Diffractive Beam Shaper for Multiwavelength Lasers for Flow Cytometry.

Illumination spot in a flow cytometer is a crucial factor determining the measurement accuracy and stability. The traditional mechanism is to precisely calibrate multiple optical components to convert circular Gaussian beams into elliptical Gaussian beams, making it difficult to shape multiwavelength lasers simultaneously. A diffractive beam shaper for multicolor lasers with high simplicity, only containing one diffractive optical element and one focusing lens is created in this work. It can produce rectangular spots, of which the number, the sizes, and the positions are accurately determined by the incident wavelengths. Demonstrated in the customized microflow cytometer, the coefficient of variations (CV) of the optical signals by the beam shaper are 3.6-6.5%, comparable to those derived from the commercial instrument with 3.3-6.3% CVs. Benefiting from the narrow rectangular spots and the flexibility of diffractively shaped lasers, the measurement of bead sizes with 4-15 µm diameters and the real-time detection of flow velocity from 0.79 to 9.50 m/s with the CV of <5% are achieved. © 2020 International Society for Advancement of Cytometry.

The Cholera Epidemics in Hamburg and What to Learn for COVID-19 (SARS-CoV-2).

© 2020 International Society for Advancement of Cytometry.

Deep Learning-Based Single-Cell Optical Image Studies.

Optical imaging technology that has the advantages of high sensitivity and cost-effectiveness greatly promotes the progress of nondestructive single-cell studies. Complex cellular image analysis tasks such as three-dimensional reconstruction call for machine-learning technology in cell optical image research. With the rapid developments of high-throughput imaging flow cytometry, big data cell optical images are always obtained that may require machine learning for data analysis. In recent years, deep learning has been prevalent in the field of machine learning for large-scale image processing and analysis, which brings a new dawn for single-cell optical image studies with an explosive growth of data availability. Popular deep learning techniques offer new ideas for multimodal and multitask single-cell optical image research. This article provides an overview of the basic knowledge of deep learning and its applications in single-cell optical image studies. We explore the feasibility of applying deep learning techniques to single-cell optical image analysis, where popular techniques such as transfer learning, multimodal learning, multitask learning, and end-to-end learning have been reviewed. Image preprocessing and deep learning model training methods are then summarized. Applications based on deep learning techniques in the field of single-cell optical image studies are reviewed, which include image segmentation, super-resolution image reconstruction, cell tracking, cell counting, cross-modal image reconstruction, and design and control of cell imaging systems. In addition, deep learning in popular single-cell optical imaging techniques such as label-free cell optical imaging, high-content screening, and high-throughput optical imaging cytometry are also mentioned. Finally, the perspectives of deep learning technology for single-cell optical image analysis are discussed. © 2020 International Society for Advancement of Cytometry.

Dengue Fever, COVID-19 (SARS-CoV-2), and Antibody-Dependent Enhancement (ADE): A Perspective.

SARS-CoV-2 pandemic and recurrent dengue epidemics in tropical countries have turned into a global health threat. While both virus-caused infections may only reveal light symptoms, they can also cause severe diseases. Here, we review the possible antibody-dependent enhancement (ADE) occurrence, known for dengue infections, when there is a second infection with a different virus strain. Consequently, preexisting antibodies do not neutralize infection, but enhance it, possibly by triggering Fcγ receptor-mediated virus uptake. No clinical data exist indicating such mechanism for SARS-CoV-2, but previous coronavirus infections or infection of SARS-CoV-2 convalescent with different SARS-CoV-2 strains could promote ADE, as experimentally shown for antibodies against the MERS-CoV or SARS-CoV spike S protein. © 2020 International Society for Advancement of Cytometry.

Live and Let Dye: Visualizing the Cellular Compartments of the Malaria Parasite Plasmodium falciparum.

Malaria remains one of the deadliest diseases worldwide and it is caused by the protozoan parasite Plasmodium spp. Parasite visualization is an important tool for the correct detection of malarial cases but also to understand its biology. Advances in visualization techniques promote new insights into the complex life cycle and biology of Plasmodium parasites. Live cell imaging by fluorescence microscopy or flow cytometry are the foundation of the visualization technique for malaria research. In this review, we present an overview of possibilities in live cell imaging of the malaria parasite. We discuss some of the state-of-the-art techniques to visualize organelles and processes of the parasite and discuss limitation and advantages of each technique. © 2019 International Society for Advancement of Cytometry.

Two-Color Analysis of Leukocytes Labeled by Modified RBCs and Their Fragments.

