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Purpose: To study the association between tear film hyperosmolarity and ocular light scatter in a cataract surgery population. Patients and Methods: Contiguous, 20-second objective scatter index (OSI) scans were recorded in hyperosmolar (≥320 mOsm/L) and normal subjects (<308 mOsm/L) with cataract nuclear opacity ≥3. OSI was measured at screening, baseline and 90 days following surgery. Along with symptoms of ocular surface disease, slit-lamp examination included corneal staining (0-3), tear film breakup time (TBUT) and evaluation of meibomian gland disease (MGD). An additional cohort of hyperosmolar subjects were measured for OSI at screening, baseline, and 5, 10, 15 and 30 minutes following instillation of 0.18% sodium hyaluronate (HA). Results: Thirty-one eyes of 31 patients were included. There was a significant difference in post-operative OSI variation when comparing hyperosmolar (0.65±0.30, N=11) to normal subjects (0.33±0.11, N=10, p=0.005). Of note, there were no significant differences in OSI variation when subjects were sorted by staining (p=0.9), TBUT (p=0.7), symptoms (p=0.7), or MGD status (p=0.9). Instillation of 0.18% HA (N=10) did not alter OSI at 5 minutes, but significant reductions in OSI of 28.8%, 38.5% and 36.7% (all p < 0.001) were observed at 10, 15 and 30 minutes. Conclusion: Hyperosmolar patients exhibited significantly increased variation in light scatter following cataract surgery that was undifferentiated by staining or TBUT. Elevated osmolarity may be indicative of light scatter equivalent to that of a grade 2-3 cataract.

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Imaging flow cytometry (IFCM) is a technique that can detect, size, and phenotype extracellular vesicles (EVs) at high throughput (thousands/minute) in complex biofluids without prior EV isolation. However, the generated signals are expressed in arbitrary units, which hinders data interpretation and comparison of measurement results between instruments and institutes. While fluorescence calibration can be readily achieved, calibration of side scatter (SSC) signals presents an ongoing challenge for IFCM. Here, we present an approach to relate the SSC signals to particle size for IFCM, and perform a comparability study between three different IFCMs using a plasma EV test sample (PEVTES). SSC signals for different sizes of polystyrene (PS) and hollow organosilica beads (HOBs) were acquired with a 405 nm 120 mW laser without a notch filter before detection. Mie theory was applied to relate scatter signals to particle size. Fluorescence calibration was accomplished with 2 µm phycoerythrin (PE) and allophycocyanin (APC) MESF beads. Size and fluorescence calibration was performed for three IFCMs in two laboratories. CD235a-PE and CD61-APC stained PEVTES were used as EV-containing samples. EV concentrations were compared between instruments within a size range of 100-1000 nm and a fluorescence intensity range of 3-10,000 MESF. 81 nm PS beads could be readily discerned from background based on their SSC signals. Fitting of the obtained PS bead SSC signals with Mie theory resulted in a coefficient of determination >0.99 between theory and data for all three IFCMs. 216 nm HOBs were detected with all instruments, and confirmed the sensitivity to detect EVs by SSC. The lower limit of detection regarding EV-size for this study was determined to be ~100 nm for all instruments. Size and fluorescence calibration of IFCM data increased cross-instrument data comparability with the coefficient of variation decreasing from 33% to 21%. Here we demonstrate - for the first time - scatter calibration of an IFCM using the 405 nm laser. The quality of the scatter-to-diameter relation and scatter sensitivity of the IFCMs are similar to the most sensitive commercially available flow cytometers. This development will support the reliability of EV research with IFCM by providing robust standardization and reproducibility, which are pre-requisites for understanding the biological significance of EVs.

