| 1. |
- Danielsen, Jonas, et al.
(författare)
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Computer Vision-Based Image Analysis of Bacteria
- 2017
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Ingår i: Bacterial Pathogenesis : Methods and Protocols - Methods and Protocols. - New York, NY : Springer New York. - 1064-3745. - 9781493966738 - 9781493966714 ; 1535, s. 161-172
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Bokkapitel (refereegranskat)abstract
- Microscopy is an essential tool for studying bacteria, but is today mostly used in a qualitative or possibly semi-quantitative manner often involving time-consuming manual analysis. It also makes it difficult to assess the importance of individual bacterial phenotypes, especially when there are only subtle differences in features such as shape, size, or signal intensity, which is typically very difficult for the human eye to discern. With computer vision-based image analysis - where computer algorithms interpret image data - it is possible to achieve an objective and reproducible quantification of images in an automated fashion. Besides being a much more efficient and consistent way to analyze images, this can also reveal important information that was previously hard to extract with traditional methods. Here, we present basic concepts of automated image processing, segmentation and analysis that can be relatively easy implemented for use with bacterial research.
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| 2. |
- de Neergaard, Therese, et al.
(författare)
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Quantification of Phagocytosis Using Flow Cytometry
- 2023. - 2
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Ingår i: Bacterial Pathogenesis : Methods and Protocols - Methods and Protocols. - 1940-6029. - 9781071632420 ; , s. 221-234
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Bokkapitel (refereegranskat)abstract
- Phagocytosis is relevant for many research fields and is often measured as a functional outcome. However, accurate quantification can be challenging, and many researchers find it difficult to study in a robust manner. There are many ways to measure phagocytosis, but what is often overlooked is the importance of experimental design and how the analysis is planned and performed. Experimental factors like reaction volume, time, and phagocyte-prey concentrations often have a large impact on the outcome, as is the choice of detection strategy with different fluorescent or colorimetric labels of prey and phagocyte. By using dose-response curve principles for both experimental design and analysis, it is possible to increase the sensitivity and robustness, leading to accurate quantification of phagocytosis that is comparable across experiments and systems.Here, we describe how to quantify phagocytosis using flow cytometry with a robust, high-throughput, and easy-to-use approach. The prey is first fluorescently double stained, followed by optional opsonization before being introduced to the phagocyte in a wide range of ratios. After incubation, data is acquired through flow cytometry. It can be assessed on both the population and single-cell level of the phagocytes, separating adhesion and internalization. As an example, we provide an experimental protocol for studying phagocytosis of opsonized Streptococcus pyogenes using the THP-1 cell line. This approach is easily incorporated into most existing phagocytosis assays and allows for reproducible results with high sensitivity.
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| 3. |
- Kumra Ahnlide, Vibha, et al.
(författare)
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Measurement of Antibody Binding Affinity on Bacterial Surfaces Using Flow Cytometry
- 2023. - 2
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Ingår i: Bacterial Pathogenesis : Methods and Protocols - Methods and Protocols. - 1940-6029. - 9781071632420 ; , s. 251-259
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Bokkapitel (refereegranskat)abstract
- Antibody binding to bacterial surfaces plays a crucial role in immunity, and a key characteristic of this protein-protein interaction is the binding affinity. Determining the affinity of an antibody binding to its antigen is the first step in predicting the function in a physiological environment where other competing protein interactions may be present. Antibody-antigen affinity is often evaluated with isolated proteins. It is informative to also be able to assess antibody binding to a bacterial surface where many antigens might be present, including multiple copies of the specific antigen the antibody recognizes, and in a context where the antigen might be in a more natural conformation. In this chapter, we present a flow cytometry-based assay to measure and calculate the cell surface binding affinity or avidity of any mono- or polyclonal antibody solution.
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| 4. |
- Shannon, Oonagh, et al.
(författare)
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Measuring Antibody Orientation at the Bacterial Surface
- 2017
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Ingår i: Bacterial Pathogenesis : Methods and Protocols - Methods and Protocols. - New York, NY : Springer New York. - 1064-3745. - 9781493966738 - 9781493966714 ; 1535, s. 331-337
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Bokkapitel (refereegranskat)abstract
- Many bacteria have the ability to interact with antibodies as a means to circumvent the immune response. This includes binding to the Fc portion of antibodies, effectively reversing the antibody orientation and thus decreasing the Fc-mediated immune signaling. Since antibody orientation at the bacterial surface has been shown to be important in human disease, it is valuable to be able to assess how antibodies are interacting with bacterial pathogens. Here, we describe a method to measure the proportion of human IgG that are bound via their Fc or Fabs to a bacterial surface. This is achieved by treating antibody-coated bacteria with the bacterial enzyme IdeS - which will cleave IgG into Fc and Fab fragments - and subsequently detect remaining fragments with fluorescent Fabs. The method is easy and fast, and the principle is most likely also applicable to other systems where distinguishing between antibody Fc and Fab binding is important.
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| 5. |
- Nordenfelt, Pontus, et al.
(författare)
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Preface
- 2023. - 2nd
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Ingår i: Bacterial Pathogenesis - Methods and Protocols. - 1064-3745. - 9781493966738 ; 2674
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 6. |
- Akbari, Elham, et al.
