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The detection of extremely rare circulating tumor cells (CTCs) in peripheral blood and simultaneously identifying their viabilities are significant for cancer diagnosis and prognosis as well as monitoring the efficacy of personalized treatment. A lens-free imaging system features high-resolution images taken over a large field of view (FOV), which has great potential for CTC detection and viability determination. But current still lens-free systems restrict the application for CTC detection in real samples due to the inherent limitations of lens-free technology: (1) the location of cells in the FOV will affect the imaging; (2) the extremely rare CTCs probably did not exist in one observation. In this paper, we realized the detection of CTCs in whole blood and the simultaneous determination of their viabilities by lens-free imaging cytometry. Our in-flow system plus a large FOV range of lens-free imaging highly increased the detection rate of rare CTCs with a high throughput of 150,000 cells per minute and improved the recognition efficiency for blood cells, living/dead CTCs by using a cell tracing–assisted deep learning algorithm. With this method, the average precision of blood cells, living/dead lung cancer cells A549, and living/dead colon cancer cells SW620 reached 98.80%, 97.88%, 97.93%, 97.72%, and 98.60%, respectively. Our system got a highly consistent result with the manual counting method using fluorescent staining (Pearson's r 99.93% for SW620) and can easily detect as few as 10 dead or living CTCs from 100,000 white blood cells (WBCs). Finally, real clinical samples were detected in our system. Both dead and living CTCs were found in all six advanced-stage cancer patients, and the number of living CTCs per million WBCs ranged from 13 to 39, more than that of the dead CTCs (5 to 25), while none of the CTCs were detected in six healthy control subjects. Moreover, we also found that CTCs died very quickly after leaving the human body, indicating that CTCs should be studied as soon as possible after sampling. Although this method is implemented for CTCs, it can also be used for the detection of other rare cells. [ABSTRACT FROM AUTHOR]

Image‐based cytometry faces challenges due to technical variations arising from different experimental batches and conditions, such as differences in instrument configurations or image acquisition protocols, impeding genuine biological interpretation of cell morphology. Existing solutions, often necessitating extensive pre‐existing data knowledge or control samples across batches, have proved limited, especially with complex cell image data. To overcome this, "Cyto‐Morphology Adversarial Distillation" (CytoMAD), a self‐supervised multi‐task learning strategy that distills biologically relevant cellular morphological information from batch variations, is introduced to enable integrated analysis across multiple data batches without complex data assumptions or extensive manual annotation. Unique to CytoMAD is its "morphology distillation", symbiotically paired with deep‐learning image‐contrast translation—offering additional interpretable insights into label‐free cell morphology. The versatile efficacy of CytoMAD is demonstrated in augmenting the power of biophysical imaging cytometry. It allows integrated label‐free classification of human lung cancer cell types and accurately recapitulates their progressive drug responses, even when trained without the drug concentration information. CytoMAD also allows joint analysis of tumor biophysical cellular heterogeneity, linked to epithelial‐mesenchymal plasticity, that standard fluorescence markers overlook. CytoMAD can substantiate the wide adoption of biophysical cytometry for cost‐effective diagnosis and screening. [ABSTRACT FROM AUTHOR]

Mechanical measurements of cells can provide unique insights into cell state and disease processes. The overall mechanical properties of cells can be heavily affected by the stiffest organelle, the nucleus. However, it is challenging to fully characterize internal nuclear structures in most cell mechanical measurement platforms. Here, we demonstrate single-cell deformability measurements of whole cells and stained nuclei in a fluorescence imaging flow cytometry platform. We also introduce bending energy derived metrics as a way to normalize measurements of cytoskeletal cortex and nuclear shape changes of cells and demonstrate the utility of relative deformability distributions to characterize populations of cells. We apply the platform to measure changes in cell biophysical properties during the process of NETosis, whereby neutrophils undergo drastic nuclear restructuring. We characterize cell size, deformability, and nuclear structure changes and their correlations in thousands of neutrophils undergoing NETosis, a process implicated in development of critical disease states, such as sepsis. This platform can aid in understanding heterogeneity in deformability in cell populations and how this may be influenced by nuclear or internal structure changes. Highlights: • Introduced a new microfluidic device to apply shear stress to cells with viscous sheath for combined high-speed fluorescence imaging and deformability cytometry • Characterized how cell deformability depends on nuclear structure in health and models of activated cells [ABSTRACT FROM AUTHOR]

Population dynamics of aquatic ciliates are controlled "bottom‐up" via food supply and "top‐down" by grazing and parasitism. While intrinsic growth rates of ciliates under saturating food conditions have been studied in some detail, mortality rates induced by starvation have received little attention thus far. To this end, we examined the response of three algivorous freshwater ciliate species to starvation using three different optical methods. Two of these methods, i.e. ciliate mortality rates (δ) estimated from (i) numerical response experiments and (ii) the rate of decline (ROD) in cell numbers, investigated the response of the ciliate population using conventional light microscopy. The third method, imaging cytometry using a FlowCAM instrument, monitored single cells during the starvation experiment. Like light microscopy, the FlowCAM approach estimated δ based on ROD in the experimental containers. However, imaging cytometry also measured the relative cellular chlorophyll a content in the ciliates' food vacuoles as a proxy for the nutritional status of the cells. The linear decline of the cellular chl. a yielded an independent estimate of δ that was similar to δ calculated from ROD. Additionally, the FlowCAM measurements revealed a high degree of phenotypic plasticity of the ciliates when exposed to starvation. [ABSTRACT FROM AUTHOR]

