of pluripotency. Thus, NT2/D1 cells differentiate into neural lineages upon exposure to all-trans retinoic acid (ATRA) and non-neural epithelial lineages upon exposure to bone morphogenetic protein-2 (BMP-2). In contrast, 27X-1 cells differentiate into extra-embryonic endodermal (ExE) cells upon treatment with either morphogen. To understand the molecular basis for the differential responses of the two cell lines, we performed gene expression profiling at the undifferentiated EC cell line state to identify constitutive differences in gene expression. NT2/D1 cells preferentially expressed transcripts associated with neurectodermal development, whereas 27X-1 cells expressed high levels of transcripts associated with mesendodermal characteristics. We then determined temporal expression profiles of 27X-1 cells during ExE differentiation upon treatment with ATRA and BMP-2 and compared the data with changes in gene expression observed during BMP-2- and ATRA-induced differentiation of NT2/D1 cells. ATRA and BMP-2 induced distinct sets of transcription factors and phenotypic markers in the two EC cell lines, underlying distinct lineage choices. Although 27X-1 differentiation yielded comprehensive gene expression profiles of parietal endodermal lineages, we were able to use the combined analysis of 27X-1 data with data derived from yolk sac tumors for the identification of transcripts associated with visceral endoderm formation. Our results demonstrate constitutive differences in the levels of pluripotency between NT2/D1 and 27X-1 cells that correlate with lineage potential. This study also demonstrates that EC cells can serve as robust models to investigate early lineage choices during both embryonic and extra-embryonic human development.

Cells from the extraembryonic endoderm of the gastrulating chick embryo adhere to one another in the absence of divalent cations. The addition of Mg2+ ions to the medium has no effect on the aggregation kinetics but the addition of Ca2+ ions increases the number of cells which aggregate and also stabilizes adhesion. Some aggregation also occurs when cells are suspended in saline devoid of Ca2+ and Mg2+ ions and supplemented with EGTA, a Ca2+ ion complexing agent, but adhesion is not stabilized. Shear sensitive and shear resistant bonds form in Ca-containing as well as in EGTA-containing saline. These results suggest that extraembryonic endoderm cells have Ca2+ indepedent and Ca2+ dependent mechanisms of adhesion.

Dissecting embryonic and extraembryonic lineage crosstalk with stem cell co-culture

Esrrb is a cell-cycle-dependent associated factor balancing pluripotency and XEN differentiation

Morphological and biochemical evidence is presented that the visceral extraembryonic endoderm of the 6.5-day mouse embryo will differentiate into parietal endoderm when cultured in contact with extraembryonic ectoderm undergoing transition into trophoblast giant cells. Egg cylinders from 6.5-day embryos were dissected into embryonic and extraembryonic halves and cultured in suspension in vitro for up to 7 days. After 4 days, the endoderm cells of the extraembryonic fragments morphologically resemble parietal endoderm, are associated with a thick basement membrane and synthesize large amounts of the matrix proteins laminin and Type IV procollagen. A similar transition in phenotype is not seen in the endoderm of embryonic fragments, nor in visceral extraembryonic endoderm cells cultured in isolation. In another series of experiments, complete egg cylinders were dissected free of visceral endoderm overlying the extraembryonic ectoderm and then cultured in vitro. The visceral endoderm cells which recolonize the surface of the extraembryonic ectoderm develop a parietal endoderm phenotype and lay down a thick basement membrane. These results suggest that the differentiation of the extraembryonic endoderm of the early mouse embryo into visceral and parietal phenotypes can be influenced by local cell-cell or cell-substrate interactions, and is not determined solely by cell lineage.

The technique of injecting genetically labelled cells into blastocysts was used in an attempt to determine whether the parietal and visceral endoderm originate from the same or different cell populations in the early embryo. When the developmental potential of 5th day primitive ectoderm and primitive endoderm cells was compared thus, only the latter were found to colonize the extraembryonic endoderm. Furthermore, single primitive endoderm cells yielded unequivocal colonization of both the parietal and the visceral endoderm in a proportion of chimaeras. However, in the majority of primitive endodermal chimaeras, donor cells were detected in the parietal endoderm only, cases of exclusively visceral colonization being rare. Visceral endoderm cells from 6th and 7th day post-implantation embryos also exhibited a striking tendency to contribute exclusively to the parietal endoderm following blastocyst injection. The above findings lend no support to a recent proposal that parietal and visceral endoderm are derived from different populations of inner cell mass cells. Rather, they suggest that the two extraembryonic endoderm layers originate from a common pool of primitive endoderm cells whose direction of differentiation depends on their interactions with non-endodermal cells.

RationaleUnbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.ObjectivesValidation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.MethodsWe analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.Measurements and Main ResultsWe observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.ConclusionCFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.

Fatty acid transport protein-4 (FATP4) is the major FATP in the small intestine. We previously demonstrated, using in vitro antisense experiments, that FATP4 is required for fatty acid uptake into intestinal epithelial cells. To further examine the physiological role of FATP4, mice carrying a targeted deletion of FATP4 were generated. Deletion of one allele of FATP4 resulted in 48% reduction of FATP4 protein levels and a 40% reduction of fatty acid uptake by isolated enterocytes. However, loss of one FATP4 allele did not cause any detectable effects on fat absorption on either a normal or a high fat diet. Deletion of both FATP4 alleles resulted in embryonic lethality as crosses between heterozygous FATP4 parents resulted in no homozygous offspring; furthermore, no homozygous embryos were detected as early as day 9.5 of gestation. Early embryonic lethality has been observed with deletion of other genes involved in lipid absorption in the small intestine, namely microsomal triglyceride transfer protein and apolipoprotein B, and has been attributed to a requirement for fat absorption early in embryonic development across the visceral endoderm. In mice, the extraembryonic endoderm supplies nutrients to the embryo prior to development of a chorioallantoic placenta. In wild-type mice we found that FATP4 protein is highly expressed by the epithelial cells of the visceral endoderm and localized to the brush-border membrane of extraembryonic endodermal cells. This localization is consistent with a role for FATP4 in fat absorption in early embryogenesis and suggests a novel requirement for FATP4 function during development.

Flow cytometry in combination with ribosomal RNA (rRNA) based fluorescence in situ hybridization is a new technique for the analysis of microbial communities. Oligonucleotide probes directed against ribosomal RNA allow the identification of species or groups of micro- organisms. Combined with flow cytometry, up to several thousand cells per second can be classified. In addition to the identification and specific enumeration of micro-organisms, further information on the distribution of cell size, DNA and ribosome content -- and therefore an assessment of activity -- within the entire community or subpopulations can be obtained. This technique is much more accurate, informative, and rapid than classical culture-dependent methods. Data of activated sludge samples hybridized with fluorescein labeled oligonucleotides and counterstained with the DNA-specific dye Hoechst 33342 are presented as examples for its applicability to complex microbial communities.

