Journal articles: 'Microscopy-Based cytometry'

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Wessels, J. T., A. C. Busse, J. Mahrt, B. Hoffschulte, G. A. Mueller, A. Tárnok, and A. Mittag. "NorthernLights in slide-based cytometry and microscopy." Cytometry Part A 9999A (2010): NA. http://dx.doi.org/10.1002/cyto.a.20863.

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Ecker, Rupert C., and Georg E. Steiner. "Microscopy-based multicolor tissue cytometry at the single-cell level." Cytometry 59A, no. 2 (2004): 182–90. http://dx.doi.org/10.1002/cyto.a.20052.

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Kummrow, A., M. Frankowski, N. Bock, C. Werner, T. Dziekan, and J. Neukammer. "Quantitative assessment of cell viability based on flow cytometry and microscopy." Cytometry Part A 83A, no. 2 (October 18, 2012): 197–204. http://dx.doi.org/10.1002/cyto.a.22213.

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Mendoza, Maria Gracia Garcia, Alex Sutton, Raymond Kong, Matthew Rodrigues, and Haley Pugsley. "A rapid and fully automated in vitro micronucleus assay using imaging flow cytometry and convolutional neural network analysis." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 172.02. http://dx.doi.org/10.4049/jimmunol.208.supp.172.02.

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Abstract Micronuclei (MN) originate from whole chromosomes or chromosome fragments that lag behind during cell division and fail to be incorporated into one of the two main nuclei. As a result, scoring MN using the well-established in vitro micronucleus assay evaluates the ability of chemicals or other agents to induce DNA damage. This technique is typically performed by manual microscopy, which can be time-consuming and prone to variability. Additionally, automated methods lack cytoplasmic visualization when using slide-scanning microscopy, and conventional flow cytometry doesn’t provide visual confirmation of MN. The ImageStream®X Mk II (ISX) imaging flow cytometer combines the high-resolution imagery of microscopy with conventional flow cytometry’s speed and statistical robustness in a single system. Previously, we developed a rapid and automated MN assay based on high-throughput image capture and feature-based image analysis using IDEAS® Software. However, the feature-based analysis was not readily applicable to multiple cell lines and chemicals, so we developed a deep learning method based on convolutional neural networks to score imaging flow cytometry data in both the cytokinesis-blocked and unblocked versions of the MN assay using Amnis® AI Software. Our current study validates our previously established assay and analyses using three different chemicals (Mitomycin C, Cyclophosphamide, and Eugenol) and three different cell lines (TK6, L5178Y, CHO-K1). Here, we demonstrate how using Amnis AI to score imagery acquired on the ISX provides a rapid and fully automated in vitro MN assay with improved accuracy, reproducibility, and time-to-results in toxicity and biodosimetry applications across multiple cell lines.

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Sláma, Petr, Zbyšek Sládek, and Dušan Ryšánek. "Application of methods for detection of apoptosis and necrosis of bovine blood neutrophil granulocytes in vitro." Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis 54, no. 5 (2006): 107–16. http://dx.doi.org/10.11118/actaun200654050107.

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The study was an in vitro analysis of bovine blood neutrophil apoptosis and necrosis based on the detection of their morphological and biochemical features. The experiment was carried out in six clinically healthy Holstein x Bohemian Red Pied crossbred virgin heifers aged 16 to 18 months. The fresh and in vitro cultivated blood were analysed by light and electron microscopy and flow cytometry. In fresh blood, non apoptotic or necrotic neutrophils were found by light microscopy. On the other hand, 3.34% apoptotic and 0.32% necrotic neutrophils were detected by flow cytometry. After four hours of incubation, 3.53% apoptotic and 1.37% necrotic neutrophils were found by light microscopy. In the same conditions, there were assessed 17.54% apoptotic and 0.58% necrotic neutrophils by flow cytometry. Correlation coefficient between light microscopy and flow cytometry in portion of apoptotic neutrophils was 0.625 (P<0.05) and correlation coefficient between light microscopy and flow cytometry in portion of necrotic neutrophils was 0.983 (P<0.05). There was found out that these detection methods (light microscopy and flow cytometry) are right combination for detection of apoptotic and necrotic bovine blood neutrophils.

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Asthana, Vishwaratn, Yuqi Tang, Adam Ferguson, Pallavi Bugga, Anantratn Asthana, Emily R. Evans, Allen L. Chen, Brett S. Stern, and Rebekah A. Drezek. "An inexpensive, customizable microscopy system for the automated quantification and characterization of multiple adherent cell types." PeerJ 6 (June 5, 2018): e4937. http://dx.doi.org/10.7717/peerj.4937.

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Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated microscopy-based cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated microscopy-based cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the microscopy-based cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity toward tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our microscopy-based cytometer is successfully able to elucidate.

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Adachi, Takahiro, and Takeshi Tsubata. "FRET-based Ca2+ measurement in B lymphocyte by flow cytometry and confocal microscopy." Biochemical and Biophysical Research Communications 367, no. 2 (March 2008): 377–82. http://dx.doi.org/10.1016/j.bbrc.2007.12.142.

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Wei, Shu-Gen, Ling-Yun Wan, Ying Wei, Li-Li He, Jin-E. Fu, and Li-Mei Pan. "Analysis of ploidy level of Artemisia annua L. based on flow cytometry and confocal laser scanning microscopy." Bangladesh Journal of Botany 50, no. 1 (March 27, 2021): 29–35. http://dx.doi.org/10.3329/bjb.v50i1.52668.

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Eighty nine Artemisia samples treated with different concentrations of colchicine were used as breeding samples, with diploid Artemisia as the control. The ploidy levels of samples were determined by flow cytometry and confocal laser scanning microscopy (CLSM). An analysis of the flow cytometry results identified three suspected tetraploid plants and seven suspected triploid plants. The results of this study may be useful for breeding new Artemisia lines.

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Heckmann, Mara, Gerald Klanert, Georg Sandner, Peter Lanzerstorfer, Manfred Auer, and Julian Weghuber. "Fluorescence microscopy-based quantitation of GLUT4 translocation." Methods and Applications in Fluorescence 10, no. 2 (January 21, 2022): 022001. http://dx.doi.org/10.1088/2050-6120/ac4998.

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Abstract Postprandial insulin-stimulated glucose uptake into target tissue is crucial for the maintenance of normal blood glucose homeostasis. This step is rate-limited by the number of facilitative glucose transporters type 4 (GLUT4) present in the plasma membrane. Since insulin resistance and impaired GLUT4 translocation are associated with the development of metabolic disorders such as type 2 diabetes, this transporter has become an important target of antidiabetic drug research. The application of screening approaches that are based on the analysis of GLUT4 translocation to the plasma membrane to identify substances with insulinomimetic properties has gained global research interest in recent years. Here, we review methods that have been implemented to quantitate the translocation of GLUT4 to the plasma membrane. These methods can be broadly divided into two sections: microscopy-based technologies (e.g., immunoelectron, confocal or total internal reflection fluorescence microscopy) and biochemical and spectrometric approaches (e.g., membrane fractionation, photoaffinity labeling or flow cytometry). In this review, we discuss the most relevant approaches applied to GLUT4 thus far, highlighting the advantages and disadvantages of these approaches, and we provide a critical discussion and outlook into new methodological opportunities.

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Lima, Ângela, Christina A. Muzny, and Nuno Cerca. "An Indirect Fluorescence Microscopy Method to Assess Vaginal Lactobacillus Concentrations." Microorganisms 12, no. 1 (January 5, 2024): 114. http://dx.doi.org/10.3390/microorganisms12010114.

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Lactobacillus species are the main colonizers of the vaginal microbiota in healthy women. Their absolute quantification by culture-based methods is limited due to their fastidious growth. Flow cytometry can quantify the bacterial concentration of these bacteria but requires the acquisition of expensive equipment. More affordable non-culturable methods, such as fluorescence microscopy, are hampered by the small size of the bacteria. Herein, we developed an indirect fluorescence microscopy method to determine vaginal lactobacilli concentration by determining the correlation between surface area bacterial measurement and initial concentration of an easily cultivable bacterium (Escherichia coli) and applying it to lactobacilli fluorescence microscopy counts. In addition, vaginal lactobacilli were quantified by colony-forming units and flow cytometry in order to compare these results with the indirect method results. The colony-forming-unit values were lower than the results obtained from the other two techniques, while flow cytometry and fluorescence microscopy results agreed. Thus, our developed method was able to accurately quantify vaginal lactobacilli.

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Chen, Feng, Jing-rang Lu, Brian J. Binder, Ying-chun Liu, and Robert E. Hodson. "Application of Digital Image Analysis and Flow Cytometry To Enumerate Marine Viruses Stained with SYBR Gold." Applied and Environmental Microbiology 67, no. 2 (February 1, 2001): 539–45. http://dx.doi.org/10.1128/aem.67.2.539-545.2001.

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ABSTRACT A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.

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Heinrichs, Mara Elena, Daniele De Corte, Bert Engelen, and Donald Pan. "An Advanced Protocol for the Quantification of Marine Sediment Viruses via Flow Cytometry." Viruses 13, no. 1 (January 13, 2021): 102. http://dx.doi.org/10.3390/v13010102.

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Viruses are highly abundant, diverse, and active components of marine environments. Flow cytometry has helped to increase the understanding of their impact on shaping microbial communities and biogeochemical cycles in the pelagic zone. However, to date, flow cytometric quantification of sediment viruses is still hindered by interference from the sediment matrix. Here, we developed a protocol for the enumeration of marine sediment viruses by flow cytometry based on separation of viruses from sediment particles using a Nycodenz density gradient. Results indicated that there was sufficient removal of background interference to allow for flow cytometric quantification. Applying this new protocol to deep-sea and tidal-flat samples, viral abundances enumerated by flow cytometry correlated well (R2 = 0.899) with counts assessed by epifluorescence microscopy over several orders of magnitude from marine sediments of various compositions. Further optimization may be needed for sediments with low biomass or high organic content. Overall, the new protocol enables fast and accurate quantification of marine sediment viruses, and opens up the options for virus sorting, targeted viromics, and single-virus sequencing.

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Sauermann, Mamatha, Florian Hahne, Christian Schmidt, Meher Majety, Heiko Rosenfelder, Stephanie Bechtel, Wolfgang Huber, Annemarie Poustka, Dorit Arlt, and Stefan Wiemann. "High-Throughput Flow Cytometry–Based Assay to Identify Apoptosis-Inducing Proteins." Journal of Biomolecular Screening 12, no. 4 (March 22, 2007): 510–20. http://dx.doi.org/10.1177/1087057107301271.

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After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry—based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers. ( Journal of Biomolecular Screening 2007:510-520)

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Vincent, Vinnyfred, Himani Thakkar, Anjali Verma, Atanu Sen, Nikhil Chandran, and Archna Singh. "A novel flow cytometry-based quantitative monocyte adhesion assay to estimate endothelial cell activation in vitro." BioTechniques 68, no. 6 (June 2020): 325–33. http://dx.doi.org/10.2144/btn-2019-0169.

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One of the earliest events in the development of atherosclerosis is endothelial activation, which is estimated in vitro at the functional level by quantifying monocyte adhesion. This involves the incubation of fluorescently labeled monocytes on top of cultured endothelial cells and quantifying the number of adhered monocytes. Currently, the quantification of adhered monocytes is done using microscopy or by lysing the cells and estimating the fluorescence. Here we present a novel flow cytometry-based method for the quantification of monocyte adhesion. This method could quantify the average number of monocytes adhered to a single endothelial cell after monocyte adhesion assay, and was also sensitive to the level of activation of endothelial cells. Flow cytometry-based quantification requires less time and effort compared with microscopy-based quantification.

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Kaur, Amandeep, Mohammad A. Haghighatbin, Conor F. Hogan, and Elizabeth J. New. "A FRET-based ratiometric redox probe for detecting oxidative stress by confocal microscopy, FLIM and flow cytometry." Chemical Communications 51, no. 52 (2015): 10510–13. http://dx.doi.org/10.1039/c5cc03394b.

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A FRET-based, ratiometric redox probe undergoes a fluorescence colour change upon reduction, and can be used to study cellular oxidative capacity using confocal microscopy, fluorescence lifetime imaging and flow cytometry.

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Milrot, Elad, Efi Makdasi, Boaz Politi, Tomer Israely, and Orly Laskar. "A Cell-Based Capture Assay for Rapid Virus Detection." Viruses 12, no. 10 (October 15, 2020): 1165. http://dx.doi.org/10.3390/v12101165.