The current methods for measuring the DNA damage response (DDR) are relatively labor-intensive and usually based on Western blotting, flow cytometry, and/or confocal immunofluorescence analyses. They require many cells and are often limited to the assessment of a single or few proteins. Here, we used the Celigo® image cytometer to evaluate the cell response to DNA-damaging agents based on a panel of biomarkers associated with the main DDR signaling pathways. We investigated the cytostatic or/and the cytotoxic effects of these drugs using simultaneous propidium iodide and calcein-AM staining. We also describe new dedicated multiplexed protocols to investigate the qualitative (phosphorylation) or the quantitative changes of eleven DDR markers (H2AX, DNA-PKcs, ATR, ATM, CHK1, CHK2, 53BP1, NBS1, RAD51, P53, P21). The results of our study clearly show the advantage of using this methodology because the multiplexed-based evaluation of these markers can be performed in a single experiment using the standard 384-well plate format. The analyses of multiple DDR markers together with the cell cycle status provide valuable insights into the mechanism of action of investigational drugs that induce DNA damage in a time- and cost-effective manner due to the low amounts of antibodies and reagents required. [ABSTRACT FROM AUTHOR]

As primary producers, phytoplankton play an integral role in global biogeochemical cycles through their production of oxygen and fixation of carbon. They also provide significant ecosystem services, by supporting secondary production and fisheries. Phytoplankton biomass and diversity have been identified by the Global Ocean Observing System (GOOS) as Essential Ocean Variables (EOVs), properties that need to be monitored to better understand and predict the ocean system. Phytoplankton identification and enumeration relies on the skills and expertise of highly trained taxonomic analysts. The training of new taxonomic analysts is intensive and requires months to years of supervised training before an analyst is able to independently and consistently apply identification skills to a sample. During the COVID-19 pandemic, access to laboratories was greatly restricted and social distancing requirements prevented supervised training. However, access to phytoplankton imaging technologies such as the Imaging FlowCytobot (IFCB), FlowCam, and PlanktoScope, combined with open online taxonomic identification platforms such as EcoTaxa, provided a means to continue monitoring, research, and training activities remotely when in-person activities were restricted. Although such technologies can not entirely replace microscopy, they have a great potential for supporting an expansion in taxonomic training, monitoring, surveillance, and research capacity. In this paper we highlight a set of imaging and collaboration tools and describe how they were leveraged during laboratory lockdowns to advance research and monitoring goals. Anecdotally, we found that the use of imaging tools accelerated the training of new taxonomic analysts in our phytoplankton analysis laboratory. Based on these experiences, we outline how these technologies can be used to increase capacity in taxonomic training and expertise, as well as how they can be used more broadly to expand research opportunities and capacity. [ABSTRACT FROM AUTHOR]

As primary producers, phytoplankton play an integral role in global biogeochemical cycles through their production of oxygen and fixation of carbon. They also provide significant ecosystem services, by supporting secondary production and fisheries. Phytoplankton biomass and diversity have been identified by the Global Ocean Observing System (GOOS) as Essential Ocean Variables (EOVs), properties that need to be monitored to better understand and predict the ocean system. Phytoplankton identification and enumeration relies on the skills and expertise of highly trained taxonomic analysts. The training of new taxonomic analysts is intensive and requires months to years of supervised training before an analyst is able to independently and consistently apply identification skills to a sample. During the COVID-19 pandemic, access to laboratories was greatly restricted and social distancing requirements prevented supervised training. However, access to phytoplankton imaging technologies such as the Imaging FlowCytobot (IFCB), FlowCam, and PlanktoScope, combined with open online taxonomic identification platforms such as EcoTaxa, provided a means to continue monitoring, research, and training activities remotely when in-person activities were restricted. Although such technologies can not entirely replace microscopy, they have a great potential for supporting an expansion in taxonomic training, monitoring, surveillance, and research capacity. In this paper we highlight a set of imaging and collaboration tools and describe how they were leveraged during laboratory lockdowns to advance research and monitoring goals. Anecdotally, we found that the use of imaging tools accelerated the training of new taxonomic analysts in our phytoplankton analysis laboratory. Based on these experiences, we outline how these technologies can be used to increase capacity in taxonomic training and expertise, as well as how they can be used more broadly to expand research opportunities and capacity.