Cytometry is the characterization and measurement of cells and cellular constituents for biological, diagnostic and therapeutic purposes, embracing the fields of cell and molecular biology, biochemistry, biophysics, cell physiology, pathology, immunology, genetics, biotechnology, plant biology and microbiology. Cytometry is based on quantitative measurements of the molecular and phenotypic properties of cells using flow and image cytometry, microarrays, and proteomics. Cytomics (molecular cell systems research) aims at the understanding of the molecular architecture and functionality of cell systems (cytomes) by single-cell analysis in combination with exhaustive bioinformatic knowledge extraction. The cytomics concept has been significantly advanced by a multitude of current developments like confocal and laser scanning microscopy, multiphoton fluorescence excitation, spectral imaging, fluorescence resonance energy transfer (FRET), fast imaging in flow, optical stretching in flow, and miniaturized flow and image cytometry within laboratories on a chip or laser microdissection, as well as the use of bead arrays [1, 2]. Data sieving or data mining of the vast amounts of collected multiparameter data for exhaustive multilevel bioinformatic knowledge extraction avoids the inadvertent loss of information from unknown molecular relations being inaccessible to an a priori hypothesis. These approaches will become powerful tools for such important fields as individualized medicine, drug discovery and drug development [3–6]. This special issue is focused on state-of-the-art research in advanced cytometry and application areas with particular emphasis on novel biophotonic methods, disease diagnosis, and monitoring of disease treatment at single-cell level in stationary and flow conditions. It seeks to advance scholarly research that spans from fundamental interactions between light, cells, vascular tissue, and labeling particles, to strategies and opportunities for preclinical and clinical research. Recent advances in slide-based cytometry for in vitro application are summarized by Gerstner et al. [7]. They show that single cell based quantitation using microscope based cytometry instruments is making its way from basic research into clinical use. Calibration and quality control are essential in every cytometric analysis. Basics of standardization and calibration in cytometry are comprehensively summarized by Mittag and colleague [8]. Leif [9] demonstrates how data formats produced by cytometric measurements can be integrated into clinical diagnostic system standards for improved data management in the clinical environment. Flow cytometry (FCM) is based both on cell detection as whole and the use of high-speed imaging of individual cells and nanoparticles. Tychinski and co-workers [10] analyze the metabolic component of cellular refractivity and its importance for optical cytometry. The refractivity decrease in response to energy depletion was demonstrated for mitochondria, chloroplasts, spores, and cancer cells using the coherent phase microscope “Airyscan”. Estimations indicated high values for the equivalent refractive index of structured water in cells. To reduce the interference from tissue light scattering background, gold nanoparticles with strong plasmon scattering resonance properties can be applied as in vivo FCM contrast agents. This technique requires an understanding of light interaction features of cells with and without nanoparticles. Tanev and coworkers [11] compare light scattering of these cells in controlled refractive index matching conditions. They analyze the optical schematics including phase contrast microscopy as a prospective modality for in vivo FCM. Jacobs et al. [12] consider some important features of the diffraction imaging of spheres and melanoma cells with a microscope objective. Using a confocal imaging-based method to reconstruct and analyze the 3D structure, they demonstrated that genetic modifications in these cells can induce morphological changes, and the modified cells can be used as an experimental model to study the correlation between 3D morphology features and diffraction image data. Galanzha et al. [13, 14] introduce in vivo FCM with photoacoustic and photothermal detection schematics. Conventional FCM provides fast multi-parameter quantification of the biological properties of individual cells at subcellular and molecular levels. Nevertheless, the invasive extraction of cells from a living system modifies their properties, preventing their long-term study in their native environment. In vivo FCM of blood and lymph vessels with natural bioflows overcomes these problems. Cells can be detected label-free, by endogenous chromophores and pigments or by advanced strongly absorbing nanoparticles as novel labels. Both approaches are demonstrated for noninvasive rapid detection and treatment of metastases in lymph flow and sentinel lymph nodes by an integrated system of multicolor photoacoustic lymph FCM/lymphography and photothermal therapy. Bykov et al. [15] employ Doppler OCT imaging of cytoplasm shuttle flow in a slime mold Physarum polycephalum. Radial contractions of the gel-like walls of the strands and the velocity distributions in the sol-like endoplasm streaming along the plasmodial strands are imaged. The motility inhibitor effect of carbon dioxide on the cytoplasm shuttle flow and strand wall contraction is shown.

