Next wave advances in single-cell analyses

Across three continents, we have come together as guest editors interested in learning and sharing what our global research community sees as critical ‘next wave’ advances in single-cell analyses. In convening this special issue of Analyst, we happily report that our field is awash in maturing and emerging approaches. The research contained in this issue communicates cutting-edge approaches addressing questions from clinical medicine to life sciences fundamentals, environmental science, material sciences and beyond.

In this special issue we peer out from a higher elevation by including a set of reviews to provide useful context for the foundations of single-cell analysis and what may be coming. To complement that vantage point, the focus of this special issue is on emerging and maturing original technical contributions for single-cell analysis including precision cytometry, imaging, spectrometry, and mass spectrometry (MS). So, please join us in taking in the map that our special issue contributors have charted for us as this collection of research highlights the future of single-cell analysis and the positive impact.

To guide readers as we look towards the horizon and see ‘next wave’ single-cell tools emerging, this special issue offers a robust set of critical reviews and focused minireviews. We find the reviews exceptionally helpful in orienting to a new or emerging field, as well as for the often-included perspective offered by the authors. First up are two reviews centering on advances in microfluidic technologies. Huang and colleagues (DOI: 10.1039/c8an01079j) present in their review the strengths and the weaknesses of a comprehensive range of microfluidic advances, including in single-cell isolation, single-cell lysis, and single-cell analysis. Complementing the tools focus, Gao and co-authors (DOI: 10.1039/c8an01186a) provide insight on a broad swath of application areas including small molecule detection, protein analysis, multidrug resistance analysis, and single-cell sequencing with a technological focus on recent (<3 years) updates on approaches to cell manipulation and detection.

Considering advances in single-molecule/single-particle resolution imaging, Liu and colleagues (DOI: 10.1039/c8an01420e) review optical microscopy optimized for bioanalysis in live cells. In particular, the team illustrates with examples the fundamental physics underpinning workhorse optical microscopy techniques, including total internal reflection fluorescence microscopy (TIRFM), super-resolution optical microscopy (SRM), and dark-field optical microscopy (DFM). A focused minireview of scanning electrochemical microscopy (SECM) by Filice and Ding (DOI: 10.1039/c8an01490f) introduces and illustrates applications of non-invasive, high-resolution mapping via electrochemical measurements in and around a cell.

In the emerging area of single-cell resolution MS, a set of reviews provide detail and perspective. First, Yin and co-workers (DOI: 10.1039/c8an01190g) review strategies used by different ionization modes to achieve single-cell analysis, and they provide needed detail on promising and powerful MS approaches (electrospray ionization MS (ESI-MS), secondary ion MS (SIMS), laser-based MS and inductively coupled plasma MS (ICP-MS)).

Next, Kempa and co-workers (DOI: 10.1039/c8an01448e) share specific interest in the impact of MS methods as part of high throughput screening (HTS) in a critical review of coupled HTS and MS workflows with low-to-no sample preparation. Simplicity in sample preparation is considered, as is the ability to provide the analytical sensitivity and efficiency required for single-cell resolution across populations of cells.

A critical review by Liu and co-workers (DOI: 10.1039/c8an01503a) discusses the exciting and daunting breadth of “omics” data needed to understand life- and disease-processes. Here, the authors open the aperture to provide a perspective on how microfluidic technologies are moving us beyond single-cell transcriptomics and genomics to encompass single-cell proteomics and metabolomics.

Diving deep into one specific emerging “omics” area, Gupta and her colleagues (DOI: 10.1039/c8an01525b) provide a minireview focused on understanding the role of lipids in single cells. The authors outline how coherent Raman scattering and image processing algorithms are elucidating the dynamics of lipid composition and the spatial distribution of lipids within a cell. As lipids play critical roles in metabolism but are not explicitly encoded in the genome, the authors discus how direct lipid profiling is a necessary complement to next generation sequencing.

Next, moving into original technical contributions, we see clinical relevance and impact of the cytometry tools of Proctor and Allbritton (DOI: 10.1039/c8an01353e). Here, the authors report on their workhorse chemical cytometry system for enzyme characterization of single cells, using capillary electrophoresis. A critical contribution in this study is that chemical cytometry is now possible with aldehyde cell fixation. By fixing cells prior to chemical cytometry, time-dependent processes are ‘frozen’ in time prior to electrophoresis, thereby reducing analytical variability. Taking sphingosine kinase activity as a case study, the report details performance with cell fixation, as well as comparison to negative controls and demonstration across a range of cell types including leukemia and lymphoma cell lines and primary leukemia cells.

One of our guest editors of this special issue finds the forum well suited to introducing single-cell mobility shift assays. Sinkala, Rosas, and Herr (DOI: 10.1039/c8an01441h) index a surface- and intracellularly-expressed protein target to the originating cellular compartment. The approach concatenates immunofluorescence (for antibody-based tagging of surface antigens) with single-cell native polyacrylamide gel electrophoresis (PAGE). Consequently, when the protein target is located on the cell surface, the species electromigrates slowly during PAGE, as surface expressed target is complexed with a large fluorescent antibody probe from immunofluorescence. In contrast, an intracellular protein target electromigrates more quickly during PAGE, having no bound antibody probe to reduce electrophoretic mobility.

Expanding the repertoire of metrology tools available to characterize cellular phenotype – with clinical relevance – Zhang and colleagues (DOI: 10.1039/c8an02100g) describe a microfluidic channel network that uses a “crossing constriction” geometry to ascertain both membrane capacitance and cytoplasmic conductivity of individual cells. Promising clinical applications are explored by resolving (with statistical significance) low-metastatic carcinoma cells (SACC-83) from high-metastatic carcinoma cells (SACC-LM), among a pooled population of approximately 100 000 cells.

Another clinically relevant, yet distinct and hybrid assay presented by Kim and co-workers (DOI: 10.1039/c8an01904e) combines measurements of magnetically and gravity induced velocity and phosphatidylserine (PS) localization to explore aging in red blood cells. Building on their surprising results, the authors discover a relationship between hemoglobin loss and the amount of PS exposed on the outer wall, leading the research team to posit a possible mechanism for red blood cell breakdown, with the additional possibility that the mechanism may arise from in vitro conditions.

With relevance to measuring the secretome of individual cells, Herrera and her colleagues (DOI: 10.1039/c8an01083h) report quantum dot (QD) signal transduction from sandwich immunoassays to detect cytokines secreted from single cells. The authors utilize two different strategies to enhance signal sensitivity in microwell features, finally showing a case study with detection as low as up to 10 000 soluble cytokine copies per microwell.

Looking forward and looking back, microscopy has played and will continue to play a crucial role in cytometry. Expanding the life sciences applications space for microscopy, Bakir and co-workers (DOI: 10.1039/c8an01591k) report on use of atomic force microscopy and near-field infrared spectroscopy for analysis of fungal cell walls. Use of the two modalities allows the authors to characterize both nanoscale architecture and chemical composition of the cell wall integrity, while applying gene deletion technology to processing of the cell wall component Galf.

Advancing their research in genomics analysis of adherent cells, Negishi and co-authors (DOI: 10.1039/c8an01456f) detail use of their previously reported photopolymerizable hydrogel encapsulation system to isolate individual adherent cancer cells from a typical cell culture dish for subsequent whole genome amplification and sequencing.

Moving us forward in single-cell genomics, Jo and colleagues (DOI: 10.1039/c8an01426d) report direct visualization of a large DNA molecule using red and green DNA-binding fluorescent proteins, thus eliminating the need for DNA amplification. Several fluorescent protein-fused DNA-binding proteins are described.

In perhaps one of the most sought after clinically relevant applications of single-cell spectroscopy, the team of Xu and colleagues (DOI: 10.1039/c8an01437j) apply Raman spectroscopy – with machine learning – to assess the diagnostic potential for chronic fatigue syndrome. