High-throughput Single-cell Analysis Research Articles

Development of High Throughput Single-Cell Analysis System for Circulating Tumor Cells Based on Digital Micromirror Device

Circulating tumor cells (CTCs) are widely known as useful biomarkers in the liquid biopsies of cancer patients. Recently, in addition to counting the number of CTCs, genetic analysis of CTCs provides critical insights into cancer metastases and add detailed clinical information that enhances patient care. It is widely known that CTCs are genetically heterogeneous, the molecular characterization of CTCs should be performed at the single-cell level. In fact, several studies have identified inter- and intra-patient heterogeneity in the mutational status of CTCs in metastatic breast and lung cancers. These studies also reported that CTCs related to drug resistance are novel therapeutic targets. Thus, genetic analysis of single CTCs has the potential to be widely applicable in personalized medicine and drug discovery. However, CTCs are extremely rare cells that only 1-100 cells are contained in 1 ml of blood, so the recovery of such extremely rare CTCs from the peripheral blood is technically challenging. So far, we have developed a CTC recovery system using a microcavity array (MCA), which demonstrates highly efficient recovery of cancer cells based on differences in cell size and deformability. Furthermore, we have proposed a novel cell manipulation method through the visualization of single cells through hydrogel encapsulation for subsequent genetic analysis of single CTCs. Subsequently, recovered CTCs can be subjected to a hydrogel-encapsulation for the following genetic analysis of single cells. Clinical studies indicated that the genetic analysis of CTCs from lung, pancreatic and gastric cancer patients was successfully achieved. However, this hydrogel encapsulation has limitations in throughput. In this study, we demonstrate a high-throughput single-cell analysis for CTCs using a multiple single-cell encapsulation system with a digital micromirror device (DMD). The novel multiple single-cell encapsulation systemwas composed of two optical systems: the wide-field fluorescence imaging system and the photopolymerization system equipped with DMD. The wide-field fluorescence imaging system using a CMOS sensor was designed to visualize 2D fluorescence imaging of the entire MCA (6.0 × 6.0 mm2). We evaluated the sensitivity of fluorescence detection of the wide-field fluorescence imaging system using the calibration slide. The limit of detection of the wide-field fluorescence imaging system was 2.1 × 102 molecules-Cy3/μm2, which is comparable to conventional fluorescence microscopy (1.0 × 102 molecules-Cy3/μm2). Generally, the expression of cellular marker protein, such as cytokeratin, was estimated to be 1.3 × 103 molecules/μm2. Therefore, our proposed system had enough sensitivity for the detection of antibody-stained CTCs. Furthermore, this wide-field fluorescence imaging system for CTC detection enables the rapid visualization of all stained cells within the field of the MCA via one-shot imaging. This rapid detection method also allows for the rapid completion of single-CTC manipulation. The photopolymerization system for single-cell encapsulation was controlled by the DMD. The curing light (λmax = 365 nm) modulated by the DMD was projected onto an MCA for hydrogel generation, and single-cell isolation by hydrogel-photopolymerization was examined at the optimized conditions. After recovery of cancer cells from whole blood onto MCA and staining by the fluorescent-labeled antibodies, cancer cells were identified by the wide-field imaging system. Then light irradiation (3 sec) was performed by the photopolymerization system. To minimize the area of the hydrogel reserved for a single cell, we newly designed the convex-type hydrogel. In the spike-in experiment, single-cell isolation rates were 97.6% (41/42) for NCI-H1975 cells (non-small cell lung cancer cell) and 94.4% (51/54) for NCI-N87 (gastric cancer cell) that were comparable with our previous study using fluorescence microscopy. With this proposed system, more than 50 single cells were encapsulated simultaneously within 1 min and isolated with an efficiency. Furthermore, single cells were successfully isolated from adjacent cells on the same microcavity without any contamination. Single cancer cell was partially encapsulated on the hydrogel, and the partial encapsulation allows us to subject them to whole genome amplification (WGA). Light irradiation and photopolymerizaion did not affect the quality of the amplified WGA products for single-cell genomic analyses. We are currently investigating single cell genetic analysis of metastatic cancer patients, and this developed multiple single-cell encapsulation system will improve the high throughput single-cell genetic analysis of CTCs in clinical samples. Our system provides an attractive application for other targets such as adherent cells, tissue samples, and microorganisms, in addition to widespread use in the isolation of CTCs for liquid biopsy.

A microfluidic cell-migration assay for the prediction of progression-free survival and recurrence time of patients with glioblastoma.

Clinical scores, molecular markers and cellular phenotypes have been used to predict clinical outcomes of patients with glioblastoma. However, their clinical use has been hampered by confounders such as patient co-morbidities, by the tumoral heterogeneity of molecular and cellular markers, and by the complexity and cost of high-throughput single-cell analysis. Here, we show that a microfluidic assay for the quantification of cell migration and proliferation can categorize patients with glioblastoma according to progression-free survival. We quantified with a composite score the ability of primary glioblastoma cells to proliferate (via the protein biomarker Ki-67) and to squeeze through microfluidic channels, mimicking aspects of the tight perivascular conduits and white-matter tracts in brain parenchyma. The assay retrospectively categorized 28 patients according to progression-free survival (short-term or long-term) with an accuracy of 86%, predicted time to recurrence, and prospectively categorized five additional patients on the basis of survival. RNA sequencing of the highly motile cells revealed differentially expressed genes that correlated with poor prognosis. Our findings suggest that cell-migration and proliferation levels can predict patient-specific clinical outcomes.

Single Cell RNA Expression Analysis Using Flow Cytometry Based on Specific Probe Ligation and Rolling Circle Amplification.

Conventional flow cytometry has been widely used for high-throughput single-cell gene expression analysis using specific antibody staining. However, this is limited by the availability of high-quality antibodies. We developed a novel flow cytometry RNA detection technique termed RCA-Flow for single-cell RNA expression analysis. We showed that it is able to analyze not only mRNAs but also microRNAs and circular RNAs that are otherwise difficult to analyze by other flow cytometry techniques. The versatility for high-throughput analysis of different types of RNA molecules makes our method possess great potential for both biomedical and clinical applications.

Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS

In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput screening via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5-25 μm in diameter) within highly monodisperse double emulsions (35 μm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS screening of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional phenotyping.

Static array of droplets and on-demand recovery for biological assays.

Microfluidics has revolutionized several research areas by providing compact yet powerful microanalytical devices that in many cases outperform conventional systems. Among different microfluidics technologies, droplet microfluidics has emerged as a powerful platform to enable analyses of biological samples and phenomena because of its simplicity and versatility. Droplet microfluidics enables high-throughput encapsulation, manipulation, and analysis of single cells while drastically reducing the cost and time required by conventional technologies. For many of these microanalysis systems, manipulation of individual droplets is extremely important as it enables multiplexed high dimensional phenotyping of the targets, going beyond surface phenotyping. One of the key manipulation steps that needs to be implemented with high precision is enabling long-term observation of droplets and recovery of a subset of these droplets for further analysis. This Perspective highlights the recent advances and provides an outlook on future developments that will enable highly complex analyses of biological samples.

VoPo leverages cellular heterogeneity for predictive modeling of single-cell data

High-throughput single-cell analysis technologies produce an abundance of data that is critical for profiling the heterogeneity of cellular systems. We introduce VoPo (https://github.com/stanleyn/VoPo), a machine learning algorithm for predictive modeling and comprehensive visualization of the heterogeneity captured in large single-cell datasets. In three mass cytometry datasets, with the largest measuring hundreds of millions of cells over hundreds of samples, VoPo defines phenotypically and functionally homogeneous cell populations. VoPo further outperforms state-of-the-art machine learning algorithms in classification tasks, and identified immune-correlates of clinically-relevant parameters.

High throughput single cell analysis of mitochondrial heteroplasmy in mitochondrial diseases

Mitochondrial heteroplasmy, which fundamentally means intracellular heterogeneity of mitochondrial DNA (mtDNA), has been measured in a group of cells, regardless of intercellular heterogeneity. Ordinal methods for mitochondrial heteroplasmy cannot discriminate between an intercellular homogenic population composed of cells with similar intracellular heterogeneity for mtDNA and an intercellular heterogenic population composed of cells with different rates of mutated mtDNA. A high-throughput method to determine mitochondrial heteroplasmy in a single cell was developed by using droplet digital PCR with TaqMan polymerase in this study. This technique revealed that there are three different cell populations of cultured fibroblasts derived from patients with mitochondrial disease carrying a mutation in the mtDNA; cells with homoplasmy of either mutated or healthy mtDNA; and cells mixed with mutated and healthy mtDNA. The presence of intercellular heterogeneity, even in uniformed cultured fibroblasts, suggests that heterogeneity should exist among different kinds of cells. The diagnosis of intercellular heterogeneity with respect to mitochondrial heteroplasmy by this methodology could provide novel insight into developing a treatment strategy for mitochondrial diseases.