Multicolor flow cytometry represents an important tool for characterization of immune cell phenotype and function. In humans, this technique is most often applied to studies of cells obtained from peripheral blood. However, in many human disease conditions, peripheral tissues are the preferred sites for investigation. Such is the case, e.g., in questions relating to the role of the immunological milieu in human liver disorders. The human liver contains large numbers of immune cells that contribute to various pathological processes 1, 2. Detailed assessment of the intrahepatic immune environment in the human liver by flow cytometry has been generally hampered by the need for large sample sizes and insufficient software tools that can provide accurate information from the, often complex, data attained. Here, we describe how the technique of ultrasound guided fine-needle liver aspiration biopsy (FNAB) sampling in combination with 22-parameter flow cytometry technology 3 and new software analysis of data 4 readily can provide high-resolution mapping of fine immune cell signatures in the human liver. The described technology significantly advances our ability to investigate intricacies of human liver immunology during physiological as well as pathological processes including drug interventions and other conditions. Most likely the techniques and data analysis described here could be adapted also to studies of immune cells from other human organs in physiological or disease settings. As a point of departure, a 22-parameter flow cytometry panel was developed covering major myeloid and lymphoid cell populations such as naïve, effector, and terminal T cells, naïve, unswitched, and switched memory B cells, immature and mature NK cells, and subpopulations of DCs (Supporting Information Table 1A). Use of this panel enabled us to theoretically collect of up to 4.2 million phenotypes (222). Such high numbers of dimensions and potential phenotypes are, however, difficult to analyze and visualize when using standard flow cytometry software 5. Additionally, multivariate relationships that are present in more than two dimensions are easily overlooked. To circumvent the latter challenge, we demonstrate here the usefulness of stochastic neighbor embedding (SNE) 5, a nonlinear dimensionality reduction analysis method, that identifies two-dimensional representations of data at the single-cell level. This technique preserves local and global geometry and has previously been reported to be superior compared to other dimensionality reduction techniques for flow cytometry data visualization 5. Here, we demonstrate its usefulness in the context of probing the total immune cell populations in human liver specimens retrieved by fine-needle aspiration biopsy (FNAB) and compare data from matched PBMC samples (Fig. 1A and Supporting Information Fig. 1A). Aiding visual interpretation of the present liver-specific traits, individual population intensities of PBMC signatures could be subtracted from FNAB signatures (Fig. 1A). When visually evaluating expression intensities of the single parameters used to generate the signatures, SNE analysis resulted in a robust classification of myeloid and lymphoid immune cells into distinct subsets (Fig. 1B). Sample batching and deferred analysis are often prerequisites when longitudinal samples are collected from human subjects, e.g., when multiple collection sites participate in a clinical study where a central facility is responsible for analysis. Such is the case, e.g., in the collection of rare samples over long periods of time or the acquisition of specimens from resource poor settings where appropriate equipment is not accessible 6. To deal with this issue, we developed a method to cryopreserve small numbers of fixed cells before actual flow cytometry staining and subsequent SNE analysis (Fig. 1C). Remarkably, compared to no fixation, prior fixation significantly improved the yields obtained after thawing of samples, thereby making further flow cytometry analysis if anything more robust. In this context, it is a well-known fact that fixation of cells by many different methods may affect mAb epitopes. Addressing this issue, we found the fixative CytoFix to be superior in preserving staining on cells. With this fixative, results were to a large degree comparable to that of unfixed cells (Fig. 1D and E, and Supporting Information Fig. 2). When analyzing rare and limited samples, it is often only possible to perform staining with a single panel. To maximize data output, the antibody panel used was designed so that it would contain several different stricto sensu lineage-specific mAbs conjugated with the same fluorochrome. This allowed us to move beyond reliance on the number of detectors available in the standard flow cytometry instrument used (Supporting Information Tables 1A and B), a strategy previously described for 8-color flow cytometry 7. No significant differences were detected between staining with each of the mAbs alone compared with staining with different mAb combinations (Supporting Information Fig. 3). Furthermore, no differences in subset frequencies were found when high and low cell numbers were compared, showing that analysis of as few as 50,000 cells is enough to characterize major and minor immune cell subsets from the samples analyzed (Fig. 1F). Taken together, we illustrate here how clinical material from FNAB, followed by fixing and cryopreserving of cells, subsequent 22-parameter flow cytometry analysis and advanced software analysis can yield results on liver immune signatures at a level previously unattainable. To illustrate the usability of the presently described strategy for high-resolution analysis of a limited numbers of cells, we evaluated the presence of distinct lymphocyte populations in FNAB samples (Fig. 2A, Supporting Information Fig. 1B). SNE generated a signature that accurately distinguished immature and mature CD4 and CD8 T cells, γδ T cells, mucosal-associated invariant T cells, subsets of NK cells, as well as mature and immature B cells (Supporting Information Fig. 1C). A residual plot display readily portrayed differences between the PBMC and FNAB samples (Fig. 2A). In this context, although SNE allows for extraction of phenotypic signatures from identified subsets 8, it is not designed to statistically determine which of the alterations between datasets significantly contribute to differentiating the sets. To do this, one can apply a recently published method that combines hierarchical clustering with group lasso regression analysis for selection of clusters distinguishing FNAB from PMBC samples 9. A similar approach has recently been used to determine peripheral blood immune signatures correlating with clinical recovery after surgery 10. Here, clustering of 17 parameters was performed and a total of 52 clusters in a hierarchical tree was generated (Fig. 2B and Supporting Information Fig. 4A and Table 1). In the subsequent group lasso regression analysis, three distinguishing clusters were identified, two out of which were more abundant in the FNAB samples as compared with PBMC samples (Fig. 2C and D and Supporting Information Table 2, Fig. 4D). The parameters used for generating the hierarchical cluster signature identified phenotypes of the three distinguishing clusters (Fig. 2E). Cluster I represented NKG2A+/−CD56bright NK cells, and clusters II and III signified discrete subsets of CD4 T cells (Fig. 2E). The three clusters were also recognizable in the SNE signature (Supporting Information Fig. 1C and 4B), indicating a robustness of the findings. Finally, and interestingly, with conventional flow cytometry analysis, only cluster I could be assessed and confirmed (Fig. 2F). In contrast, the two CD4 T cell clusters were present in multivariate space not allowing identification by standard flow cytometric analysis (Fig. 2E). Taken together, we illustrate how state-of-the-art methodologies, technologies, and software can be applied to fine human liver immunophenotyping at a level not previously attainable, based on small number of cells obtained with a minimally invasive procedure (i.e. FNAB). With the complexity of flow cytometry data continuously increasing new approaches to handle data, here exemplified by the use of SNE and hierarchical clustering with automated identification of distinguishing immune cell subsets, likely will facilitate unbiased analysis and find previously unrecognized multivariate cellular phenotypes. These techniques, and other techniques such as the imaging methods histocytometry and laser-scanning microscopy, which allow for investigation of the spatial segregation of cells in tissue 11, 12, should expand the horizons of human liver immunology research and advance our ability to interrogate the intricacies of immune responses in this organ. Finally, the strategies described above are most certainly not limited to the liver, but could readily be extended to other human organs and tissues. We thank M. Holm, UPPMAX, Uppsala for writing data pre- and postprocessing code, which enabled usage of Barnes-Hut SNE. This work was funded by the International Research Training Group 1273 supported by the German Research Foundation (to H.W.), the Center Research Grants 738 (project B2) and 900 (project A5) supported by the DFG (H.W.), the Deutsches Zentrum für Infektionsforschung (H.W.), the Swedish Research Council (N.K.B. and H.G.L.), Swedish Cancer Society (N.K.B. and H.G.L.), the Karolinska Institutet (N.K.B. and H.G.L.), the Swedish Society for Medical Research (N.K.B.), the Cancer Research Foundations of Radiumhemmet (N.K.B), the Swedish Society of Medicine (N.K.B.), Novo Nordisk's Foundation (N.K.B.), Jeansson's Foundation (N.K.B.), Bengt Ihre's Foundation (N.K.B.), Åke Olsson's Foundation (N.K.B.), Groschinsky's Foundation (N.K.B.), Hedlunds' Foundation (N.K.B.), KI-DKFZ Joint Project Grant (N.K.B. and H.G.L.), and Julin's Foundation (N.K.B.). The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Table 1A. Instrument settings and staining panel Table 1B. Lineage restriction of markers used in double Table 2. Cluster sizes (frequency) of FNAB and PBMC samples for the 52 clusters identified with Citrus Figure s1 Figure s2 Figure s3 Figure s4 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Regulation of laminin and COUP-TF expression in extraembryonic endodermal cells.

Introduction: Cutaneous leishmaniasis (CL) is a significant public health problem leading to permanently disfiguring skin lesions caused by Leishmania parasites. Lesion severity stems from an excessive host inflammatory response that prevents healing. Materials and Methods: Here, we characterized the transcriptional and translational responses of lymphatic endothelial cells (LECs) during murine CL using single-cell RNA sequencing data combined with flow cytometry and invivo puromycin incorporation to assess translational activity. Results: We identified upregulation of antigen presentation pathways including MHC-I, MHC-II, and immunoproteasome transcripts in dermal LECs from Leishmania major-infected mice compared to naive controls. LECs also exhibited increased expression of guanylate binding proteins and interferon-inducible genes, indicative of immune activation. Moreover, our findings demonstrate that LECs in leishmanial lesions displayed heightened translational activity relative to LECs from uninflamed ears, and LEC translational activity was highest in activated LECs. Furthermore, LEC translational activity exceeded that of other cell types within the lesion microenvironment. Validating the transcriptomic data, LECs in lesions expressed elevated MHC-II and programmed death-ligand 1 (PDL-1), supporting their potential role in antigen presentation. Functional assays using DQ-ovalbumin confirmed that LECs from leishmanial lesions efficiently uptake and process antigens, highlighting their capability as antigen presenting cells in the inflamed dermal microenvironment. Conclusion: Overall, our study reveals the activation status of LECs in leishmanial lesions, shedding light on their potential role in shaping local immunity and inflammation in a variety of skin diseases.

Long-chain saturated and polyunsaturated fatty acids of two new thraustochytrid isolates cultured from Taiwan mangroves, Aurantiochytrium sp. IMB169 and Aurantiochytrium sp. IMB171, were characterized through their cell growth and development in relation to their intracellular lipid accumulation using transmission electron microscopy. Flow cytometry in combination with the lipophilic fluorescent dye BODIPY 505/515 was used to stain and characterize intracellular lipid bodies in the two isolates. The transmission electron microscopy and flow cytometry analyses revealed a progressive accumulation of lipid products in IMB169 and IMB171. Further, selective BODIPY stained cells were successfully separated and enriched using flow cytometry at single cell level. Among the two isolates, IMB169 was found to produce a high level of docosahexaenoic acid. The qualitative and analytical results obtained using electron microscopy and flow cytometry studies were validated by gas chromatography (GC). In addition, a quantitative baseline was established using cell growth, flow cytometry and GC analyses for developing an efficient bioprocessing methodology to selectively enrich thraustochytrids phenotypes with desirable characteristics.