Perspectives on label-free microscopy of heterogeneous and dynamic biological systems.

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.

Interferon-gamma producing CD4+ T cells quantified by flow cytometry as early markers for Mycobacterium avium ssp. paratuberculosis infection in cattle.

Current diagnostic methods for Johne's disease in cattle allow reliable detection of infections with Mycobacterium avium ssp. paratuberculosis (MAP) not before animals are 2 years of age. Applying a flow cytometry-based approach (FCA) to quantify a MAP-specific interferon-gamma (IFN-γ) response in T cell subsets, the present study sought to monitor the kinetics of the cell-mediated immune response in experimentally infected calves. Six MAP-negative calves and six calves, orally inoculated with MAP at 10 days of age, were sampled every 4 weeks for 52 weeks post-inoculation (wpi). Peripheral blood mononuclear cells (PBMC) were stimulated with either purified protein derivatives (PPD) or whole cell sonicates derived from MAP (WCSj), M. avium ssp. avium or M. phlei for 6 days followed by labeling of intracellular IFN-γ in CD4+ and CD8+ T cells. No antigen-specific IFN-γ production was detectable in CD8+ cells throughout and the responses of CD4+ cells of MAP-infected and control calves were similar up to 12 wpi. However, the mean fluorescence intensity (MFI) for the detection of IFN-γ in CD4+ cells after WCSj antigen stimulation allowed for a differentiation of animal groups from 16 wpi onwards. This approach had a superior sensitivity (87.8%) and specificity (86.8%) to detect infected animals from 16 wpi onwards, i.e., in an early infection stage, as compared to the IFN-γ release assay (IGRA). Quantification of specific IFN-γ production at the level of individual CD4+ cells may serve, therefore, as a valuable tool to identify MAP-infected juvenile cattle.

Flow cytometer for a dilution-free measurement approach with sample recollection.

Blood testing using flow cytometry is a common and rapid method for the initial screening and diagnosis of patients. Measurements are often combined with other scientific techniques, and analyzed samples are commonly diluted and discarded afterward. When the sample is recollected instead, sample dilution is a challenge when the sample is intended or needed for additional measurements. Therefore, it is advantageous to recollect the undiluted sample. In order to enable measurements of the same undiluted sample aliquot, we designed and constructed a purpose-built flow cytometer. Our instrument employs syringes, acoustic focusing, and an open fluidics system to recollect and reuse the unadulterated sample. The cytometer is compact, which reduces sample consumption. It acquires forward, sideward, and fluorescence signals, offering opportunities for diverse measurement approaches. In particular, our cytometer has been designed to be ready for additional downstream analysis of cells, e.g., applying mass spectrometry, magnetic resonance spectroscopy, or other analytical tools. This study presents results on instrument performance, a comparison with a cytometer that uses standard hydrodynamic focusing, and a proof of concept for multiple measurements.

Evaluating Plasmodium falciparum automatic detection and parasitemia estimation: A comparative study on thin blood smear images.

Malaria is a deadly disease that is transmitted through mosquito bites. Microscopists use a microscope to examine thin blood smears at high magnification (1000x) to identify parasites in red blood cells (RBCs). Estimating parasitemia is essential in determining the severity of the Plasmodium falciparum infection and guiding treatment. However, this process is time-consuming, labor-intensive, and subject to variation, which can directly affect patient outcomes. In this retrospective study, we compared three methods for measuring parasitemia from a collection of anonymized thin blood smears of patients with Plasmodium falciparum obtained from the Clinical Department of Parasitology-Mycology, National Reference Center (NRC) for Malaria in Paris, France. We first analyzed the impact of the number of field images on parasitemia count using our framework, MALARIS, which features a top-classifier convolutional neural network (CNN). Additionally, we studied the variation between different microscopists using two manual techniques to demonstrate the need for a reliable and reproducible automated system. Finally, we included thin blood smear images from an additional 102 patients to compare the performance and correlation of our system with manual microscopy and flow cytometry. Our results showed strong correlations between the three methods, with a coefficient of determination between 0.87 and 0.92.

Multiplex, high-throughput method to study cancer and immune cell mechanotransduction.

Studying cellular mechanoresponses during cancer metastasis is limited by sample variation or complex protocols that current techniques require. Metastasis is governed by mechanotransduction, whereby cells translate external stimuli, such as circulatory fluid shear stress (FSS), into biochemical cues. We present high-throughput, semi-automated methods to expose cells to FSS using the VIAFLO96 multichannel pipetting device custom-fitted with 22 G needles, increasing the maximum FSS 94-fold from the unmodified tips. Specifically, we develop protocols to semi-automatically stain live samples and to fix, permeabilize, and intracellularly process cells for flow cytometry analysis. Our first model system confirmed that the pro-apoptotic effects of TRAIL therapeutics in prostate cancer cells can be enhanced via FSS-induced Piezo1 activation. Our second system implements this multiplex methodology to show that FSS exposure (290 dyn cm-2) increases activation of murine bone marrow-derived dendritic cells. These methodologies greatly improve the mechanobiology workflow, offering a high-throughput, multiplex approach.

A specific natural killer cells phenotypic signature associated to long term elite control of HIV infection.

Elite controllers (ECs) are an exceptional group of people living with HIV (PLWH) that control HIV replication without therapy. Among the mechanisms involved in this ability, natural killer (NK)-cells have recently gained much attention. We performed an in-deep phenotypic analysis of NK-cells to search for surrogate markers associated with the long term spontaneous control of HIV. Forty-seven PLWH (22 long-term EC [PLWH-long-term elite controllers (LTECs)], 15 noncontrollers receiving antiretroviral treatment [ART] [PLWH-onART], and 10 noncontrollers cART-naïve [PLWH-offART]), and 20 uninfected controls were included. NK-cells homeostasis was analyzed by spectral flow cytometry using a panel of 15 different markers. Data were analyzed using FCSExpress and R software for unsupervised multidimensional analysis. Six different subsets of NK-cells were defined on the basis of CD16 and CD56 expression, and the multidimensional analysis revealed the existence of 68 different NK-cells clusters based on the expression levels of the 15 different markers. PLWH-offART presented the highest disturbance of NK-cells homeostasis and this was not completely restored by long-term ART. Interestingly, long term spontaneous control of HIV (PLWH-LTEC group) was associated with a specific profile of NK-cells homeostasis disturbance, characterized by an increase of CD16dimCD56dim subset when compared to uninfected controls (UC) group and also to offART and onART groups (p < 0.0001 for the global comparison), an increase of clusters C16 and C26 when compared to UC and onART groups (adjusted p-value < 0.05 for both comparisons), and a decrease of clusters C10 and C20 when compared to all the other groups (adjusted p-value < 0.05 for all comparisons). These findings may provide clues to elucidate markers of innate immunity with a relevant role in the long-term control of HIV.

Cell metabolism: Functional and phenotypic single cell approaches.

Several metabolic pathways are essential for the physiological regulation of immune cells, but their dysregulation can cause immune dysfunction. Hypermetabolic and hypometabolic states represent deviations in the magnitude and flexibility of effector cells in different contexts, for example in autoimmunity, infections or cancer. To study immunometabolism, most methods focus on bulk populations and rely on in vitro activation assays. Nowadays, thanks to the development of single-cell technologies, including multiparameter flow cytometry, mass cytometry, RNA cytometry, among others, the metabolic state of individual immune cells can be measured in a variety of samples obtained in basic, translational and clinical studies. Here, we provide an overview of different single-cell approaches that are employed to investigate both mitochondrial functions and cell dependence from mitochondria metabolism. Moreover, besides the description of the appropriate experimental settings, we discuss the strengths and weaknesses of different approaches with the aim to suggest how to study cell metabolism in the settings of interest.

Spectral analysis and sorting of microbial organisms using a spectral sorter.

This chapter discusses the problems related to the application of conventional flow cytometers to microbiology. To address some of those limitations, the concept of spectral flow cytometry is introduced and the advantages over conventional flow cytometry for bacterial sorting are presented. We demonstrate by using ThermoFisher's Bigfoot spectral sorter where the spectral signatures of different stains for staining bacteria are demonstrated with an example of performing unmixing on spectral datasets. In addition to the Bigfoot's spectral analysis, the special biosafety features of this instrument are discussed. Utilizing these biosafety features, the sorting and patterning at the single cell level is optimized using non-pathogenic bacteria. Finally, the chapter is concluded by presenting a novel, label free, non-destructive, and rapid phenotypic method called Elastic Light Scattering (ELS) technology for identification of the patterned bacterial cells based on their unique colony scatter patterns.

Harmonized analysis of PBMC by mass cytometry.