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The proliferation and dissemination of antimicrobial-resistant bacteria is an increasingly global challenge and is attributed mainly to the excessive or improper use of antibiotics. Currently, the gold-standard phenotypic methodology for detecting resistant strains is agar plating, which is a time-consuming process that involves multiple subculturing steps. Genotypic analysis techniques are fast, but they require pure starting samples and cannot differentiate between viable and non-viable organisms. Thus, there is a need to develop a better method to identify and prevent the spread of antimicrobial resistance. This work presents a novel method for detecting and identifying antibiotic-resistant strains by combining a cell sorter for bacterial detection and an elastic-light-scattering method for bacterial classification. The cell sorter was equipped with safety mechanisms for handling pathogenic organisms and enabled precise placement of individual bacteria onto an agar plate. The patterning was performed on an antibiotic-gradient plate, where the growth of colonies in sections with high antibiotic concentrations confirmed the presence of a resistant strain. The antibiotic-gradient plate was also tested with an elastic-light-scattering device where each colony's unique colony scatter pattern was recorded and classified using machine learning for rapid identification of bacteria. Sorting and patterning bacteria on an antibiotic-gradient plate using a cell sorter reduced the number of subculturing steps and allowed direct qualitative binary detection of resistant strains. Elastic-light-scattering technology is a rapid, label-free, and non-destructive method that permits instantaneous classification of pathogenic strains based on the unique bacterial colony scatter pattern. KEY POINTS: • Individual bacteria cells are placed on gradient agar plates by a cell sorter • Laser-light scatter patterns are used to recognize antibiotic-resistant organisms • Scatter patterns formed by colonies correspond to AMR-associated phenotypes.

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BACKGROUND: Tuberculosis is a chronic infectious disease caused by mycobacterium tuberculosis (MTB) and is the ninth leading cause of death worldwide. It is still difficult to distinguish active TB from latent TB,but it is very important for individualized management and treatment to distinguish whether patients are active or latent tuberculosis infection. METHODS: A total of 220 subjects, including active TB patients (ATB, n = 97) and latent TB patients (LTB, n = 113), were recruited in this study .46 features about blood routine indicators and the VCS parameters (volume, conductivity, light scatter) of neutrophils(NE), monocytes(MO), and lymphocytes(LY) were collected and was constructed classification model by four machine learning algorithms(logistic regression(LR), random forest(RF), support vector machine(SVM) and k-nearest neighbor(KNN)). And the area under the precision-recall curve (AUPRC) and the area under the receiver operating characteristic curve (AUROC) to estimate of the model's predictive performance for dentifying active and latent tuberculosis infection. RESULTS: After verification,among the four classifications, LR and RF had the best performance (AUROC = 1, AUPRC = 1), followed by SVM (AUROC = 0.967, AUPRC = 0.971), KNN (AUROC = 0.943, AUPRC = 0.959) in the training set. And LR had the best performance (AUROC = 0.977, AUPRC = 0.957), followed by SVM (AUROC = 0.962, AUPRC = 0.949), RF (AUROC = 0.903, AUPRC = 0.922),KNN(AUROC = 0.883, AUPRC = 0.901) in the testing set. CONCLUSIONS: The machine learning algorithm classifier based on leukocyte VCS parameters is of great value in identifying active and latent tuberculosis infection.

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The analysis of small particles, including extracellular vesicles and viruses, is contingent on their ability to scatter sufficient light to be detected. These detection methods include flow cytometry, nanoparticle tracking analysis, and single particle reflective image sensing. To standardize measurements and enable orthogonal comparisons between platforms, a quantifiable limit of detection is required. The main parameters that dictate the amount of light scattered by particles include size, morphology, and refractive index. To date, there has been a lack of accessible techniques for measuring the refractive index of nanoparticles at a single-particle level. Here, we demonstrate two methods of deriving a small particle refractive index using orthogonal measurements with commercially available platforms. These methods can be applied at either a single-particle or population level, enabling the integration of diameter and scattering cross section values to derive the refractive index using Mie theory.

