Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug ...discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.
Ever since its invention half a century ago, flow cytometry has been a major tool for single‐cell analysis, fueling advances in our understanding of a variety of complex cellular systems, in ...particular the immune system. The last decade has witnessed significant technical improvements in available cytometry platforms, such that more than 20 parameters can be analyzed on a single‐cell level by fluorescence‐based flow cytometry. The advent of mass cytometry has pushed this limit up to, currently, 50 parameters. However, traditional analysis approaches for the resulting high‐dimensional datasets, such as gating on bivariate dot plots, have proven to be inefficient. Although a variety of novel computational analysis approaches to interpret these datasets are already available, they have not yet made it into the mainstream and remain largely unknown to many immunologists. Therefore, this review aims at providing a practical overview of novel analysis techniques for high‐dimensional cytometry data including SPADE, t‐SNE, Wanderlust, Citrus, and PhenoGraph, and how these applications can be used advantageously not only for the most complex datasets, but also for standard 14‐parameter cytometry datasets.
PURPOSE AND APPROPRIATE SAMPLE TYPES
This 40‐color flow cytometry‐based panel was developed for in‐depth immunophenotyping of the major cell subsets present in human peripheral blood. Sample ...availability can often be limited, especially in cases of clinical trial material, when multiple types of testing are required from a single sample or timepoint. Maximizing the amount of information that can be obtained from a single sample not only provides more in‐depth characterization of the immune system but also serves to address the issue of limited sample availability. The panel presented here identifies CD4 T cells, CD8 T cells, regulatory T cells, γδ T cells, NKT‐like cells, B cells, NK cells, monocytes and dendritic cells. For each specific cell type, the panel includes markers for further characterization by including a selection of activation and differentiation markers, as well as chemokine receptors. Moreover, the combination of multiple markers in one tube might lead to the discovery of new immune phenotypes and their relevance in certain diseases. Of note, this panel was designed to include only surface markers to avoid the need for fixation and permeabilization steps. The panel can be used for studies aimed at characterizing the immune response in the context of infectious or autoimmune diseases, monitoring cancer patients on immuno‐ or chemotherapy, and discovery of unique and targetable biomarkers. Different from all previously published OMIPs, this panel was developed using a full spectrum flow cytometer, a technology that has allowed the effective use of 40 fluorescent markers in a single panel. The panel was developed using cryopreserved human peripheral blood mononuclear cells (PBMC) from healthy adults (Table 1). Although we have not tested the panel on fresh PBMCs or whole blood, it is anticipated that the panel could be used in those sample preparations without further optimization. @ 2020 Cytek Biosciences, Inc. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.
This OMIP describes the first 40‐color fluorescent panel using full spectrum flow cytometry to broadly phenotype much of the cellular composition of the human peripheral immune system. The panel has been thoroughly optimized to ensure high‐quality data and well‐resolved populations, enabling the description of most canonical subsets of T cells, B cells, NK cells, monocytes, and dendritic cells. This panel will be particularly useful for studies in which sample availability is limited or unique biomarker signatures are sought.
Imaging Flow Cytometry Barteneva, Natasha S.; Fasler-Kan, Elizaveta; Vorobjev, Ivan A.
The journal of histochemistry and cytochemistry,
10/2012, Letnik:
60, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Imaging flow cytometry (IFC) platforms combine features of flow cytometry and fluorescent microscopy with advances in data-processing algorithms. IFC allows multiparametric fluorescent and ...morphological analysis of thousands of cellular events and has the unique capability of identifying collected events by their real images. IFC allows the analysis of heterogeneous cell populations, where one of the cellular components has low expression (<0.03%) and can be described by Poisson distribution. With the help of IFC, one can address a critical question of statistical analysis of subcellular distribution of proteins in a cell. Here the authors review advantages of IFC in comparison with more traditional technologies, such as Western blotting and flow cytometry (FC), as well as new high-throughput fluorescent microscopy (HTFM), and discuss further developments of this novel analytical technique.
Rationale Immunoglobulin E (IgE) plays a central role in allergic diseases due to its ability to bind to high-affinity receptors on mast cells and induce degranulation upon allergen crosslinking. GO ...analysis of IgE DEG demonstrated that IgE cells expressed genes associated with N-glycosylation, the IRE1-XBP1 endoplasmic reticulum unfolded protein response, and mitochondrial oxidative phosphorylation, all indicative of a plasma cell program.
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