Mass cytometry permits the high dimensional analysis of cellular systems at single-cell resolution with high throughput in various areas of biomedical research. Here, we provide a state-of-the-art protocol for the analysis of human peripheral blood mononuclear cells (PBMC) by mass cytometry. We focus on the implementation of measures promoting the harmonization of large and complex studies to aid robustness and reproducibility of immune phenotyping data.

Measuring response to therapy in AML: Difference from normal flow cytometry vs RQ-PCR.

Multiple technologies have been used to monitor response to therapy in acute myeloid leukemia (AML) to improve detection of leukemia over the standard of practice, morphologic counting of blasts. The two techniques most frequently used in a routine clinical setting, flow cytometry and RQ-PCR, differ in their targets, sensitivity, and ability to detect residual disease. Both flow cytometry and RQ-PCR detect the expression of abnormal gene products, at the protein level or RNA level, respectively. Flow cytometry can be applied to a broad range of AML cases while RQ-PCR is limited to specific genetic abnormalities identified in subsets of AML. This article compares the results when both techniques were used in a reference laboratory to monitor AML over the course of treatment, comparing quantitative and qualitative results.

Immunophenotyping challenging tissue types using high-dimensional full spectrum flow cytometry.

Technological advancements in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system, have led to the development of large flow cytometry panels, reaching up to 40 markers at the single-cell level. Full spectrum flow cytometry, that measures the full emission range of all the fluorophores present in the panel instead of only the emission peaks is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With the capacity to use larger and more complex staining panels, optimized protocols are required for the best panel design, panel validation and high-dimensional data analysis outcomes. In addition, for ex vivo experiments, tissue preparation methods for single-cell analysis should also be optimized to ensure that samples are of the highest quality and are truly representative of tissues in situ. Here we provide optimized step-by-step protocols for full spectrum flow cytometry panel design, tissue digestion and panel optimization to facilitate the analysis of challenging tissue types.

Molecular cytometry for comprehensive immune profiling.

Molecular cytometry refers to a group of high-parameter technologies for single-cell analysis that share the following traits: (1) combined (multimodal) measurement of protein and transcripts, (2) medium throughput (10-100K cells), and (3) the use of oligonucleotide-tagged antibodies to detect protein expression. The platform can measure over 100 proteins and either hundreds of targeted genes or the whole transcriptome, on a cell-by-cell basis. It is currently one of the most powerful technologies available for immune monitoring. Here, we describe the technology platform (which includes CITE-Seq, REAP-Seq, and AbSeq), provide guidance for its optimization, and discuss advantages and limitations. Finally, we provide some vignettes from studies that demonstrate the application and potential insight that can be gained from molecular cytometry studies.

Analysis of the multiparametric cell cycle data.

This chapter was originally written in 2011. The idea was to give some history of cell cycle analysis before and after flow cytometry became widely accessible; provide references to educational material for single parameter DNA content analysis, introduce and discuss multiparameter cell cycle analysis in a methodological style, and in a casual style, discuss aspects of the work over the last 40years that we have given thought, performing some experiments, but didn't publish. It feels like there is a linear progression that moves from counting cells for growth curves, to counting labeled mitotic cells by autoradiography, to DNA content analysis, to cell cycle states defined by immunofluorescence plus DNA content analysis, to extraction of cell cycle expression profiles, and finally to probability state modeling, which should be the "right" way to analyze cytometric cell cycle data. This is the sense of this chapter. In 2023, we have updated it, but the exciting, expansive aspects brought about by spectral and mass cytometry are still young and developing, and thus have not been vetted, reviewed, and presented in mature form.

Single-cell multi-parametric characterization of microbiota by flow cytometry.

It has become evident, that the microbes colonizing the human body have a great impact on health and disease. Investigations of microbiota currently primarily rely on culturomics, high-throughput sequencing and metaproteomics which have considerably advanced our knowledge regarding the role of the microbiota in our environment and for our health. While single-cell phenotyping of immune cells and other somatic cells by flow cytometry has become widely used, the detailed analysis of bacterial cells such as the human microbiota on the single-cell level, is lagging behind. Here, we outline a protocol for the single-cell characterization of bacterial cells from complex microbiota samples, such as stool, by multi-parametric flow cytometry. Our protocol describes the isotype-specific detection of host-antibody coating of intestinal bacteria ex vivo, which together with quantitative DNA staining and light scatter detection comprise an individual's microbiota fingerprint. Cryoconservation and appropriate staining controls ensure reliable, reproducible data generation and analysis. We have automated the analysis of the multi-dimensional data using a segmentation approach by self-organizing map (SOM) algorithm for downstream comparative analyses. Our protocol can be adapted to integrate further phenotypic markers and uses the power of analytical cytometry for the characterization of bacteria on the single-cell level.