Porcine epidemic diarrhea virus (PEDV) is an emerging porcine coronavirus that causes a tremendous economic burden on the swine industry. The assessment of PEDV-neutralizing antibody levels provides a valuable tool to assess and predict herd immunity. We evaluated the performance of a PEDV imaging cytometry–based high-throughput neutralization test (HTNT) and compared the HTNT to a fluorescent focus neutralization (FFN) assay using serum samples from pigs of known PEDV infection status (n = 159). Estimates of diagnostic sensitivity and specificity for HTNT and FFN assays derived from receiver-operator characteristic (ROC) curve analyses showed that both PEDV FFN and HTNT provided excellent diagnostic performance. However, in the laboratory, imaging cytometry provided an objective and semi-automated approach that removed human subjectivity from the testing process and reduced the read-time of a 96-well plate to < 4 min. In addition, imaging cytometry facilitated the rapid collection and long-term storage of test images and data for further evaluation or client consultation. For PEDV and other pathogens, imaging cytometry could provide distinct advantages over classic virus neutralization or FFN assays for the detection and quantitation of neutralizing antibody. [ABSTRACT FROM AUTHOR]

High parameter imaging is an important tool in the life sciences for both discovery and healthcare applications. Imaging Mass Cytometry (IMC) and Multiplexed Ion Beam Imaging (MIBI) are two relatively recent technologies which enable clinical samples to be simultaneously analyzed for up to 40 parameters at subcellular resolution. Importantly, these “Mass Cytometry Imaging” (MCI) modalities are being rapidly adopted for studies of the immune system in both health and disease. In this review we discuss, first, the various applications of MCI to date. Second, due to the inherent challenge of analyzing high parameter spatial data, we discuss the various approaches that have been employed for the processing and analysis of data from MCI experiments.

Microalgae play a key role in aquatic ecology, and methods providing species determination and enumeration can provide critical information about—for instance—harmful algae blooms (HABs) or spreading of invasive species. A crucial step in current methods is the use of sedimentation. This provides the enrichment needed to achieve statistical counts of sometimes rare species within reasonable timeframes, but it comes with the drawback of aggregating the sample. This is a real challenge for computer-aided identification as particle aggregates can often be erroneously classified. In this paper, we propose an alternative method based on flow-through imaging aided by acoustic-focussing, as this provides better input-data for automated counting-methods while simultaneously removing the need for manual sample preparation. We demonstrate that by acoustically focussing microalgae and other particulates in a fast-flowing water sample, it is possible to analyse up to 8 mL sample per minute with sufficient image quality to discriminate the invasive species Ostreopsis ovata from other particulates in samples taken directly from the Mediterranean. We also showcase the ability to achieve sharp images in flow-through at magnifications up to × 50. [ABSTRACT FROM AUTHOR]

High parameter imaging is an important tool in the life sciences for both discovery and healthcare applications. Imaging Mass Cytometry (IMC) and Multiplexed Ion Beam Imaging (MIBI) are two relatively recent technologies which enable clinical samples to be simultaneously analyzed for up to 40 parameters at subcellular resolution. Importantly, these "Mass Cytometry Imaging" (MCI) modalities are being rapidly adopted for studies of the immune system in both health and disease. In this review we discuss, first, the various applications of MCI to date. Second, due to the inherent challenge of analyzing high parameter spatial data, we discuss the various approaches that have been employed for the processing and analysis of data from MCI experiments. [ABSTRACT FROM AUTHOR]

Molecular analysis of rare single cells like circulating tumor cells (CTCs) from whole blood patient samples bears multiple challenges. One of those challenges is the efficient and ideally loss‐free isolation of CTCs over contaminating white and red blood cells. While there is a multitude of commercial and non‐commercial systems available for the enrichment of CTCs their cell output does not deliver the purity most molecular analysis methods require. Here we describe the ALS CellCelector™ which can solve this challenge allowing the retrieval of 100% pure single CTCs from blood processed by different upstream enrichment techniques. It is a multifunctional, extremely flexible system for automated screening of cell culture plates, Petri dishes, and microscope slides. Fixed or live single cells or multicellular clusters detected during screening can be picked out of those plates automatically. The complete scan and picking process is fully documented hence allowing highest standardization and reproducibility of all processes. Use of CellCelector allowed the isolation of pure single tumor cells or clusters from liquid biopsies of breast, prostate, ovarian, colorectal, lung, and brain cancers for their subsequent molecular analysis. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. [ABSTRACT FROM AUTHOR]