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MicroRNAs are likely to be a next-generation clinical biomarker for many diseases. While gold-standard technologies, e.g., reverse transcription-quantitative polymerase chain reaction (RT-qPCR), exist for microRNA detection, there is a need for rapid and low-cost testing. Here, an emulsion loop-mediated isothermal amplification (eLAMP) assay was developed for miRNA that compartmentalizes a LAMP reaction and shortens the time-to-detection. The miRNA was a primer to facilitate the overall amplification rate of template DNA. Light scatter intensity decreased when the emulsion droplet got smaller during the ongoing amplification, which was utilized to moitor the amplification non-invasively. A custom low-cost device was designed and fabricated using a computer cooling fan, a Peltier heater, an LED, a photoresistor, and a temperature controller. It allowed more stable vortexing and accurate light scatter detection. Three miRNAs, miR-21, miR-16, and miR-192, were successfully detected using the custom device. Specifically, new template and primer sequences were developed for miR-16 and miR-192. Zeta potential measurements and microscopic observations confirmed emulsion size reduction and amplicon adsorption. The detection limit was 0.01 fM, corresponding to 2.4 copies per reaction, and the detection could be made in 5 min. Since the assays were rapid and both template and miRNA + template could eventually be amplified, we introduced the success rate (compared to the 95% confidence interval of the template result) as a new measure, which worked well with lower concentrations and inefficient amplifications. This assay brings us one step closer to allowing circulating miRNA biomarker detection to become commonplace in the clinical world.

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Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis.

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BACKGROUND: Flow cytometry immunophenotyping (FCM) is a benchmark test for integrated diagnosis of myelodysplastic syndromes (MDS). Our department's FCM-MDS-score follows international guidelines and additionally includes the maturing erythroid (mEry) side scatter (SSC)/lymphocyte SSC ratio (mErySSCr), often increased in MDS patients. A recent exploratory computational flow analysis study highlighted mErySSC as the top feature for separating MDS from non-MDS. Thus, we sought to systematically evaluate the diagnostic accuracy of mErySSCr in conventional diagnostic FCM as used currently in-house. METHODS: Historical MDS (n = 93), chronic myelomonocytic leukemia (CMML; n = 27) and non-neoplastic cytopenia (n = 57) cohorts were created. Differences between these cohorts and LG-MDS entities were mapped and the mErySSCr cut-off was refined. Prospective bone marrows (n = 213) received for marrow failure work-up were used to determine the sensitivity and specificity of mErySSCr, both as a sole parameter and as a component of the MDS-score. RESULTS: Low-grade (LG)-MDS mErySSCr differed more prominently from controls (p = <0.0001) than high-grade (HG)-MDS (p = 0.024). CMML and controls had a similar mErySSCr. As sole parameter, mErySSCr specificity was 91.1% (n = 112 non-MDS diagnoses) and sensitivity was 36% for LG-MDS (n = 36) and 25% for new HG-MDS diagnoses (n = 16). The specificity of the MDS-score was similar if mErySSCr was omitted (81.3% with and 82.1% without). The MDS-score sensitivity for new HG-MDS diagnoses and CMML (n = 17) was 100%, and was not affected by mErySSCr. The score sensitivity for LG-MDS however, dropped from 86.1% to 72.2% when mErySSCr was excluded. CONCLUSION: mErySSCr increases the diagnostic accuracy of flow-based MDS scoring in our setting, particularly for LG-MDS.

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Doublet discrimination is usually based on pulse analysis of light scatter parameters. A combination of two pulse parameters (Area, A; Height, H; or Width, W) can be used to discriminate a pulse originated in a single cell from a pulse originated from cells stuck together. Fluorescence signals can be also used to discriminate aggregates, being essential to identify cells in the G2/M phase from doublets in the G0/G1 phase in cell cycle/DNA applications. The most used method combines FSC-A versus FSC-H, whereas other strategies combine FSC-H versus FSC-W, SSC-H versus SSC-A and SSC-H versus SSC-W. However, when studying activated or proliferating cells, scatter discrimination can be difficult. In this study, we have compared the use of light scattering with fluorescence measurement techniques for successful doublet discrimination for single cells. Effective use of FSC and SSC height, area and width are commonly used to eliminate aggregates. However, fluorescence-based methods using viable DNA stains provide a good compromise between performance and accurate manual gating methods, especially for highly concentrated cell products and pathological specimens. Viable DNA dyes, such as Vybrant™ DyeCycle™ Violet stain or Hoechst 33342, can be used to detect nucleated cells in blood and in bone marrow, or to discriminate cell aggregates and debris based on no-lyse no-wash assays, where scatter degradation is a dominant component of the measured data, which increases with event rate.

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BACKGROUND: The lack of appropriate mesenchymal stem cells (MSCs) selection methods has given the challenges for standardized harvesting, processing, and phenotyping procedures of MSCs. Genetic engineering coupled with high-throughput proteomic studies of MSC surface markers arises as a promising strategy to identify stem cell-specific markers. However, the technical limitations are the key factors making it less suitable to provide an appropriate starting material for the screening platform. A more accurate, easily accessible approach is required to solve the issues. METHODS: This study established a high-throughput screening strategy with forward versus side scatter gating to identify the adipogenesis-associated markers of bone marrow-derived MSCs (BMSCs) and tonsil-derived MSCs (TMSCs). We classified the MSC-derived adipogenic differentiated cells into two clusters: lipid-rich cells as side scatter (SSC)-high population and lipid-poor cells as SSC-low population. By screening the expression of 242 cell surface proteins, we identified the surface markers which exclusively found in lipid-rich subpopulation as the specific markers for BMSCs and TMSCs. RESULTS: High-throughput screening of the expression of 242 cell surface proteins indicated that CD49f and CD146 were specific for BMSCs and TMSCs. Subsequent immunostaining confirmed the consistent specific expression of CD49f and CD146 and in BMSCs and TMSCs. Enrichment of MSCs by CD49f and CD146 surface markers demonstrated that the simultaneous expression of CD49f and CD146 is required for adipogenesis and osteogenesis of mesenchymal stem cells. Furthermore, the fate decision of MSCs from different sources is regulated by distinct responses of cells to differentiation stimulations despite sharing a common CD49f+CD146+ immunophenotype. CONCLUSIONS: We established an accurate, robust, transgene-free method for screening adipogenesis associated cell surface proteins. This provided a valuable tool to investigate MSC-specific markers. Additionally, we showed a possible crosstalk between CD49f and CD146 modulates the adipogenesis of MSCs.

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light scatter artefacts are a methodological problem in testing residual visual capacities (RVCs), for instance blindsight, in patients with homonymous visual field defects (HVFDs). The term light scatter artefact describes the phenomenon that light from targets directed towards the HVFD can stray into the sighted visual field. This might enable an observer to respond correctly to information directed at her blind field despite the fact that she is unable to process that information in the blind field itself. In this manuscript, we present a review of the relevance of light scatter in visual neuroscience, discuss factors that influence the impact of light scatter and evaluate means to test for light scatter artefacts. Furthermore, we present findings from an empirical study that was aimed at developing tests for RVCs that are free of light scatter artefacts. Previous studies on light scatter only used small sample sizes and equipment that is no longer in use. Hence, their results cannot be generalized to future experiments making it necessary to run laborious light scatter tests for every new study on RVCs. To avoid this, we hereby start a pool of stimuli and paradigms which demonstrably do not elicit light scatter artefacts. To this end, we investigated 21 healthy young participants in three frequently used RVC-paradigms: (1) temporal 2AFC task, (2) movement direction discrimination, and (3) redundant target paradigm. For each paradigm, we applied the blind-spot method. But first, we had to establish that our testing paradigm was sufficiently sensitive to detect light scatter artefacts. For this, we used conditions that are known to produce strong light scatter and a paradigm that is very sensitive to such effects. Specifically, we presented white targets on a black background in a dark room. The stimuli were presented to observers' blind spot. To check for light scatter artefacts, we used a target-detection task in a temporal 2AFC format. We obtained clear light scatter artefacts. Participants produced reliably above-chance detection performance under these conditions. The other two luminance conditions, measured in an illuminated room, did not produce light scatter artefacts. Accuracy in the temporal 2AFC task was at chance level for white targets on a grey background at the blind-spot position. Additionally, black targets on a grey background avoided light scatter artefacts in all three RVC-paradigms. In future, researchers can apply these stimulus and illumination conditions when using one of the three above paradigms in their studies. Using these conditions, they will be able to avoid light scatter artefacts without having to perform their own blind-spot tests.

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Flow cytometry is a single-cell technology that measures scatter and fluorescence to establish a set of unique cellular properties. Flow cytometry is used in many areas of science, in particular biotechnology and medicine, but also in industrial applications. Flow cytometry can identify multiple phenotypic subsets from a mixture, select a single cell and even isolate that cell by a process called cell sorting. The field is currently undergoing dramatic changes. We are moving rapidly from the polychromic flow cytometry that has been the go-to technology for 45 years to spectral flow cytometry, which is now the most significant change in nearly half a century of flow cytometry. With change comes opportunity. Even spectral flow cytometry will morph into second-generation spectral flow cytometry within 5 years. New, exciting features will open up molecular diagnostics and physiology to flow cytometry.

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An increasing number of Arcobacter species (including several regarded as emerging human foodborne pathogens) have been isolated from shellfish, an important food commodity. A method to distinguish these species and render viable isolates for further analysis would benefit epidemiological and ecological studies. We describe a method based on Elastic Light Scatter analysis (ELSA) for the detection and discrimination of eleven shellfish-associated Arcobacter species. Although substantive differences in the growth rates of some taxa were seen, ELSA was able to differentiate all the species studied, apart from some strains of A. butzleri and A. cryaerophilus, which were nonetheless distinguished from all other species examined. ELSA appears to be a promising new approach for the detection and identification of Arcobacter species in shellfish and may also be applicable for studies in other foods and matrices.

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Isolation of the pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis from foods typically rely on slow (10-21 day) "cold enrichment" protocols before confirmed results are obtained. We describe an approach that yields results in 39 h that combines an alternative enrichment method with culture on a non-selective medium, and subsequent identification of suspect colonies using elastic light scatter (ELS) analysis. A prototype database of ELS profiles from five Yersinia species and six other bacterial genera found in pork mince was established, and used to compare similar profiles of colonies obtained from enrichment cultures from pork mince samples seeded with representative strains of Y. enterocolitica and Y. pseudotuberculosis. The presumptive identification by ELS using computerised or visual analyses of 83/90 colonies in these experiments as the target species was confirmed by partial 16S rDNA sequencing. In addition to seeded cultures, our method recovered two naturally occurring Yersinia strains. Our results indicate that modified enrichment combined with ELS is a promising new approach for expedited detection of foodborne pathogenic yersiniae.

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BACKGROUND: Study utility of seven automated VCS parameters (V-volume, C-conductivity and S-scatter) in leukocytes as an objective read-out of dysplasia in Myelodysplastic Syndromes (MDS). METHODS: Peripheral blood was analyzed by Beckman-Coulter DxH800 hematology analyzer in 43 patients with low-grade, high-grade MDS and 21 control individuals. The differences in mean (MN) and standard deviation (SD) of each parameter were examined. The optimal sensitivity and specificity to predict MDS were determined by statistical analysis. RESULTS: In neutrophils, all means of the light scatters were significantly lower in high-grade MDS than in the control group. Mean median angle light scatter (MN-MALS-NE) and mean upper median angle light scatter (MN-UMALS-NE) were significantly different between low-grade MDS and control patients. MN-MALS-NE as a MDS predictor revealed 63% sensitivity and 67% specificity with a cutoff value of ≤133. SDs of each parameter in neutrophils differed significantly among three groups. SD of neutrophil upper median angle light scatter (SD-UMALS-NE) had 77% sensitivity and 82% specificity (cutoff value of ≥11.16) to predict MDS. CONCLUSIONS: MDS patients have a significant decrease with a linear trend in VCS parameters in neutrophils, indicating cell dysplasia. The degree of the heterogeneity measured by SD is the most predictive of MDS.

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Flow cytometry is a popular technique used for both clinical and research purposes. It involves laser-based technology to characterize cells based on size, shape, and complexity. Additionally, flow cytometers are equipped with the ability to take fluorescence measurements at multiple wavelengths. This capability makes the flow cytometer a practical resource in the utilization of fluorescently conjugated antibodies, fluorescent proteins, DNA binding dyes, viability dyes, and ion indicator dyes. As the technology advances, the number of parameters a flow cytometer can measure has increased tremendously, and now some has the capacity to analyze 30-50 or more parameters on a single cell. Here, we describe the basic principles involved in the mechanics and procedures of flow cytometry along with an insight into applications of flow cytometry techniques for biomedical and allergic disease research.