(författare)
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SEPARATION OF CLUSTERS OF GROUP A STREPTOCOCCI USING DETERMINISTIC LATERAL DISPLACEMENT
- 2021
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Ingår i: MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - 9781733419031 ; , s. 1201-1202
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Konferensbidrag (refereegranskat)abstract
- Differences in morphologies of bacteria and bacteria clusters are known to influence their pathogenicity. However, it is difficult to separate cells and cell clusters based on morphology using standard cell biological methods, making studies of the underlying mechanisms difficult. Here we report a simple label-free method for the continuous separation of clusters of group A streptococci, based on cluster size and morphology, using Deterministic Lateral Displacement (DLD). In general, this opens up for the generation of cell populations with heterogenicity in cluster size and physical properties.
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| 7. |
- Akbari, Elham, et al.
(författare)
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SEPARATION OF SINGLETS AND CLUSTERS OF GROUP A STREPTOCOCCI USING DETERMINISTIC LATERAL DISPLACEMENT AND FILTER SONICATION
- 2022
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Ingår i: MicroTAS 2022 - 26th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - 9781733419048 ; , s. 306-307
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Konferensbidrag (refereegranskat)abstract
- Differences in morphologies of bacteria and bacteria clusters are thought to contribute to their virulence and colonization. However, the conventional standard cell biological methods cannot separate bacteria and bacteria clusters based on their morphologies and sizes, making studies of the underlying mechanisms difficult. Here we report a simple label-free method for the continuous separation of singlets and clusters, of group A streptococci, based on their size and morphology, using Deterministic Lateral Displacement and filter-sonication. In general, this opens up for the generation of cell populations with heterogenicity in cluster size and physical properties.
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| 8. |
- Alamiri, Feiruz, et al.
(författare)
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Role of serotype and virulence determinants of Streptococcus pyogenes biofilm bacteria in internalization and persistence in epithelial cells in vitro.
- 2023
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Ingår i: Frontiers in cellular and infection microbiology. - 2235-2988. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes causes a multitude of local and systemic infections, the most common being pharyngitis in children. Recurrent pharyngeal infections are common and are thought to be due to the re-emergence of intracellular GAS upon completion of antibiotic treatment. The role of colonizing biofilm bacteria in this process is not fully clear. Here, live respiratory epithelial cells were inoculated with broth-grown or biofilm bacteria of different M-types, as well as with isogenic mutants lacking common virulence factors. All M-types tested adhered to and were internalized into epithelial cells. Interestingly, internalization and persistence of planktonic bacteria varied significantly between strains, whereas biofilm bacteria were internalized in similar and higher numbers, and all strains persisted beyond 44 hours, showing a more homogenous phenotype. The M3 protein, but not the M1 or M5 proteins, was required for optimal uptake and persistence of both planktonic and biofilm bacteria inside cells. Moreover, the high expression of capsule and SLO inhibited cellular uptake and capsule expression was required for intracellular survival. Streptolysin S was required for optimal uptake and persistence of M3 planktonic bacteria, whereas SpeB improved intracellular survival of biofilm bacteria. Microscopy of internalized bacteria showed that planktonic bacteria were internalized in lower numbers as individual or small clumps of bacteria in the cytoplasm, whereas GAS biofilm bacteria displayed a pattern of perinuclear localization of bacterial aggregates that affected actin structure. Using inhibitors targeting cellular uptake pathways, we confirmed that planktonic GAS mainly uses a clathrin-mediated uptake pathway that also required actin and dynamin. Clathrin was not involved in biofilm internalization, but internalization required actin rearrangement and PI3 kinase activity, possibly suggesting macropinocytosis. Together these results provide a better understanding of the potential mechanisms of uptake and survival of various phenotypes of GAS bacteria relevant for colonization and recurrent infection.
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| 9. |
- André, Oscar, et al.
(författare)
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Data-driven microscopy allows for automated context-specific acquisition of high-fidelity image data
- 2023
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Ingår i: Cell reports methods. - : Elsevier BV. - 2667-2375. ; 3:3
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Tidskriftsartikel (refereegranskat)abstract
- Light microscopy is a powerful single-cell technique that allows for quantitative spatial information at subcellular resolution. However, unlike flow cytometry and single-cell sequencing techniques, microscopy has issues achieving high-quality population-wide sample characterization while maintaining high resolution. Here, we present a general framework, data-driven microscopy (DDM) that uses real-time population-wide object characterization to enable data-driven high-fidelity imaging of relevant phenotypes based on the population context. DDM combines data-independent and data-dependent steps to synergistically enhance data acquired using different imaging modalities. As a proof of concept, we develop and apply DDM with plugins for improved high-content screening and live adaptive microscopy for cell migration and infection studies that capture events of interest, rare or common, with high precision and resolution. We propose that DDM can reduce human bias, increase reproducibility, and place single-cell characteristics in the context of the sample population when interpreting microscopy data, leading to an increase in overall data fidelity.
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| 10. |
- André, Oscar, et al.
(författare)
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Data-driven microscopy allows for automated targeted acquisition of relevant data with higher fidelity
- 2022
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Light microscopy is a powerful single-cell technique that allows for quantitative spatial information at subcellular resolution. However, unlike flow cytometry and single-cell sequencing techniques, microscopy has issues achieving high-quality population-wide sample characterization while maintaining high resolution. Here, we present a general framework, data-driven microscopy (DDM), that uses population-wide cell characterization to enable data-driven high-fidelity imaging of relevant phenotypes. DDM combines data-independent and data-dependent steps to synergistically enhance data acquired using different imaging modalities. As proof-of-concept, we apply DDM with plugins for improved high-content screening and live adaptive microscopy. DDM also allows for easy correlative imaging in other systems with a plugin that uses the spatial relationship of the sample population for automated registration. We believe DDM will be a valuable approach for reducing human bias, increasing reproducibility, and placing singlecell characteristics in the context of the sample population when interpreting microscopy data, leading to an overall increase in data fidelity.
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