The potential of quantitative phase imaging (QPI) with digital holographic microscopy (DHM) for quantification of cell culture quality was explored. Label-free QPI of detached single cells in suspension was performed by Michelson interferometer-based self-interference DHM. Two pancreatic tumor cell lines were chosen as cellular model and analyzed for refractive index, volume, and dry mass under varying culture conditions. Firstly, adequate cell numbers for reliable statistics were identified. Then, to characterize the performance and reproducibility of the method, we compared results from independently repeated measurements and quantified the cellular response to osmolality changes of the cell culture medium. Finally, it was demonstrated that the evaluation of QPI images allows the extraction of absolute cell parameters which are related to cell layer confluence states. In summary, the results show that QPI enables label-free imaging cytometry, which provides novel complementary integral biophysical data sets for sophisticated quantification of cell culture quality with minimized sample preparation. © 2017 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Fluorescence in situ hybridization (FISH) is a microscopy technique which uses a fluorescent probe to detect DNA sequences and is generally performed on metaphase spreads or interphase nuclei of intact cells on a slide. In a diagnostic laboratory, cells are hybridized with fluorescent probes and up to 200 cells counted for the number of cells with probe 'spots.' Recent modifications to standard FISH include immuno-FISH, where chromosomal abnormalities are detected only in cells by their phenotype, and S-FISH where probe hybridization is performed on whole cells in suspension. Here we describe the development of an immuno-S-FISH method that combines immunophenotyping and FISH analysis of cells in suspension followed by analysis on an imaging flow cytometer. This single platform technique couples microscopy with flow cytometry and 'spot' detection of bound FISH probe. Automated immuno-S-FISH enables large numbers of analyzed cells to be identified by phenotype and assessed for specific chromosomal determinants by FISH. This novel robust method enables quantitative cell population analysis and 'spot' counting for large numbers of cells. We report method optimization of this imaging immuno-S-FISH flow cytometry protocol which has capability for many clinical applications. © 2016 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Histological techniques based on tissue colorations (e.g., hematoxylin-eosin, Sirius red) and immunostaining remain gold standard methodologies for diagnostic or phenotyping purposes in liver disease research and clinical hepatology. With the development of -omics technologies, greater information can be extracted from tissue sections. We describe a sequential immunostaining protocol consisting of repetitive cycles of immunostaining and chemically induced antibody stripping that can be readily applied to various formalin-fixed tissues (liver or other organs, mouse or human) and does not require specific equipment or commercial kits. Importantly, the combination of antibodies can be adapted according to specific clinical or scientific needs., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Immunotherapies for the treatment of cancer have spurred the development of new drugs that seek to harness the ability of T cells to recognize and kill malignant cells. There is a substantial need to evaluate how these experimental drugs influence T cell functional outputs in co-culture systems that contain cancerous cells. We describe an imaging cytometry-based platform that can simultaneously quantify activated T cells and the capacity of these T cells to kill cancer cells. Our platform was developed using the Nur77-GFP reporter system because GFP expression provides a direct readout of T cell activation that is induced by T cell antigen receptor (TCR) signaling. We combined the Nur77-GFP reporter system with a cancer cell line that displays a TCR-specific antigen and evaluated the relationship between T cell activation and cancer cell death. We demonstrate that imaging cytometry can be used to quantify the number of activated cytotoxic CD8+ T cells (CTLs) and the capacity of these CTLs to recognize and kill adherent MC38 cancer cells. We tested whether this platform could evaluate heterogenous lymphocyte populations by quantifying the proportion of antigen-specific activated T cells in co-cultures that contain unresponsive lymphocytes. The effects of a SRC family kinase inhibitor on CTL activation and MC38 cell death were also determined. Our findings demonstrate that the Nur77-GFP reporter system can be used to evaluate the effects of diverse treatment conditions on T cell-cancer co-cultures in a microtiter plate-based format by imaging cytometry. We anticipate the combined analysis of T cell activation with T cell-mediated cancer cell death can be used to rapidly assess immuno-oncology drug candidates and T cell-based therapeutics. [ABSTRACT FROM AUTHOR]

Circulating tumor cell ( CTC) enumeration is important clinically for identifying prognostic and predictive factors in patients with solid cancers. The CellSearch device (Veridex) is an immunomagnetic CTC selection and enumeration system used in clinical practice. The ImageStream (Amnis) combines the strengths of flow cytometry and fluorescent microscopy in a single platform and has potential application for CTC counting. The performance in CTC enumeration was compared between the ImageStream and CellSearch systems. Various numbers of PANC-1 tumor cells were spiked into 7.5 mL of peripheral blood from a healthy donor. Before cell analysis by the ImageStream, tumor cell enrichment was performed by immunomagnetic selection with anti-Ep PCAM. Anti- CD45 and anti- CK markers were used to discriminate between tumor cells and leukocytes. The ratios of tumor cells recovered from each dilution were calculated for both methods. The Wilcoxon rank test was applied to compare the results of the two methods and the reference value. The results of the two tested methods differed significantly from the reference value, but did not differ between them. Nevertheless, lower level of trueness and precision was observed in ImageStream when fewer numbers of CTCs were analyzed. Our results suggest that ImageStream platform for CTC enumeration has a potential value for the early diagnosis of disseminated disease, but needs an improvement of precision for the enumeration of low number of CTC. [ABSTRACT FROM AUTHOR]