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Use of flow cytometry to analyze small particles has been implemented for several decades. More recently, small particle analysis has become increasingly utilized owing to the increased sensitivity of conventional and commercially available flow cytometers along with growing interest in small particles such as extracellular vesicles (EVs). Despite an increase in small particle flow cytometry utilization, a lack of standardization in data reporting has resulted in a growing body of literature regarding EVs that cannot be easily interpreted, validated, or reproduced. Methods for fluorescence and light scatter standardization are well established, and the reagents to perform these analyses are commercially available. Here, we describe FCMPASS , a software package for performing fluorescence and light scatter calibration of small particles while generating standard reports conforming to the MIFlowCyt-EV standard reporting framework. This article covers the workflow of implementing calibration using FCMPASS as follows: acquisition of fluorescence and light scatter calibration materials, cataloguing the reference materials for use in the software, creating cytometer databases and datasets to associate calibration data and fcs files, importing fcs files for calibration, inputting fluorescence calibration parameters, inputting light scatter calibration parameters, and applying the calibration to fcs files. Published 2020. U.S. Government. Basic Protocol 1: Acquisition and gating of light scatter calibration materials Basic Protocol 2: Acquisition and gating of fluorescence calibration materials Alternate Protocol: Cross-calibration of fluorescence reference materials Basic Protocol 3: Cataloguing light scatter calibration materials Basic Protocol 4: Cataloguing fluorescence calibration materials Basic Protocol 5: Creating cytometer databases and datasets Basic Protocol 6: Importing fcs files Basic Protocol 7: Fluorescence calibration Basic Protocol 8: Light scatter calibration Basic Protocol 9: Performing and reporting fcs file calibration.

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Flow cytometers have been utilized for the analysis of submicron-sized particles since the late 1970s. Initially, virus analyses preceded extracellular vesicle (EV), which began in the 1990s. Despite decades of documented use, the lack of standardization in data reporting has resulted in a growing body of literature that cannot be easily interpreted, validated, or reproduced. This has made it difficult for objective assessments of both assays and instruments, in-turn leading to significant hindrances in scientific progress, specifically in the study of EVs, where the phenotypic analysis of these submicron-sized vesicles is becoming common-place in every biomedical field. Methods for fluorescence and light scatter standardization are well established and the reagents to perform these analyses are commercially available. However, fluorescence and light scatter calibration are not widely adopted by the small particle community as methods to standardize flow cytometry (FCM) data. In this proof-of-concept study carried out as a resource for use at the CYTO2019 workshop, we demonstrate for the first-time simultaneous fluorescence and light scatter calibration of small particle data to show the ease and feasibility of this method for standardized FCM data reporting. This data was acquired using standard configuration commercial flow cytometers, with commercially available materials, published methods, and freely available software tools. We show that application of light scatter, fluorescence, and concentration calibration can result in highly concordant data between FCM platforms independent of instrument collection angle, gain/voltage settings, and flow rate; thus, providing a means of cross comparison in standard units. © 2020 International Society for Advancement of Cytometry.

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Single-cell analysis of microbial population heterogeneity is a fast growing research area in microbiology due to its potential to identify and quantify the impact of subpopulations on microbial performance in, for example, industrial biotechnology, environmental biology, and pathogenesis. Although several tools have been developed, determination of population heterogenity in anaerobic bacteria, especially spore-forming clostridia species has been amply studied. In this study we applied single cell analysis techniques such as flow cytometry (FCM) and fluorescence-assisted cell sorting (FACS) on the spore-forming succinate producer Pseudoclostridium thermosuccinogenes. By combining FCM and FACS with fluorescent staining, we differentiated and enriched all sporulation-related morphologies of P. thermosuccinogenes. To evaluate the presence of metabolically active vegetative cells, a blend of the dyes propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) tested best. Side scatter (SSC-H) in combination with metabolic indicator cFDA dye provided the best separation of sporulation populations. Based on this protocol, we successfully determined culture heterogeneity of P. thermosuccinogenes by discriminating between mature spores, forespores, dark and bright phase endospores, and vegetative cells populations. Henceforth, this methodology can be applied to further study sporulation dynamics and its impact on fermentation performance and product formation by P. thermosuccinogenes.

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