Single-cell Metabolomics Analysis by Microfluidics and Mass Spectrometry: Recent New Advances

Metabolomics at the single-cell level can analyze cellular metabolites, study the phenotypic variations in individual cells and reveal phenomena and mechanisms that cannot be observed by analyzing large number of cells. It also provides an efficient way to study the dynamically changing metabolites and intermediates in single living cells. Microfluidics enables high-throughput analysis of single cells without disturbing cell metabolism as much as possible. Mass spectrometry is a promising tool for single-cell metabolomics and its innovation provides rich information of metabolites for single-cell analysis. In this mini-review, the recent new advances of single-cell metabolomics based on microfluidics and mass spectrometry in last five years are reviewed and prospected.

Reimagining Flow Cytometric Cell Sorting.

In this review, a brief history of this unrivaled technology, flow cytometry, is provided, highlighting its past and present advances, with particular focus on "flow cell" technologies. Flow cytometry has truly revolutionized high-throughput single cell analysis, which has tremendous implications, from laboratory to the clinic. This technology embodies what is truly referred to as cross fertile research, merging the physical with the life sciences. This review introduces the recent notable advancements in flow cell technology. This advancement sees the complete removal of liquid sheath flow, which has advanced the technology with the possibility of both the reduction in its foot print, while also simplifying the flow cells explored in cytometry. Interestingly, the novel sheathless flow cell technology demonstrated herein has the flexibility for handling both heterogeneous cell populations and whole organisms, thus demonstrating a versatile flow cell technology for both flow cytometry and fluorescent-activated cell sorting.

High-Throughput Single Cell Analysis Reveals the Heterogeneity of QDots-Induced Response in Macrophages

The ability to assess the responses of cells exposed to nanoparticles at the single cell level will lead to mechanistic understanding of their toxicity and safety. Here, we demonstrated the heterogeneity of cell uptake and cellular responses in a QDots-exposed macrophage using a high-content screening (HCS) analysis. The dynamic uptake and intracellular location of QDots were characterized by HCS due to its fluorescence. We further verified the association between fluorescence intensity and mass concentration of intracellular QDots using combined methods. The heterogeneity of multiple cell responses, including apoptosis, autophagy, calcium influx, mitochondria membrane potential, nucleus size, and reactive oxygen species generation, was analyzed for fingerprinting of the cytotoxicity of QDots at the single cell level. On the basis of the heterogeneity and the established dose–response relationship, we illustrated that ROS generation was the most sensitive parameter to intracellular QDots. This study makes an important methodological contribution to nanotoxicology with much higher throughput and accuracy at the single cell level.

Multiplex Stimulated Raman Scattering Imaging Cytometry Reveals Lipid-Rich Protrusions in Cancer Cells under Stress Condition.

SummaryIn situ measurement of cellular metabolites is still a challenge in biology. Conventional methods, such as mass spectrometry or fluorescence microscopy, would either destroy the sample or introduce strong perturbations to target molecules. Here, we present multiplex stimulated Raman scattering (SRS) imaging cytometry as a label-free single-cell analysis platform with chemical specificity and high-throughput capabilities. Using SRS imaging cytometry, we studied the metabolic responses of human pancreatic cancer cells under stress by starvation and chemotherapeutic drug treatments. We unveiled protrusions containing lipid droplets as a metabolic marker for stress-resistant cancer cells. Furthermore, by spectroscopic SRS mapping, we unveiled that triglyceride in lipid droplets are used for local energy production through lipolysis, autophagy, and β-oxidation. Our findings demonstrate the potential of targeting lipid metabolism for selective treatment of stress-resistant cancers. Collectively, these results highlight SRS imaging cytometry as a powerful label-free tool for biological discoveries with a high-throughput, high-content capacity.

Parallel Sampling of Nanoliter Droplet Arrays for Noninvasive Protein Analysis in Discrete Yeast Cultivations by MALDI-MS.

Miniaturization of cell-based assays enables the analysis of secreted compounds from low cell numbers down to a single cell. Droplet microfluidics is a well-established tool for high-throughput single-cell analysis. Nevertheless, the integration of label-free bioanalytical techniques like mass spectrometry is still ongoing. For example, without additional separation steps, droplet-enclosed cells do not survive the analysis. Cell separation techniques for droplets have been reported, but could not yet be coupled to mass spectrometric analysis. Here, we present a simple approach for high-throughput cell separation in parallel in nanoliter droplets and demonstrate that it can be used for qualitative analysis of protein secretion by the yeast Komagataella phaffii. Using a custom-made droplet spotter, we generated an array of 200 droplets of nanoliter volumes on a glass plate, each containing approximately 500 cells. After cultivation for 24 h, a second plate was placed above the droplet array and brought in contact with the droplets. All droplets were sampled in parallel by plate-based droplet splitting. The nanoliter samples of the supernatant could be interfaced with mass spectrometry and we were able to detect the protein brazzein (his-tagged, 7445 Da) in all but two droplets. Additionally, we show that the cells were viable after the cell separation and a sample from one spot could be transferred to a cultivation tube. An advantage of our protocol is that each cell suspension is directly linked to the analysis result by its position. Furthermore, we demonstrate that our method is capable of splitting around 6000 droplets in a few seconds. In the future, additional processing steps on a small scale, such as desalting and protein digestion, could be developed and will enable structural proteomics in nanoliter volumes.

Cyto-Mine: An Integrated, Picodroplet System for High-Throughput Single-Cell Analysis, Sorting, Dispensing, and Monoclonality Assurance.

The primary goal of bioprocess cell line development is to obtain high product yields from robustly growing and well-defined clonal cell lines in timelines measured in weeks rather than months. Likewise, high-throughput screening of B cells and hybridomas is required for most cell line engineering workflows. A substantial bottleneck in these processes is detecting and isolating rare clonal cells with the required characteristics. Traditionally, this was achieved by the resource-intensive method of limiting dilution cloning, and more recently aided by semiautomated technologies such as cell sorting (e.g., fluorescence-activated cell sorting) and colony picking. In this paper we report on our novel Cyto-Mine Single Cell Analysis and Monoclonality Assurance System, which overcomes the limitations of current technologies by screening hundreds of thousands of individual cells for secreted target proteins, and then isolating and dispensing the highest producers into microtiter plate wells (MTP). The Cyto-Mine system performs this workflow using a fully integrated, microfluidic Cyto-Cartridge. Critically, all reagents and Cyto-Cartridges used are animal component-free (ACF) and sterile, thus allowing fast, robust, and safe isolation of desired cells.

Improving Single-Cell Encapsulation Efficiency and Reliability through Neutral Buoyancy of Suspension.

Single-cell analysis is of critical importance in revealing cell-to-cell heterogeneity by characterizing individual cells and identifying minority sub-populations of interest. Droplet-based microfluidics has been widely used in the past decade to achieve high-throughput single-cell analysis. However, to maximize the proportion of single-cell emulsification is challenging due to cell sedimentation and aggregation. The purpose of this study was to investigate the influence of single-cell encapsulation and incubation through the use of neutral buoyancy. As a proof of concept, OptiPrep™ was used to create neutrally buoyant cell suspensions of THP-1, a human monocytic leukemia cell line, for single-cell encapsulation and incubation. We found that using a neutrally buoyant suspension greatly increased the efficiency of single-cell encapsulation in microdroplets and eliminated unnecessary cell loss. Moreover, the presence of OptiPrep™ was shown to not affect cellular viability. This method significantly improved the effectiveness of single-cell study in a non-toxic environment and is expected to broadly facilitate single-cell analysis.

T-bet+ Memory B Cells Link to Local Cross-Reactive IgG upon Human Rhinovirus Infection

SUMMARYHuman rhinoviruses cause the common cold and exacerbate chronic respiratory diseases. Although infection elicits neutralizing antibodies, these do not persist or cross-protect across multiple rhinovirus strains. To analyze rhinovirus-specific B cell responses in humans, we developed techniques using intact RV-A16 and RV-A39 for high-throughput high-dimensional single-cell analysis, with parallel assessment of antibody isotypes in an experimental infection model. Our approach identified T-bet+ B cells binding both viruses that account for ~5% of CXCR5− memory B cells. These B cells infiltrate nasal tissue and expand in the blood after infection. Their rapid secretion of heterotypic immunoglobulin G (IgG) in vitro, but not IgA, matches the nasal antibody profile post-infection. By contrast, CXCR5+ memory B cells binding a single virus are clonally distinct, absent in nasal tissue, and secrete homotypic IgG and IgA, mirroring the systemic response. Temporal and spatial functions of dichotomous memory B cells might explain the ability to resolve infection while rendering the host susceptible to re-infection.

High-throughput single-cell analysis of exosome mediated dual drug delivery, in vivo fate and synergistic tumor therapy.

Exosomes could serve as delivery platforms, owing to their good biocompatibility, stability, and long blood circulation time. Tracking the biological fate of exosomes in vivo is essential for evaluating their functions, delivery efficacy, and biosafety, and it is invaluable for guiding exosome-based therapy. Here, we merged a single-cell technique, mass cytometry, with in vivo uptake analysis to comprehensively reveal the fate of exosomes at the single-cell level. In tandem with multivariate cellular phenotyping, in vivo uptake of exosomes labeled with heavy metal-containing tags was quantified in a high-throughput manner. Interestingly, an organ-dependent uptake landscape of exosomes by diverse cell types was distinctly demonstrated, which implied that cancer cells seemed to preferably take up more released drugs from the exosomes. Using these cellular insights, the administration method of drug-loaded exosomes was optimized to elevate their accumulation in tumor sites and minimize their spread into healthy organs. Dual drug-loaded exosomes were locally administered and superior synergistic tumor treatment effects were achieved in a solid tumor model. The disclosure of exosome cellular distribution, together with the successful engineering of exosomes with multiple anticancer capacities, provides a new level of insight into optimizing and enhancing exosome-based drug delivery and synergistic tumor therapy.

A droplet-based gradient microfluidic to monitor and evaluate the growth of Chlorella vulgaris under different levels of nitrogen and temperatures

Algae are potential sources for different industrial products. However, biofuel production from algae still needs to reach valuable economical approaches. Select algae and optimize culture conditions considered main challenges for biofuel production. Traditional batch cultures' techniques are time and labor consuming. PDMS-based Microfluidics is time-and labor-saving technique with high throughput single cell analysis. We present gradient-based microfluidics as tools for studying algal growth under different cultivation conditions. Applying our device provides the opportunity to generate droplets with different concentrations of medium nutrients' in 200s which keeps reagent and time for preparing such solutions using conventional techniques. Chlorella vulgaris was encapsulated inside uniformly droplets with different NO3−-N concentrations of 2.46, 6.69, 10.91, and 15.14 mΜ/L. separated devices were exposed to different temperatures of 20, 27, and 37°C. The photosynthetic energy conversion for each cell per droplet was calculated. It was observed that the higher the NO3− concentration, the higher the growth kinetics. According to the presented results, the optimum nitrate and temperature conditions were 15.14 mΜ/L and 37°C. Our device is considered the third effort for applying a gradient chip in algal biotechnology field. It is unique and simple in use with high competence results.

Functional Precision Medicine in AML: Technical Performance Evaluation for in Vitro Diagnostics Using High-Throughput Image-Based Screening of Primary Patient Cells

Introduction: Identification of the right drug, for the right patient, at the right time is the defining goal for precision and personalized medicine programs. Many tools are currently being pursued for this including next-gen sequencing and other -omics approaches to determine target availability (Moscow et al, 2018 Nat Rev Clin Oncol.), functional drug response profiling in viable patient samples (Snijder et al, 2017 Lancet Haem), or a combination of both (Schmidl et al, 2019 Nat Chem Bio). Of those, functional precision medicine approaches are only just beginning to gain clinical interest, and because of this, nearly all programs lack systematic testing in prospective clinical studies (Letai, 2017 Nat Rev Med). One of the first functional precision medicine approaches to be tested in a small prospective clinical study was the use of high-throughput image-based screening and single cell analysis of drug response in primary tissues ("Pharmacoscopy") as a tool to prospectively rank drug options for patients with late-stage hematological indications (Snijder et al 2017, Lancet Haem, NCT03096821). In this study, taking Pharmacoscopy data into account for treatment selection led to a higher overall response rate (88% vs. 24%) and longer progression free survival (22.6 vs. 5.7 weeks) compared to the previous line of therapy. In order for functional precision medicine programs to reach widespread routine use, ultimately the development of fully-certified in vitro diagnostic test products will be required. This requires amongst others a systematic understanding of i) the technical robustness of functional drug testing approaches and ii) a detailed understanding of how pre-analytical sample handling influences assay results. Here, we set out to investigate two crucial questions that arose in the course of systematic assay development for AML: a) do CD34+ cells derived from peripheral blood and bone marrow AML patients respond differently to short term ex vivo treatment with small molecule drugs and b) does the overall response of these cells change upon cryopreservation? These issues are of high relevance even outside the setting of functional precision medicine development programs given worldwide biobanking efforts, and the lack of robust systematic model comparison. Methods: Fresh patient blood and bone marrow samples of newly diagnosed as well as relapsed and refractory patients with AML were collected at Medical University of Graz under appropriate ethics approval. Samples were divided into two parts and one part used immediately, the other cryopreserved as viable cells. The response of CD34+ cells against 140 different small molecule drugs (two concentrations and three technical replicates) was evaluated using single-cell high content microscopy (Allcyte's "Pharmacoscopy" platform) in both peripheral blood and bone marrow as well as freshly used and previously viably frozen samples. Drug response was determined by fitting to generalised linear and mixed models. Further, RNA is isolated from additional patient samples before and after biobanking for Nanostring analysis. Results: Overall, across all tested patient samples, we observed a high correlation between drug response tested in blood and bone marrow samples from this group of patient samples, indicating, at least for AML, no strong niche dependence for the drugs tested. Additionally, we also observed a high correlation between drug response tested in fresh and frozen tissues (Figure 1). The differences that did appear were, in particular, in drugs targeting metabolic and cell-growth dependent functions, amongst others. Conclusions: Understanding how biobanking affects the ex vivo drug response of primary human tissues is paramount to being able to understand how far translational efforts can go using tissues and functional drug response assays. We provide systematic data supporting the use of cryopreserved samples for the functional analysis of drug response in AML. This is a fundamental question associated with the use of biobanks for investigations of primary patient material. Figure 1 Disclosures Krall: Allcyte: Employment, Equity Ownership. Meszaros:Allcyte: Employment. Vilagos:Allcyte: Employment. Sill:Astellas: Other: Advisory board; Novartis: Other: Advisory board; Astex: Other: Advisory board; AbbVie: Other: Advisory board. Vladimer:Allcyte: Employment, Equity Ownership, Patents &amp; Royalties: EP3198276A1.

Using single-cell transcriptomics to understand functional states and interactions in microbial eukaryotes.

Understanding the diversity and evolution of eukaryotic microorganisms remains one of the major challenges of modern biology. In recent years, we have advanced in the discovery and phylogenetic placement of new eukaryotic species and lineages, which in turn completely transformed our view on the eukaryotic tree of life. But we remain ignorant of the life cycles, physiology and cellular states of most of these microbial eukaryotes, as well as of their interactions with other organisms. Here, we discuss how high-throughput genome-wide gene expression analysis of eukaryotic single cells can shed light on protist biology. First, we review different single-cell transcriptomics methodologies with particular focus on microbial eukaryote applications. Then, we discuss single-cell gene expression analysis of protists in culture and what can be learnt from these approaches. Finally, we envision the application of single-cell transcriptomics to protist communities to interrogate not only community components, but also the gene expression signatures of distinct cellular and physiological states, as well as the transcriptional dynamics of interspecific interactions. Overall, we argue that single-cell transcriptomics can significantly contribute to our understanding of the biology of microbial eukaryotes. This article is part of a discussion meeting issue 'Single cell ecology'.

Interfacial Nanoinjection-Based Nanoliter Single-Cell Analysis.

Single-cell analysis offers unprecedented resolution for the investigation of cellular heterogeneity and the capture of rare cells from large populations. Here, described is a simple method named interfacial nanoinjection (INJ), which can miniaturize various single-cell assays to be performed in nanoliter water-in-oil droplets on standard microwell plates. The INJ droplet handler can adjust droplet volumes for multistep reactions on demand with high precision and excellent monodispersity, and consequently enables a wide range of single-cell assays. Importantly, INJ can be coupled with fluorescence-activated cell sorting (FACS), which is currently the most effective and accurate single-cell sorting and isolation method. FACS-INJ pipelines for high-throughput plate well-based single-cell analyses, including single-cell proliferation, drug-resistance testing, polymerase chain reaction (PCR), reverse-transcription PCR, and whole-genome sequencing are introduced. This FACS-INJ pipeline is compatible with a wide range of samples and can be extended to various single-cell analysis applications in microbiology, cell biology, and biomedical diagnostics.