We have developed a non-destructive imaging flow cell-sorting system using an ultra-high-speed camera (shutter speed of 1/10,000 s) with a real-time image analysis unit and a poly(methyl methacrylate) (PMMA)-based disposable microfluidic chip for single-cell-based on-chip cellomics. It has a 3-D micropipetting device that supports fully automated sorting and collection of samples. The entire fluidic system is implemented in a disposable plastic chip, enabling biological samples to be lined up in a laminar flow using hydrodynamic focusing. Its optical system enables direct observation-based cell identification using specific image indexes and phase-contrast/fluorescence microscopy, real-time image processing. It has a non-destructive, wider dynamic range, sorting procedure using mild electrostatic force in a laminar flow; agarose gel electrodes are used to prevent electrode loss and electrolysis bubble formation. The microreservoir used for recultivating collected target cells is contamination-free. An integrated ultra-high-speed droplet polymerase chain reaction measurement module is used for DNA/mRNA analysis of the collected target cells. This system was used to separate cardiomyocyte cells from a mixture of various cells. All the operations were automated using the 3-D micropipetting device. The results demonstrate that this imaging flow cell-sorting system is practically applicable for biological research and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Apoptosis and necrosis play an important role in various aspects of preclinical pharmaceutical drug discovery and validation. The ability to quickly determine the cytotoxic effect of chemical compounds on cancer cells allows researchers to efficiently identify potential drug candidates for further development in the pharmaceutical discovery pipeline. Recently, a new imaging cytometry system has been developed by Nexcelom Bioscience LLC (Lawrence, MA, USA) for fluorescence-based cell population analysis. Currently, fluorescence-based cell death assays have not been demonstrated by the Cellometer system, which can potentially provide a quick, simple, and inexpensive alternative method for smaller biomedical research laboratories. In this study, we demonstrate for the first time the use of Cellometer imaging cytometry for necrosis/apoptosis detection by studying the dose-response effect of heat and drug-induced cell death in Jurkat cells labeled with annexin V-FITC (apoptotic) and propidium iodide (necrotic). The experimental results were evaluated to validate the imaging cytometric capabilities of the Cellometer system as compared to the conventional flow cytometry. Similar cell population results were obtained from the two methods. The ability of Cellometer to rapidly and cost-effectively perform fluorescent cell-based assays has the potential of improving research efficiency, especially where a flow or laser scanning cytometer is not available or in situations where a rapid analysis of data is desired. [ABSTRACT FROM AUTHOR]

Worldwide awareness of fossil-fuel depletion and global warming has been increasing over the last 30 years. Numerous countries, including the USA and Brazil, have introduced large-scale industrial fermentation facilities for bioethanol, biobutanol, or biodiesel production. Most of these biofuel facilities perform fermentation using standard baker's yeasts that ferment sugar present in corn mash, sugar cane, or other glucose media. In research and development in the biofuel industry, selection of yeast strains (for higher ethanol tolerance) and fermentation conditions (yeast concentration, temperature, pH, nutrients, etc.) can be studied to optimize fermentation performance. Yeast viability measurement is needed to identify higher ethanol-tolerant yeast strains, which may prolong the fermentation cycle and increase biofuel output. In addition, yeast concentration may be optimized to improve fermentation performance. Therefore, it is important to develop a simple method for concentration and viability measurement of fermenting yeast. In this work, we demonstrate an imaging cytometry method for concentration and viability measurements of yeast in corn mash directly from operating fermenters. It employs an automated cell counter, a dilution buffer, and staining solution from Nexcelom Bioscience to perform enumeration. The proposed method enables specific fluorescence detection of viable and nonviable yeasts, which can generate precise results for concentration and viability of yeast in corn mash. This method can provide an essential tool for research and development in the biofuel industry and may be incorporated into manufacturing to monitor yeast concentration and viability efficiently during the fermentation process. [ABSTRACT FROM AUTHOR]

We have developed a novel imaging cytometry system using a poly(methyl methacrylate (PMMA)) based microfluidic chip. The system was contamination-free, because sample suspensions contacted only with a flammable PMMA chip and no other component of the system. The transparency and low-fluorescence of PMMA was suitable for microscopic imaging of cells flowing through microchannels on the chip. Sample particles flowing through microchannels on the chip were discriminated by an image-recognition unit with a high-speed camera in real time at the rate of 200 event/s, e.g., microparticles 2.5 μm and 3.0 μm in diameter were differentiated with an error rate of less than 2%. Desired cells were separated automatically from other cells by electrophoretic or dielectrophoretic force one by one with a separation efficiency of 90%. Cells in suspension with fluorescent dye were separated using the same kind of microfluidic chip. Sample of 5 μL with 1 × 106 particle/mL was processed within 40 min. Separated cells could be cultured on the microfluidic chip without contamination. The whole operation of sample handling was automated using 3D micropipetting system. These results showed that the novel imaging flow cytometry system is practically applicable for biological research and clinical diagnostics. [ABSTRACT FROM AUTHOR]

Abstract: Rapid assessment of immune or stem cells, which are now widely applied in the clinical setting of cancer treatment, is necessary to speed their development and to determine their quality. We have evaluated immature dendritic cells (iDC) by semiautomated imaging cytometry which provides detailed assessment at a single cell level. Nuclear translocation of NF-κB was studied by imaging analysis as well as electrophoretic mobility shift assay with an excellent correlation (r=0.981) over a broad range of lipopolysaccharide (LPS) concentrations. Imaging analysis was time saving (5 h vs. 3 days), and required 30- to 100-fold less cells per analysis. Single cell information revealed remarkable heterogeneity between individual iDC and permitted detection of responses to 40 pg/ml of LPS. In IL-1β/IFNγ activated iDC, STAT1 responses preceded NF-κB responses, and the expression of both was strongly correlated in individual cells (p&#60;0.001). IFNγ amplified IL-1-induced NF-κB responses. NF-κB responses to IL-1β, CD40L, and LPS were donor-dependent (n=7), correlated with the quality of iDC preparations (p=0.002), and IL-12 p70 production (p=0.010). NF-κB measurements in iDC within mixed cell cultures (iDC, NK, K562) demonstrated that these strategies are applicable for analyses of complex cell–cell interactions. Imaging analysis is a method that could be valuable for quality control of cell therapy preparations. [Copyright &y& Elsevier]