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Routine methods for virus detection in clinical specimens rely on a variety of sensitive methods, such as genetic, cell culture and immuno-based assays. It is imperative that the detection assays would be reliable, reproducible, sensitive and rapid. Isolation of viruses from clinical samples is crucial for deeper virus identification and analysis. Here we introduce a rapid cell-based assay for isolation and detection of viruses. As a proof of concept several model viruses including West Nile Virus (WNV), Modified Vaccinia Ankara (MVA) and Adenovirus were chosen. Suspended Vero cells were employed to capture the viruses following specific antibody labeling which enables their detection by flow cytometry and immuno-fluorescence microscopy assays. Using flow cytometry, a dose response analysis was performed in which 3.6e4 pfu/mL and 1e6 pfu/mL of MVA and WNV could be detected within two hours, respectively. When spiked to commercial pooled human serum, detection sensitivity was slightly reduced to 3e6 pfu/mL for WNV, but remained essentially the same for MVA. In conclusion, the study demonstrates a robust and rapid methodology for virus detection using flow cytometry and fluorescence microscopy. We propose that this proof of concept may prove useful in identifying future pathogens.

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Hinzmann, M., M. Lopes-Lima, F. Cerca, A. Correia, J. Machado, and M. Vilanova. "Identification of distinct haemocyte populations from the freshwater bivalves swan mussel (Anodonta cygnea) and duck mussel (Anodonta anatina) using wheat-germ agglutinin." Canadian Journal of Zoology 95, no. 12 (December 2017): 937–47. http://dx.doi.org/10.1139/cjz-2017-0006.

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Haemocytes play a major role in molluscs immunity. Functional studies are, however, impaired by limited available experimental tools to identify and sort distinct haemocyte populations. Therefore, using nonlethal methods, we aimed at evaluating whether lectin staining combined with flow cytometry could be used to distinguish circulating haemocyte populations from two freshwater bivalves of the family Unionidae, the duck mussel (Anodonta anatina (L., 1758)) and the swan mussel (Anodonta cygnea (L., 1758)). Based on classical classification, haemocytes were distinguished as granulocytes and hyalinocytes and cytological features were visualized using transmission microscopy and staining techniques. Size, granularity, viability, and surface staining using lectins as specific probes were analysed by flow cytometry and fluorescence microscopy. The microscopic proportions of granulocytes and hyalinocytes significantly differed, being of 70% and 30% for A. cygnea and of 85% and 15% for A. anatina, respectively. Two haemocyte populations were sorted by flow cytometry based on size and granularity and confirmed as granulocytes and hyalinocytes. Interestingly, two different granulocyte populations could be further discriminated in A. cygnea according to their binding affinity to wheat-germ agglutinin (WGA), whereas granulocytes of A. anatina all stained similarly. Our results show that WGA labelling combined with flow cytometry can be used to better discriminate Anodonta haemocyte populations and obtain purified populations for functional studies.

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Hjelm, Anna, Bill Söderström, David Vikström, Wouter S. P. Jong, Joen Luirink, and Jan-Willem de Gier. "Autotransporter-Based Antigen Display in Bacterial Ghosts." Applied and Environmental Microbiology 81, no. 2 (November 14, 2014): 726–35. http://dx.doi.org/10.1128/aem.02733-14.

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ABSTRACTBacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage ϕX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered theEscherichia coliautotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using theMycobacterium tuberculosisvaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorateE. coliandSalmonellaghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E inE. coliandSalmonellacan be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.

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Zhao, Xiaohui, Leqi Ding, Jingsheng Yan, Jin Xu, and Hao He. "Constructing an In Vitro and In Vivo Flow Cytometry by Fast Line Scanning of Confocal Microscopy." Sensors 23, no. 6 (March 21, 2023): 3305. http://dx.doi.org/10.3390/s23063305.

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Composed of a fluidic and an optical system, flow cytometry has been widely used for biosensing. The fluidic flow enables its automatic high-throughput sample loading and sorting while the optical system works for molecular detection by fluorescence for micron-level cells and particles. This technology is quite powerful and highly developed; however, it requires a sample in the form of a suspension and thus only works in vitro. In this study, we report a simple scheme to construct a flow cytometry based on a confocal microscope without any modifications. We demonstrate that line scanning of microscopy can effectively excite fluorescence of flowing microbeads or cells in a capillary tube in vitro and in blood vessels of live mice in vivo. This method can resolve microbeads at several microns and the results are comparable to a classic flow cytometer. The absolute diameter of flowing samples can be indicated directly. The sampling limitations and variations of this method is carefully analyzed. This scheme can be easily accomplished by any commercial confocal microscope systems, expands the function of them, and is of promising potential for simultaneous confocal microscopy and in vivo detection of cells in blood vessels of live animals by a single system.

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George, Thaddeus, Anne Spurkland, Vibeke Sundvold-Gjerstadt, Brandon Burbach, Yoji Shimizu, Brian Hall, and Sherree Friend. "Quantitative analysis of immune synapse formation using imaging flow cytometry. (130.18)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 130.18. http://dx.doi.org/10.4049/jimmunol.184.supp.130.18.

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Abstract Antigen-specific immune responses are initiated via direct cellular contact between an antigen presenting cell (APC) and an effector cell. Early events in the interaction between these two cells involve reorganization of the actin cytoskeleton and recruitment of adhesive and signaling molecules to the immunological synapse (IS), which ultimately results in effector cell activation. Because manual image acquisition and quantitative image analysis are time consuming processes, microscopic analyses of protein recruitment to the IS have remained either qualitative or statistically limited, despite the rich information content inherent in high resolution digital images. Here we describe an objective, statistically robust microscopy-based method for quantifying molecular recruitment to the IS using multispectral imaging flow cytometry, which enables quantitative image analysis of large populations of automatically collected images. The analysis technique uses morphology-based features to identify conjugates, followed by measurement of fluorescence specifically at the conjugate junction. Two models are described, including: 1) actin recruitment to the IS following T cell activation by artificial APC coated with anti-CD3; 2) antigen-specific recruitment of ADAP to the IS within TCR transgenic T cells. These data outline an objective and statistically robust method to rapidly quantify molecular recruitment to the immune synapse using automated high speed microscopy.

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Ghosh, Sayari, Ishita Chakraborty, Monojit Chakraborty, Ashis Mukhopadhyay, Raghwendra Mishra, and Debasish Sarkar. "Evaluating the morphology of erythrocyte population: An approach based on atomic force microscopy and flow cytometry." Biochimica et Biophysica Acta (BBA) - Biomembranes 1858, no. 4 (April 2016): 671–81. http://dx.doi.org/10.1016/j.bbamem.2016.01.021.

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Besse, Laetitia, Typhaine Rumiac, Anne Reynaud-Angelin, Cédric Messaoudi, Marie-Noëlle Soler, Sarah A. E. Lambert, and Vincent Pennaneach. "Protocol for automated multivariate quantitative-image-based cytometry analysis by fluorescence microscopy of asynchronous adherent cells." STAR Protocols 4, no. 3 (September 2023): 102446. http://dx.doi.org/10.1016/j.xpro.2023.102446.

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Kiepś, Jakub, Wojciech Juzwa, and Radosław Dembczyński. "Imaging Flow Cytometry Demonstrates Physiological and Morphological Diversity within Treated Probiotic Bacteria Groups." International Journal of Molecular Sciences 24, no. 7 (April 6, 2023): 6841. http://dx.doi.org/10.3390/ijms24076841.

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Probiotic bacteria can be introduced to stresses during the culturing phase as an alternative to the use of protectants and coating substances during drying. Accurate enumeration of the bacterial count in a probiotic formulation can be provided using imaging flow cytometry (IFC). IFC overcomes the weak points of conventional, commonly used flow cytometry by combining its statistical power with the imaging content of microscopy in one system. Traditional flow cytometers only collect the fluorescence signal intensities, while IFC provides many more steps as it correlates the data on the measured parameters of fluorescence light with digitally processed images of the analyzed cells. As an alternative to standard methods (plate cell counts and traditional flow cytometry) IFC provides additional insight into the physiology and morphology of the cell. The use of complementary dyes (RedoxSensorTM Green and propidium iodide) allows for the designation of groups based on their metabolic activity and membrane damage. Additionally, cell sorting is incorporated to assess each group in terms of growth on different media (MRS-Agar and MRS broth). Results show that the groups with intermediate metabolic activity and some degree of cellular damage correspond with the description of viable but nonculturable cells.

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Felip, Marisol, Stefan Andreatta, Ruben Sommaruga, Viera Straskrábová, and Jordi Catalan. "Suitability of Flow Cytometry for Estimating Bacterial Biovolume in Natural Plankton Samples: Comparison with Microscopy Data." Applied and Environmental Microbiology 73, no. 14 (May 18, 2007): 4508–14. http://dx.doi.org/10.1128/aem.00733-07.

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ABSTRACT The relationship between flow cytometry data and epifluorescence microscopy measurements was assessed in bacterioplankton samples from 80 lakes to estimate bacterial biovolume and cell size distribution. The total counts of 4′,6′-diamidino-2-phenylindole-stained cells estimated by both methods were significantly related, and the slope of their linear regression was not significantly different from 1, indicating that both methods produce very similar estimates of bacterial abundance. The relationships between side scatter (SSC) and 4′,6′-diamidino-2-phenylindole fluorescence and cell volume (microscopy values) were improved by binning of the data in three frequency classes for each, but further increases in the number of classes did not improve these relationships. Side scatter was the best cell volume predictor, and significant relationships were observed between the SSC classes and the smallest (R 2 = 0.545, P < 0.001, n = 80) and the largest (R 2 = 0.544, P < 0.001, n = 80) microscopy bacterial-size classes. Based on these relationships, a reliable bacterial biomass estimation was obtained from the SSC frequency classes. Our study indicates that flow cytometry can be used to properly estimate bacterioplankton biovolume, with an accuracy similar to those of more time-consuming microscopy methods.

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Veloz, Jorge Jesús, Marysol Alvear, and Luis A. Salazar. "Evaluation of Alternative Methods to Assess the Biological Properties of Propolis on Metabolic Activity and Biofilm Formation in Streptococcus mutans." Evidence-Based Complementary and Alternative Medicine 2019 (August 18, 2019): 1–8. http://dx.doi.org/10.1155/2019/1524195.

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Several biological activities have been reported for the Chilean propolis, among their antimicrobial and antibiofilm properties, due to its high polyphenol content. In this study, we evaluate alternative methods to assess the effect of Chilean propolis on biofilm formation and metabolic activity of Streptococcus mutans (S. mutans), a major cariogenic agent in oral cavity. Biofilm formation was studied by using crystal violet and by confocal microscopy. The metabolic activity of biofilm was evaluated by MTT and by flow cytometry analysis. The results show that propolis reduces biofilm formation and biofilm metabolic activity in S. mutans. When the variability of the methods to measure biofilm formation was compared, the coefficient of variation (CV) fluctuated between 12.8 and 23.1% when using crystal violet methodology. On the other hand, the CV ranged between 2.2 and 3.3% with confocal microscopy analysis. The CV for biofilm’s metabolic activity measured by MTT methodology ranged between 5.0 and 11.6%, in comparison with 1.9 to 3.2% when flow cytometry analysis was used. Besides, it is possible to conclude that the methods based on colored compounds presented lower precision to study the effect of propolis on biofilm properties. Therefore, we recommend the use of flow cytometry and confocal microscopy in S. mutans biofilm analysis.

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Pugsley, Haley Renee, Sherree L. Friend, Raymond K. Kong, and Philip Morrissey. "Illuminating T cell – APC interactions using multispectral imaging flow cytometry." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 146.6. http://dx.doi.org/10.4049/jimmunol.198.supp.146.6.

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Abstract Interaction between antigen-specific T cells and antigen presenting cells (APC) cognate ligand involve reorganization of the cytoskeleton and recruitment of adhesive and signaling molecules to the site of intercellular contact. Sustained adhesion of T cells to APCs and formation of the immunological synapse after T cell receptor stimulation are required for the antigen-specific response. One way to measure an immunological synapse is by fluorescently labeling the molecules that have been recruited to the synapse and imaging by fluorescence microscopy. However, immunological synapses are rare and therefore difficult to analyze objectively and statistically by traditional microscopy methods. To overcome these problems, we employed the Amnis brand ImageStream to objectively collect imagery of large numbers of cells. In this study, Raji B cells loaded with Staphylococcal enterotoxin B (SEB) were incubated with human T cells to create T cell-APC conjugates. Cells were stained in various combinations for CD3, CD19, Actin, LFA-1, Lck and NFkB. Using multispectral imaging flow cytometry we demonstrate image-based parameters that were used to assess the frequency of conjugates with an organized immunological synapse, evaluate the specific location of the adhesion and signaling molecules LFA-1 and Lck within the immunological synapse complex and measure the nuclear localization of NFkB in the T cell-APC conjugates.