Rapid Preparation of Living Drosophila Pupal Macrophages for Ex Vivo Imaging.

The fruit fly Drosophila is a well-established invertebrate model that enables in vivo imaging of innate immune cell (e.g., macrophage) migration and signaling at high spatiotemporal resolution within the intact, living animal. While optimized methods already exist to enable flow cytometry-based macrophage isolation from Drosophila at various stages of development, there remains a need for more rapid and gentle methods to isolate living macrophages for downstream ex vivo applications. Here, we describe techniques for rapid and direct isolation of living macrophages from mature Drosophila pupae and their downstream ex vivo preparation for live imaging and immunostaining. This strategy enables straightforward access to physiologically relevant innate immune cells, both circulating and tissue-resident populations, for subsequent imaging of signal transduction.

A Pipeline for Dynamic Analysis of Mitochondrial Content in Developing T Cells: Bridging the Gap Between High-Throughput Flow Cytometry and Single-Cell Microscopy Analysis.

Analyzing the dynamics of mitochondrial content in developing T cells is crucial for understanding the metabolic state during T cell development. However, monitoring mitochondrial content in real-time needs a balance of cell viability and image resolution. In this chapter, we present experimental protocols for measuring mitochondrial content in developing T cells using three modalities: bulk analysis via flow cytometry, volumetric imaging in laser scanning confocal microscopy, and dynamic live-cell monitoring in spinning disc confocal microscopy. Next, we provide an image segmentation and centroid tracking-based analysis pipeline for automated quantification of a large number of microscopy images. These protocols together offer comprehensive approaches to investigate mitochondrial dynamics in developing T cells, enabling a deeper understanding of their metabolic processes.

Diagnosis of rare entity CD138 negative plasma cell multiple myeloma: the synergy between laboratory and clinician makes the difference.

Plasma cell multiple myeloma (MM) is a multiform clinical entity characterized by different laboratory hallmarks. This case shows a rare entity of plasma cell myeloma: the entire plasma cell population lack the CD138 expression. In this case, a careful analysis of laboratory finding, particular flow cytometry gating strategies and the use of other ancillary laboratory tests, guide the clinicians to correct diagnosis. The correct evaluation of pre-analytical phase and the correct gating strategy are the necessary conditions to produce robust and solid flow cytometric results. The diagnostic implications of CD138-negative plasma cell are strictly linked to stem cell-like clonogenic features, such as possible more aggressive clinical behaviour and increasing probability of chemotherapy resistance. At this time, clinical laboratory remains the main reference point to MM diagnosis.

Detection of clonal plasma cells in POEMS syndrome using multiparameter flow cytometry.

POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein [M-protein], and skin changes) is a rare systemic disorder characterized by various symptoms caused by underlying plasma cell (PC) dyscrasia. Detection of monoclonal PCs is mandatory for the diagnosis of POEMS syndrome; however, the usefulness of EuroFlow-based next-generation flow cytometry (EuroFlow-NGF) in POEMS syndrome for detecting monoclonal PCs in bone marrow (BM) and the gating strategy suitable for flow cytometry study of POEMS syndrome remain unknown. We employed EuroFlow-NGF-based single-tube eight-color multiparameter flow cytometry (MM-flow) and established a new gating strategy (POEMS-flow) to detect the monoclonal PCs in POEMS syndrome, gating CD38 broadly from dim to bright and CD45 narrowly from negative to dim compared to MM-flow. MM-flow detected monoclonal PCs in 9/25 (36.0%) cases, including 2/2 immunofixation electrophoresis (IFE)-negative cases (100%). However, POEMS-flow detected monoclonal PCs in 18/25 cases (72.0%), including 2/2 IFE-negative cases (100%). POEMS-flow detected monoclonal PCs with immunophenotypes of CD19- in 17/18 (94.4%). In six cases where post-treatment samples were available, the size of the clones was significantly reduced after the treatment (P = 0.031). POEMS-flow can enhance the identification rate of monoclonal PCs in POEMS syndrome and become a valuable tool for the diagnosis of POEMS syndrome.

Preparation and application of fluorescent monoclonal antibodies recognizing goat CD4+CD25+ regulatory T cells.

Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: • A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. • C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. • The frequency of Treg cells was positively correlated with the viral load during orf virus infection.