Abstract Background MicroRNAs (miRNAs) are regulatory molecules that play an important role in many physiological processes, including cell growth, differentiation, and apoptosis. In addition to modulating normal cellular functions, it has also been reported that miRNAs are involved in the development of many pathologies, including cardiovascular diseases, cancer, inflammation, and neurodegeneration. Methods for the sensitive detection and measurement of specific miRNAs and their cellular targets are essential for both basic research endeavours, as well as diagnostic efforts aimed at understanding the role of miRNAs in disease processes. Findings In this study, we describe a novel, imaging cytometry-based protocol that allows for simultaneous visualisation and quantification of miRNAs and their putative targets. We validated this methodology in a neuronal cell line by examining the relationship of the miRNA miR-124 and its known target, cyclin dependent kinase 6 (CDK6). We found that ectopic overexpression of miR-124 resulted in the downregulation of CDK6, decreased cellular proliferation, and induced cellular morphological changes. Conclusions This method is suitable for analysing the expression and cellular localisation of miRNAs and target proteins in small cell subsets within a heterogeneous cell suspension. We believe that our cytometry-based methodology will be easily adaptable to miRNA studies in many areas of biomedical research including neuroscience, stem cell biology, immunology, and oncology.

Significance: The use of optofluidic time-stretch flow cytometry enables extreme-throughput cell imaging but suffers from the difficulties of capturing and processing a large amount of data. As significant amounts of continuous image data are generated, the images require identification with high speed. Aim: We present an intelligent cell phenotyping framework for high-throughput optofluidic time-stretch microscopy based on the XGBoost algorithm, which is able to classify obtained cell images rapidly and accurately. The applied image recognition consists of density-based spatial clustering of applications with noise outlier detection, histograms of oriented gradients combining gray histogram fused feature, and XGBoost classification. Approach: We tested the ability of this framework against other previously proposed or commonly used algorithms to phenotype two groups of cell images. We quantified their performances with measures of classification ability and computational complexity based on AUC and test runtime. The tested cell image datasets were acquired from high-throughput imaging of over 20,000 drug-treated and untreated cells with an optofluidic time-stretch microscope. Results: The framework we built beats other methods with an accuracy of over 97% and a classification frequency of 3000 cells / s. In addition, we determined the optimal structure of training sets according to model performances under different training set components. Conclusions: The proposed XGBoost-based framework acts as a promising solution to processing large flow image data. This work provides a foundation for future cell sorting and clinical practice of high-throughput imaging cytometers. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) is an emerging porcine coronavirus that causes a tremendous economic burden on the swine industry. The assessment of PEDV-neutralizing antibody levels provides a valuable tool to assess and predict herd immunity. We evaluated the performance of a PEDV imaging cytometry-based high-throughput neutralization test (HTNT) and compared the HTNT to a fluorescent focus neutralization (FFN) assay using serum samples from pigs of known PEDV infection status ( n  = 159). Estimates of diagnostic sensitivity and specificity for HTNT and FFN assays derived from receiver-operator characteristic (ROC) curve analyses showed that both PEDV FFN and HTNT provided excellent diagnostic performance. However, in the laboratory, imaging cytometry provided an objective and semi-automated approach that removed human subjectivity from the testing process and reduced the read-time of a 96-well plate to < 4 min. In addition, imaging cytometry facilitated the rapid collection and long-term storage of test images and data for further evaluation or client consultation. For PEDV and other pathogens, imaging cytometry could provide distinct advantages over classic virus neutralization or FFN assays for the detection and quantitation of neutralizing antibody.

Among all environmental pollutants, the toxic heavy metal cadmium is considered as a human carcinogen. Cadmium may induce cell death by apoptosis in various cell types, although the underlying mechanisms are still unclear. In this paper we show how a label‐free digital holography (DH)‐based technique is able to quantify the evolution of key biophysical parameters of cells during the exposure to cadmium for the first time. Murine embryonic fibroblasts NIH 3T3 are chosen here as cellular model for studying the cadmium effects. The results demonstrate that DH is able to retrieve the temporal evolution of different key parameters such as cell volume, projected area, cell thickness and dry mass, thus providing a full quantitative characterization of the cell physical behaviour during cadmium exposure. Our results show that the label‐free character of the technique would allow biologists to perform systematic and reliable studies on cell death process induced by cadmium and we believe that more in general this can be easily extended to others heavy metals, thus avoiding the time‐consuming, expensive and invasive label‐based procedures used nowadays in the field. In fact, pollution by heavy metals is severe issue that needs rapid and reliable methods to be settled. According to the International agency for Research on Cancer, cadmium (Cd) is considered as a human carcinogen. Cadmium may induce cell death by apoptosis in various cell types, although the underlying mechanisms are still unclear. In this work, we present a label‐free digital holography (DH)‐based technique as an in vitro cytotoxicity assay, which overcomes the limitations of conventional in vitro test based on colour or fluorescence read outs. [ABSTRACT FROM AUTHOR]

Activation of Ataxia Telangiectasia Mediated protein kinase (ATM) by its phosphorylation on serine 1981 and phosphorylation of histone H2AX on serine 139 (γH2AX) are the key events reporting DNA damage, primarily formation of DNA double strand breaks. These events are detected immunocytochemically in individual cells using phospho-specific Abs. The protocols are presented that describe the methodology of immunofluorescent labeling of cells in conjunction with specific staining of cellular DNA. Flow- and imaging-cytometry, the latter exemplified as laser scanning cytometry, is used to quantify intensity of cellular fluorescence reporting activation of ATM and induction of γH2AX with respect to cellular DNA content, which in turn reports the cell cycle phase. Different protocols are presented for analysis of cells either grown in suspension or attached to surface of culture vessels. Examples of ATM activation and H2AX phosphorylation in response to DNA damage in leukemic HL-60 cells by DNA topoisomerase I inhibitor topotecan, and in lung carcinoma A549 cells by hydrogen peroxide, are presented.

Current research in cancer therapy is beginning to shift toward the use of combinational drug treatment regimens. However, the efficient delivery of drug combinations is governed by a number of complex factors in the clinical setting. Therefore, the ability to synchronize the pharmacokinetics of the individual therapeutic agents present in combination not only to allow for simultaneous tumor accumulation but also to allow for a synergistic relationship at the intracellular level could prove to be advantageous. In this work, we report the development of a novel folic acid-targeted liposomal formulation simultaneously co-loaded with C6 ceramide and doxorubicin [FA-(C6+Dox)-LP]. In vitro cytotoxicity assays showed that the FA-(C6+Dox)-LP was able to significantly reduce the IC50 of Dox when compared to that after the treatment with the doxorubicin-loaded liposomes (Dox-LP) as well as the untargeted drug co-loaded (C6+Dox)-LP on HeLa, A2780-ADR, and H69-AR cells. The analysis of the cell cycle distribution showed that while the C6 liposomes (C6-LP) did not cause cell cycle arrest, all the Dox-containing liposomes mediated cell cycle arrest in HeLa cells in the G2 phase at Dox concentrations of 0.3 and 1 μM and in the S phase at the higher concentrations. It was also found that this arrest in the S phase precedes the progression of the cells to apoptosis. The targeted FA-(C6+Dox)-LP were able to significantly enhance the induction of apoptotic events in HeLa cell monolayers as compared to the other treatment groups. Next, using time-lapse phase holographic imaging microscopy, it was found that upon treatment with the FA-(C6+Dox)-LP, the HeLa cells underwent rapid progression to apoptosis after 21 h as evidenced by a drastic drop in the average area of the cells after loss of cell membrane integrity. Finally, upon evaluation in a HeLa spheroid cell model, treatment with the FA-(C6+Dox)-LP showed significantly higher levels of cell death compared to those with C6-LP and Dox-LP. Overall, this study clearly shows that the co-delivery of C6 ceramide and Dox using a liposomal platform significantly correlates with an antiproliferative effect due to cell cycle regulation and subsequent induction of apoptosis and thus warrants its further evaluation in preclinical animal models.

Aptamers compete with antibodies in many applications, in which high-affinity and specificity ligands are needed. In this regard, fluorescence-tagged aptamers have gained applications in flow and imaging cytometry for detecting cells expressing distinct antigens. Here we present prospective methods, as a starting point, for using these high-affinity ligands for cytometry applications.

Many intracellular signal transduction events involve the reversible shuttling of proteins between the cytoplasm and the nucleus. Study of these processes requires imaging information on the protein localization in a given cell and a large number of measurements to obtain sufficient statistics on the protein localization in the whole population. The protocol describes method for quantitative imaging flow cytometry analysis of intracellular distribution of NF-kappaB in ARPE-19 cells stained with specific fluorochrome-conjugated antibodies. The described technique alone or in combination with standard flow cytometry methods can be applied to study any protein undergoing translocation from cytoplasm into the nucleus in a variety of cell lines as well as in heterogeneous primary cell populations.

Immune cells need to communicate with each other via direct cell contact formation. The contact zone has similar functions as a neuronal synapse and is therefore named immune synapse. Supramolecular activation clusters consisting of a variety of surface receptors and cytoplasmic proteins are formed within the immune synapse, which are pivotal for T-cell activation. Thus, a malfunction of immune synapse formation has detrimental effects on the healthiness of the individual.Classical confocal microscopy to analyze the supramolecular cluster formation and maturation of the immune synapse between primary human T-cells and antigen-presenting cells is time consuming and the number of cells that can be analyzed is limited. Therefore, we have established an InFlow microscopy approach for the analysis of immune synapses. InFlow microscopy is a hybrid method combining fluorescence microscopy and flow cytometry. Our InFlow microscopy method allows quantifying protein distribution in immune synapses of several hundred or even thousand cell couples in one sample. Importantly, comparisons of different samples with a strong statistical power are possible with InFlow microcopy.

The ability to image large numbers of cells at high resolution enhances flow cytometric analysis of cells and cell populations. In particular, the ability to image intracellular features adds a unique aspect to analyses, and can enable correlation between molecular phenomena resulting in alterations in cellular phenotype. Unicellular microalgae are amenable to high-throughput analysis to capture the diversity of cell types in natural samples, or diverse cellular responses in clonal populations, especially using imaging cytometry. Using examples from our laboratory, we review applications of imaging cytometry, specifically using an Amnis(®) ImageStream(®)X instrument, to characterize photosynthetic microalgae. Some of these examples highlight advantages of imaging flow cytometry for certain research objectives, but we also include examples that would not necessarily require imaging and could be performed on a conventional cytometer to demonstrate other concepts in cytometric evaluation of microalgae. We demonstrate the value of these approaches for (1) analysis of populations, (2) documentation of cellular features, and (3) analysis of gene expression.

A quantum dot method for highly efficient labelling of single adenoviral particles is developed. The technique has no impact on viral fitness and allows the imaging and tracking of virus binding and internalisation events using a variety of techniques including imaging cytometry and confocal microscopy. The method is applied to characterise the tropism of different adenoviral vectors., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cell patterning on photo-responsive materials are a promising tool for preparing unique single-cell arrays. However, most conventional single-cell arrays on such smart materials can be applied only to adherent cells and limit cellular functions such as extension and migration within the patterned adhesive surfaces. In this study, a versatile single cell array that works with both non-adherent and adherent cells was constructed using a photo-cleavable polyethylene glycol (PEG)-lipid/collagen surface. On this single-cell array, cells behaved similar to their native functions without limitation from the patterned surface. Furthermore, quantitative imaging analyses of cellular motility and morphological changes were performed in a high-throughput manner., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements utilizing cyan and yellow fluorescent proteins as a FRET pair. Membrane labeling with Cy5 dye is used for cell identification and contouring. Pixel-by-pixel and single-cell FRET efficiencies are calculated to estimate the extent of the molecular interactions and their distribution in the cell populations examined. We also present a non-high-throughput donor photobleaching FRET application, for obtaining the required instrument parameters for ratiometric FRET. In the biological model presented, HeLa cells are transfected with the ECFP- or EYFP-tagged Fos and Jun nuclear proteins, which heterodimerize to form active AP1 transcription factor., (Copyright © 2014 John Wiley & Sons, Inc.)

Endothelial cells in the peripheral circulation are rare events that require technically rigorous approaches for detection by flow cytometry. Visualization of these cells has been even more demanding, as this has historically required extensive enrichment and processing prior to attempting imaging. As a result, few, if any, examples exist on images of peripheral blood circulating endothelial cells (CECs) that include verification of the cell lineage both phenotypically and genomically. In this study, we have devised a method whereby CECs can be directly visualized after lysis of red blood cells and staining, without pre-enrichment or additional processing. Peripheral blood is stained with CD45, CD146, CD3, Hoechst, and DAPI to permit identification of CD146 positive, nonleukocyte, nucleated, and live cells that fit the description of CECs. These cells are imaged using the Amnis ImageStream(X), an imaging flow cytometer. Genomic verification of the endothelial nature of these cells is accomplished by using an aliquot of the same stained samples for sorting CECs using similar gating strategies. This proof of principle of direct imaging of CECs by imaging flow cytometry will permit studies to be conducted heretofore not possible, as the ImageStream(X) has the capability of detecting additional fluorochromes other than those used to identify the CECs. Such potential investigations include antigen colocalization or capping, autophagy and apoptosis, morphologic changes in response to therapy, and others. Thus, this method will enable a broad range of novel studies to be conducted using CECs as surrogates of the endothelium., (Published 2013 Wiley-Periodicals, Inc.)

To push the 100-plex envelope of suspension array technology, we have developed fully automated methods to acquire multispectral images of multiplexed quantum-dot (QD) encoded microspheres, to segment them in the images, to classify them based on their color code, and to quantify the multiplexed assays. Instead of coding microspheres with two colors and n levels, microspheres were coded with n colors and two levels (present or absent), thus transforming the classification problem from analog to digital. Images of multiplexed microspheres, sedimented at the bottom of microwells, were acquired through a tunable filter at the peak luminescence wavelength of each QD coding species in the system and the assay label wavelength. Another image of the light scattered from microspheres was captured in the excitation bandwidth that was utilized to localize microspheres in multispectral luminescence images. Objects in the acquired images are segmented and luminescence from each identified microsphere in each channel is recorded, based on which the "color code" of each microsphere is determined by applying a mathematical model and a classification algorithm. Our image analysis procedures could identify and classify microspheres with more than 97% accuracy, and the assay CVs were under 20%. These proof-of-principle results demonstrate that highly multiplexed quantification of specific proteins is possible with this rapid, small-sample volume format.