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Woodrow, Charles J., Chirapat Wangsing, Kanlaya Sriprawat, Peter R. Christensen, Francois Nosten, Laurent Rénia, Bruce Russell, and Benoît Malleret. "Comparison between Flow Cytometry, Microscopy, and Lactate Dehydrogenase-Based Enzyme-Linked Immunosorbent Assay for Plasmodium falciparum Drug Susceptibility Testing under Field Conditions." Journal of Clinical Microbiology 53, no. 10 (August 12, 2015): 3296–303. http://dx.doi.org/10.1128/jcm.01226-15.

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Flow cytometry is an objective method for conductingin vitroantimalarial sensitivity assays with increasing potential for application in field sites. We examinedin vitrosusceptibility to seven anti-malarial drugs for 40 freshP. falciparumfield isolates via a flow cytometry method (FCM), a colorimetric LDH-based ELISA(DELI), and standard microscopic slide analysis of growth. For FCM, 184/280 (66%) assays met analytical acceptance criteria, compared to 166/280 (59%) for DELI. There was good agreement between FCM and microscopy, while DELI tended to produce higher half-maximal inhibition constants (IC50s) than FCM, with an overall bias of 2.2-fold (Bland-Altman comparison). Values for artesunate and dihydroartemisinin were most affected. Paradoxical increases in signal at very high concentrations of mefloquine and related compounds were more marked with the DELI assay, suggesting that off-target effects on LDH production may be responsible. Loss of FCM signal due to reinvasion or slow growth was observed in a small number of samples. These results extend previous work on use of flow cytometry to determine antimalarial susceptibility in terms of the number of samples, range of drugs, and comparison with other methods.

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Ecker, Rupert C., Radu Rogojanu, Marc Streit, Katja Oesterreicher, and Georg E. Steiner. "An improved method for discrimination of cell populations in tissue sections using microscopy-based multicolor tissue cytometry." Cytometry Part A 69A, no. 3 (2006): 119–23. http://dx.doi.org/10.1002/cyto.a.20219.

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Doucette, Jaimee, Ziyan Zhao, Rory J. Geyer, Melanie M. Barra, Marcy J. Balunas, and Adam Zweifach. "Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening." Journal of Biomolecular Screening 21, no. 6 (February 23, 2016): 535–47. http://dx.doi.org/10.1177/1087057116634007.

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Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)–activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections.

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Nikitayev, V. G., A. N. Pronichev, N. N. Tupitsin, V. Yu Selchuk, V. V. Dmitrieva, A. D. Palladina, E. V. Polyakov, K. A. Liberis, and V. I. Tsyplyak. "Integrated information and measurement system for the diagnosis of acute leukemia and minimal residual disease based on computer microscopy, flow laser cytofluorimetry and artificial intelligence." Journal of Physics: Conference Series 2058, no. 1 (October 1, 2021): 012033. http://dx.doi.org/10.1088/1742-6596/2058/1/012033.

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Abstract The article considers a new integrated information and measurement system for the diagnosis of acute leukemia and minimal residual disease based on computer microscopy and flow laser cytometry. The system is based on combining the results of computer microscopy in the analysis of bone marrow preparations and the results of flow laser cytofluorimetry. A special feature of the system is the use of artificial intelligence technologies in the recognition of images of bone marrow cells in the computer microscopy subsystem. The work was the result of joint work of the Department of Computer Medical Systems of the National Research Nuclear University "MEPhI" and the Laboratory of Hematopoietic Immunology of the National Medical Research Center of Oncology named after N. N. Blokhin.

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Saedi-Marghmaleki, Mojtaba, Mohammad-Taghi Moradi, Payam Ghasemi-Dehkordi, Leyla Hashemi, and Ali Karimi. "Evaluation of lentiviral vector-based green fluorescent protein expression in human gastric cancer cell line." Journal of Shahrekord University of Medical Sciences 21, no. 5 (October 30, 2019): 204–9. http://dx.doi.org/10.34172/jsums.2019.36.

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Background and aims : Human immunodeficiency virus type 1 (HIV-1) based-lentivirus vector is one of the most promising viral vectors for gene delivery in different cell lines including gastric cell lines. Therefore, the aim of this study was to produce a lentivirus vector for transduction and expression of green fluorescent protein (GFP) in human gastric cancer cell line, AGS. Materials and Methods: In this piece of work, Escherichia coli HB101 was transformed with plasmids psPAX2, pTD, and pMD2.G, following the purification of which their DNA was extracted along with their quantity and quality evaluated to be used in the next experiments. Subsequently, to produce the vector, the packaging cells were transfected with the plasmids and the vector containing supernatant was collected and purified using ultracentrifuge. ELISA was used to confirm the construction of the vector. Fluorescent microscopy and flow cytometry were used to check the expression of GFP in the cell line and to calculate the percentage of GFP expression, respectively. Results: In this study, the results of ELISA confirmed the construction of the plasmid used in this study. AGS cells were infected with viruses produced to detect the viral activity and GFP expression was evaluated by fluorescence microscopy and flow cytometry after 72 hours. Based on the results of flow cytometry, GFP was expressed in over 90% of transduced AGS cells. Conclusion: The results of this study showed that lentiviral vector is a highly efficient vector for expression of GFP gene in AGS cell line.

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Voitova, A. A., M. D. Dmitrieva, V. A. Richter, and E. V. Kuligina. "Comparative Analysis of the Binding Efficiency of Tumor-Targeting Phage Particles to Cancer Cells and Health Brain Cells." Biotekhnologiya 36, no. 6 (2020): 61–67. http://dx.doi.org/10.21519/0234-2758-2020-36-6-61-67.

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The binding efficiency of tumor-targeting phage particles, which exhibited tumor-addressing peptides and were obtained by phage display, to the human glioblastma cell line U-87 MG and health brain cells DKM- 5 has been evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA). The specific binding of the selected bacteriophages to U-87 MG cells was shown by fluorescence microscopy. Based on the obtained data, tumor-targeting phage particles were selected that provide the most efficient binding to the U-87 MG cells in vitro for further studies in order to create targeted antitumor compounds. phage display, tumor-addressing peptides, glioblastoma, flow cytometry, ELISA The authors are grateful to S. I. Baiborodin, Head of the Microscopic Analysis of Biological Objects Core Facilities Center, SB RAS, for assistance in conducting confocal microscopy. This study was supported by the Russian Science Fund (grant no. 19-44-02006).

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Johnson, Suzanne M., Antonia Banyard, Christopher Smith, Aleksandr Mironov, and Martin G. McCabe. "Large Extracellular Vesicles Can be Characterised by Multiplex Labelling Using Imaging Flow Cytometry." International Journal of Molecular Sciences 21, no. 22 (November 18, 2020): 8723. http://dx.doi.org/10.3390/ijms21228723.

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Extracellular vesicles (EVs) are heterogeneous in size (30 nm–10 µm), content (lipid, RNA, DNA, protein), and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs from medulloblastoma cells and examine EV marker expression and diameter using imaging flow cytometry. Our approach permits the characterisation of each large EVs as an individual event, decorated with multiple fluorescently conjugated markers with the added advantage of visualising each event to ensure robust gating strategies are applied. Methods: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and tunable resistance pulse sensing (TRPS). Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. Results: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. Conclusions: We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9, and LAMP1) on individual EVs within a widely heterogeneous population. Taken together, data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring.

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Ploeger, Lennert S., André Huisman, Jurryt van der Gugten, Dionne M. van der Giezen, Jeroen A. M. Beliën, Abdelhadi Y. Abbaker, Hub F. J. Dullens, et al. "Implementation of Accurate and Fast DNA Cytometry by Confocal Microscopy in 3D." Analytical Cellular Pathology 27, no. 4 (January 1, 2005): 225–30. http://dx.doi.org/10.1155/2005/289216.

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Background: DNA cytometry is a powerful method for measuring genomic instability. Standard approaches that measure DNA content of isolated cells may induce selection bias and do not allow interpretation of genomic instability in the context of the tissue. Confocal Laser Scanning Microscopy (CLSM) provides the opportunity to perform 3D DNA content measurements on intact cells in thick histological sections. Because the technique is technically challenging and time consuming, only a small number of usually manually selected nuclei were analyzed in different studies, not allowing wide clinical evaluation. The aim of this study was to describe the conditions for accurate and fast 3D CLSM cytometry with a minimum of user interaction to arrive at sufficient throughput for pilot clinical applications. Methods: Nuclear DNA was stained in 14 μm thick tissue sections of normal liver and adrenal stained with either YOYO-1 iodide or TO-PRO-3 iodide. Different pre-treatment strategies were evaluated: boiling in citrate buffer (pH 6.0) followed by RNase application for 1 or 18 hours, or hydrolysis. The image stacks obtained with CLSM at microscope magnifications of ×40 or ×100 were analyzed off-line using in-house developed software for semi-automated 3D fluorescence quantitation. To avoid sectioned nuclei, the top and bottom of the stacks were identified from ZX and YZ projections. As a measure of histogram quality, the coefficient of variation (CV) of the diploid peak was assessed. Results: The lowest CV (10.3%) was achieved with a protocol without boiling, with 1 hour RNase treatment and TO-PRO-3 iodide staining, and a final image recording at ×60 or ×100 magnifications. A sample size of 300 nuclei was generally achievable. By filtering the set of automatically segmented nuclei based on volume, size and shape, followed by interactive removal of the few remaining faulty objects, a single measurement was completely analyzed in approximately 3 hours. Conclusions: The described methodology allows to obtain a largely unbiased sample of nuclei in thick tissue sections using 3D DNA cytometry by confocal laser scanning microscopy within an acceptable time frame for pilot clinical applications, and with a CV small enough to resolve smaller near diploid stemlines. This provides a suitable method for 3D DNA ploidy assessment of selected rare cells based on morphologic characteristics and of clinical samples that are too small to prepare adequate cell suspensions.

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Pugsley, Haley Renee, Bryan R. Davidson, and Philip Morrissey. "Immunophenotyping extracellular vesicles using the CellStream flow cytometer." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 131.5. http://dx.doi.org/10.4049/jimmunol.202.supp.131.5.

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Abstract Extracellular vesicles are membrane derived structures that include exosomes, microvesicles and apoptotic bodies. The importance of extracellular vesicles as key mediators of intercellular communication is not well understood. Exosomes have been shown to transfer molecules between cells, potentially transmitting signals. Exosomes are released under normal physiological conditions; however, they are also believed to serve as mediators in the pathogenesis of neurological, vascular, haematological and autoimmune diseases as well as cancer. Quantifying and characterizing extracellular vesicles in a reproducible and reliable manner is challenging due to their small size (exosomes range from 30 to 100 nm in diameter). Extracellular vesicle analysis can be done using high-magnification microscopy; however, this technique has a very low throughput. Attempts to analyze extracellular vesicles using traditional PMT based flow cytometers has been hampered by the limit of detection of such small particles and their low refractive index. To overcome these limitations, we have employed the Amnis CellStream that has the advantage of high throughput flow cytometry with higher sensitivity to small particles due to the CCD based, time-delay-integration image capturing system. Data will be presented using the CellStream flow cytometer to immunophenotype extracellular vesicles derived from red blood cells and platelets.

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Perez-Feito, Rafael, Jordan Peccia, and Daniel R. Noguera. "Comparison between Direct Microscopy and Flow Cytometry for rRNA-Based Quantification of Candidatus Accumulibacter phosphatis in Activated Sludge." Water Environment Research 78, no. 2 (February 2006): 181–88. http://dx.doi.org/10.2175/106143005x89634.

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Streit, Marc, Rupert C. Ecker, Katja Österreicher, Georg E. Steiner, Horst Bischof, Christine Bangert, Tamara Kopp, and Radu Rogojanu. "3D parallel coordinate systems—A new data visualization method in the context of microscopy-based multicolor tissue cytometry." Cytometry Part A 69A, no. 7 (2006): 601–11. http://dx.doi.org/10.1002/cyto.a.20288.

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Köhler, Stephan, Safia Ouahrani-Bettache, Marion Layssac, Jacques Teyssier, and Jean-Pierre Liautard. "Constitutive and Inducible Expression of Green Fluorescent Protein in Brucella suis." Infection and Immunity 67, no. 12 (December 1, 1999): 6695–97. http://dx.doi.org/10.1128/iai.67.12.6695-6697.1999.

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ABSTRACT A gene fusion system based on plasmid pBBR1MCS and the expression of green fluorescent protein was developed for Brucella suis, allowing isolation of constitutive and inducible genes. Bacteria containing promoter fusions of chromosomal DNA togfp were visualized by fluorescence microscopy and examined by flow cytometry. Twelve clones containing gene fragments induced inside J774 murine macrophages were isolated and further characterized.

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Gasparri, Fabio, Paolo Cappella, and Arturo Galvani. "Multiparametric Cell Cycle Analysis by Automated Microscopy." Journal of Biomolecular Screening 11, no. 6 (June 7, 2006): 586–98. http://dx.doi.org/10.1177/1087057106289406.

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Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics’s ArrayScan™ reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells.

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Ukah, Obiaara, Maritza Puray-Chavez, Philip Tedbury, Alon Herschhorn, Joseph Sodroski, and Stefan Sarafianos. "Visualization of HIV-1 RNA Transcription from Integrated HIV-1 DNA in Reactivated Latently Infected Cells." Viruses 10, no. 10 (September 30, 2018): 534. http://dx.doi.org/10.3390/v10100534.

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We have recently developed the first microscopy-based strategy that enables simultaneous multiplex detection of viral RNA (vRNA), viral DNA (vDNA), and viral protein. Here, we used this approach to study the kinetics of latency reactivation in cells infected with the human immunodeficiency virus (HIV). We showed the transcription of nascent vRNA from individual latently integrated and reactivated vDNA sites appearing earlier than viral protein. We further demonstrated that this method can be used to quantitatively assess the efficacy of a variety of latency reactivating agents. Finally, this microscopy-based strategy was augmented with a flow-cytometry-based approach, enabling the detection of transcriptional reactivation of large numbers of latently infected cells. Hence, these approaches are shown to be suitable for qualitative and quantitative studies of HIV-1 latency and reactivation.

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Kim, Mee-Sook, Ned B. Klopfenstein, Geral I. McDonald, Kathiravetpillai Arumuganathan, and Anne K. Vidaver. "Use of flow cytometry, fluorescence microscopy, and PCR-based techniques to assess intraspecific and interspecific matings of Armillaria species." Mycological Research 105, no. 2 (February 2001): 153–63. http://dx.doi.org/10.1017/s0953756200003555.

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Bode-Lesniewska, Beata. "Flow Cytometry and Effusions in Lymphoproliferative Processes and Other Hematologic Neoplasias." Acta Cytologica 60, no. 4 (2016): 354–64. http://dx.doi.org/10.1159/000448325.

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Cytopathologists are regularly confronted with lymphocyte-rich effusions, and the definite decision of whether the lymphocytosis is of a purely reactive nature or a presentation of an indolent lymphoma may be an extremely difficult one based on microscopy alone. Flow cytometry (FC) offers many advantages in terms of its application in body cavity fluids, and it has proven to be very useful both in the setting of a known disease and for new lymphoma diagnoses. In this paper, the studies published in recent years dealing with the applications of FC in body cavity effusions in the context of hematologic neoplasia are reviewed, stressing the integrative diagnostic approach. The incorporation of microscopical, immunophenotypical, and molecular findings from examinations of the cellular content of effusions and the interpretation of results in relation to the current WHO classification of hematolymphoid malignancies give cytopathologists new perspectives on advanced and clinically highly relevant diagnostics.

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Li, Weizhe, Ronald N. Germain, and Michael Y. Gerner. "Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D)." Proceedings of the National Academy of Sciences 114, no. 35 (August 14, 2017): E7321—E7330. http://dx.doi.org/10.1073/pnas.1708981114.

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Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Ce3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Ce3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques.

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Nizamudeen, Zubair Ahmed, Rachael Xerri, Christopher Parmenter, Kiran Suain, Robert Markus, Lisa Chakrabarti, and Virginie Sottile. "Low-Power Sonication Can Alter Extracellular Vesicle Size and Properties." Cells 10, no. 9 (September 14, 2021): 2413. http://dx.doi.org/10.3390/cells10092413.

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Low-power sonication is widely used to disaggregate extracellular vesicles (EVs) after isolation, however, the effects of sonication on EV samples beyond dispersion are unclear. The present study analysed the characteristics of EVs collected from mesenchymal stem cells (MSCs) after sonication, using a combination of transmission electron microscopy, direct stochastic optical reconstruction microscopy, and flow cytometry techniques. Results showed that beyond the intended disaggregation effect, sonication using the lowest power setting available was enough to alter the size distribution, membrane integrity, and uptake of EVs in cultured cells. These results point to the need for a more systematic analysis of sonication procedures to improve reproducibility in EV-based cellular experiments.

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Picot, Stéphane, Thomas Perpoint, Christian Chidiac, Alain Sigal, Etienne Javouhey, Yves Gillet, Laurent Jacquin, et al. "Diagnostic accuracy of fluorescence flow-cytometry technology using Sysmex XN-31 for imported malaria in a non-endemic setting." Parasite 29 (2022): 31. http://dx.doi.org/10.1051/parasite/2022031.

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Malaria diagnosis based on microscopy is impaired by the gradual disappearance of experienced microscopists in non-endemic areas. Aside from the conventional diagnostic methods, fluorescence flow cytometry technology using Sysmex XN-31, an automated haematology analyser, has been registered to support malaria diagnosis. The aim of this prospective, monocentric, non-interventional study was to evaluate the diagnostic accuracy of the XN-31 for the initial diagnosis or follow-up of imported malaria cases compared to the reference malaria tests including microscopy, loop mediated isothermal amplification, and rapid diagnostic tests. Over a one-year period, 357 blood samples were analysed, including 248 negative and 109 positive malaria samples. Compared to microscopy, XN-31 showed sensitivity of 100% (95% CI: 97.13–100) and specificity of 98.39% (95% CI: 95.56–100) for the initial diagnosis of imported malaria cases. Moreover, it provided accurate species identification asfalciparumor non-falciparumand parasitaemia determination in a very short time compared to other methods. We also demonstrated that XN-31 was a reliable method for patient follow-up on days 3, 7, and 28. Malaria diagnosis can be improved in non-endemic areas by the use of dedicated haematology analysers coupled with standard microscopy or other methods in development, such as artificial intelligence for blood slide reading. Given that XN-31 provided an accurate diagnosis in 1 min, it may reduce the time interval before treatment and thus improve the outcome of patient who have malaria.

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Moyetta, Natalia R., Fabián O. Ramos, Jimena Leyria, Lilián E. Canavoso, and Leonardo L. Fruttero. "Morphological and Ultrastructural Characterization of Hemocytes in an Insect Model, the Hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae)." Insects 12, no. 7 (July 14, 2021): 640. http://dx.doi.org/10.3390/insects12070640.

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Hemocytes, the cells present in the hemolymph of insects and other invertebrates, perform several physiological functions, including innate immunity. The current classification of hemocyte types is based mostly on morphological features; however, divergences have emerged among specialists in triatomines, the insect vectors of Chagas’ disease (Hemiptera: Reduviidae). Here, we have combined technical approaches in order to characterize the hemocytes from fifth instar nymphs of the triatomine Dipetalogaster maxima. Moreover, in this work we describe, for the first time, the ultrastructural features of D. maxima hemocytes. Using phase contrast microscopy of fresh preparations, five hemocyte populations were identified and further characterized by immunofluorescence, flow cytometry and transmission electron microscopy. The plasmatocytes and the granulocytes were the most abundant cell types, although prohemocytes, adipohemocytes and oenocytes were also found. This work sheds light on a controversial aspect of triatomine cell biology and physiology setting the basis for future in-depth studies directed to address hemocyte classification using non-microscopy-based markers.

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Pollard, Peter C. "Enumerating Viruses by Using Fluorescence and the Nature of the Nonviral Background Fraction." Applied and Environmental Microbiology 78, no. 18 (July 6, 2012): 6615–18. http://dx.doi.org/10.1128/aem.01268-12.

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ABSTRACTBulk fluorescence measurements could be a faster and cheaper way of enumerating viruses than epifluorescence microscopy, flow cytometry, or transmission electron microscopy (TEM). However, since viruses are not imaged, the background fluorescence compromises the signal, and we know little about its nature. In this paper the size ranges of nucleotides that fluoresce in the presence of SYBR gold were determined for wastewater and a range of freshwater samples using a differential filtration method. Fluorescence excitation-emission matrices (FEEMs) showed that >70% of the SYBR fluorescence was in the <10-nm size fraction (background) and was not associated with intact viruses. This was confirmed using TEM. The use of FEEMs to develop a fluorescence-based method for counting viruses is an approach that is fundamentally different from the epifluorescence microscopy technique used for enumerating viruses. This high fluorescence background is currently overlooked, yet it has had a most pervasive influence on the development of a simple fluorescence-based method for quantifying viral abundance in water.

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Lai, Ning, Emily Lyettefi, Alnoor Pirani, Tim Smith, Leo Chan, and Bo Lin. "A novel imaging cytometry method for quantitative cell viability assays (65.13)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 65.13. http://dx.doi.org/10.4049/jimmunol.186.supp.65.13.

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Abstract Accurate measurement of viability is essential for many cell based research. The traditional cell viability analysis instrumentation includes microscopy, flow cytometry and plate reader. However, these systems are often expensive to purchase and maintain. Also, the operational processes are often tedious or complicated. Here we present a novel Cellometer Vision system, which incorporates image based cell counting and fluorescence detection in a compact and easy-to-use instrument. This method utilizes both bright-field and fluorescence imaging of a disposable cell counting chamber to quickly provide concentration and viability measurements of cell populations. In this poster, we demonstrate the application of Cellometer Vision system for cell viability assays that test for different characteristics of cells death. Jurkat cells were treated with cytotoxic compounds and heat treatment. Cell viability was assessed by mitochondria function assay using JC-1 dye, apoptosis assay using Annexin-V, membrane integrity assay using trypan blue and propidium iodide, metabolic assay using calcium AM and alamrBlue, and cell proliferation assay. Differences in cell viabilities were observed from different assays for cells intermediate stages of cell death. Cellometer Vision provides a simple, rapid and cost-effective tool for cell viability measurements. This novel imaging cytometry method is demonstrated here to support viability assays that test different stages of the cell death process.

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Li, Jiao, Juhee Ahn, Donghong Liu, Shiguo Chen, Xingqian Ye, and Tian Ding. "Evaluation of Ultrasound-Induced Damage to Escherichia coli and Staphylococcus aureus by Flow Cytometry and Transmission Electron Microscopy." Applied and Environmental Microbiology 82, no. 6 (January 8, 2016): 1828–37. http://dx.doi.org/10.1128/aem.03080-15.

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ABSTRACTAs a nonthermal sterilization technique, ultrasound has attracted great interest in the field of food preservation. In this study, flow cytometry and transmission electron microscopy were employed to investigate ultrasound-induced damage toEscherichia coliandStaphylococcus aureus. For flow cytometry studies, single staining with propidium iodide (PI) or carboxyfluorescein diacetate (cFDA) revealed that ultrasound treatment caused cell death by compromising membrane integrity, inactivating intracellular esterases, and inhibiting metabolic performance. The results showed that ultrasound damage was independent of initial bacterial concentrations, while the mechanism of cellular damage differed according to the bacterial species. For the Gram-negative bacteriumE. coli, ultrasound worked first on the outer membrane rather than the cytoplasmic membrane. Based on the double-staining results, we inferred that ultrasound treatment might be an all-or-nothing process: cells ruptured and disintegrated by ultrasound cannot be revived, which can be considered an advantage of ultrasound over other nonthermal techniques. Transmission electron microscopy studies revealed that the mechanism of ultrasound-induced damage was multitarget inactivation, involving the cell wall, cytoplasmic membrane, and inner structure. Understanding of the irreversible antibacterial action of ultrasound has great significance for its further utilization in the food industry.

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Anishchenko, Evgeniya, Carmela Vigorito, Luigi Mele, Patrizia Lombari, Alessandra Perna, and Diego Ingrosso. "Novel Applications of Lead Acetate and Flow Cytometry Methods for Detection of Sulfur-Containing Molecules." Methods and Protocols 2, no. 1 (February 1, 2019): 13. http://dx.doi.org/10.3390/mps2010013.

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Hydrogen sulfide (H2S) is the most recently established gaseous vasodilator, enzymatically produced from cysteine metabolism, involved in a number of pathophysiological processes. However, its accurate detection in vivo is critical due to its volatility and tendency to form sulfane sulfur derivatives, thus limiting the data interpretation of its biological roles. We developed new applications of the simple and rapid method to measure H2S release in cell culture systems, based on the lead acetate strip test. This test, previously prevalently used in microbiology, was compared with the agar trap method, applied, in parallel, on both cell cultures and cell-free samples. Sulfane sulfur represents the major species derived from intracellular H2S. Various fluorescent probes are available for quantitation of H2S derivatives intracellularly. We present here an alternative to the classic imaging method for sulfane sulfur evaluation, running on a flow cytometer, based on SSP4 probe labeling. Flow cytometry turned out to be more direct, fully quantitative and less time-consuming compared to microscopy and more precise with respect to the fluorescence multi-plate reader assay. The new application methods for H2S determination appear to be fully suitable for the analysis of H2S release and sulfane sulfur content in biological samples.

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Journal articles on the topic 'Microscopy-Based cytometry'

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