Red blood cells (RBCs) are attractive tools for surface modification to adhere specifically to molecules, cellular fragments (e.g., microvesicles), or whole cells for potential use in bioanalytical assays or as a delivery vehicle in targeted therapy. Within this study, we have loaded RBCs with fluorochrome-conjugated antibodies (Ab) against CD45 and CD22 leukocyte markers and evaluated the conjugation process by microscopy. We have assessed the potential application of RBCs fragments generated from conjugated RBCs for targeting Cyto-Trol control cells by flow cytometric (FCM) approaches. Based on their scattering and fluorescence characteristics (FITC and PE expression), modified RBCs and their fragments, Cyto-Trol cells, and clusters of both were distinguished by two color FCM analysis. Fragments with anti-human Kallestad Ab as a nonspecific FITC conjugate had less than 20% binding to Cyto-Trol controls compared to CD45-FITC Ab conjugate with nearly 100% binding capacity. Cyto-Trol-microvesicle-clusters were more than 45% positive for either FITC or PE. Anti-CD22-PE modified RBCs fragments were also useful in staining and showing about 19.5% positively stained events in the Cyto-Trol region. The proof-of-concept shows, that specific antibody can be attached to RBCs, and generated fragments can be useful to stain target cells for FCM analysis. © 2018 International Society for Advancement of Cytometry.

Fast RBC loading by fluorescent antibodies and nuclei staining dye and their potential bioanalytical applications.

This study was designed to load different antibodies (Abs) and a fluorescent dye onto the red blood cell (RBC) surface. We have used fluorescein isothiocyanate (FITC)-conjugate anti-human Ab, CD22-PE (B-cell marker-phycoerythrin Ab), and 4',6-diamidino-2-phenylindole (DAPI) for insertion over the RBC surface. In a first step, conjugation experiments were performed: in dimethyl sulfoxide (DMSO), RBCs were conserved and modified by succinic anhydride to create an additional -COOH group, and then activated with 3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide (EDC-NHS) in 2-(N-morpholino) ethanesulfonic acid hydrate buffer for insertion of labeled Abs or DAPI. In a second step, fluorescence signals were evaluated by microscopy and the mean fluorescence intensities of cell lysates were measured by spectrofluorometry. The results showed clear evidence for adsorption of FITC- and PE-labeled Abs to activated conserved RBCs. DAPI was adsorbed well also to DMSO-conserved RBCs without the need for an activation step. The DMSO conservation step was enough to create reactive RBCs for insertion of specific Abs and fluorescent dyes. The additional modification by succinic anhydride and activation with EDC-NHS resulted in two- to seven-fold increase in fluorescence signals, indicating a much higher RBC loading capacity. These Ab- and fluorescent dye-functionalized RBCs have potentially high application in developing new biomedical diagnostic and in vitro assay techniques.

Effect of confounding factors on a phospho-flow assay of ribosomal S6 protein for therapeutic drug monitoring of the mTOR-inhibitor everolimus in heart transplanted patients.

CONTEXT: Several assays of monitoring immune cell function have been developed to enhance therapeutic drug monitoring. OBJECTIVE: An in vitro-validated whole-blood assay of phosphorylated ribosomal protein S6 (pS6RP) was evaluated for confounders to monitor the mTOR-inhibitor everolimus (ERL). MATERIALS AND METHODS: Whole blood samples from 87 heart transplant recipients were analyzed for pS6RP-expression in CD3-positive T-cells by phospho-flow analysis. RESULTS: ERL blood concentration, laboratory parameters, co-medications, demographic and clinical data were reviewed. CONCLUSION: Evaluating the pS6RP-assay revealed that pS6RP is influenced by cyclosporine A (CsA) blood concentration, duration of ERL treatment, co-medication with thiazide diuretics and different metabolic parameters.

Inflammation in tissue engineering: The Janus between engraftment and rejection.

Tissue engineering (TE) for tissue and organ regeneration or replacement is generally performed with scaffold implants, which provide structural and molecular support to in vitro seeded or in vivo recruited cells. TE implants elicit the host immune response, often resulting in engraftment impediment or rejection. Besides this negative effect, however, the immune system components also yield a positive influence on stem cell recruitment and differentiation, allowing tissue regeneration and healing. Thus, a balanced cooperation between proinflammatory and proresolution players of the immune response is an essential element of implant success. In this context, macrophage plasticity plays a fundamental role. Therefore modulating the immune response, instead of immune suppressing the host, might be the best way to successfully implant TE tissues or organs. In particular, it is becoming evident that the scaffold, immune, and stem cells are linked by a three-way interaction, and many efforts are being made for scaffold-appropriate design and functionalization in order to drive the inflammation process toward regeneration, vascularization, and implant success. This review discusses current and potential strategies for inflammation modulation to aid engraftment and regeneration, supporting the concept that quality, and not quantity, of inflammation might influence implant success.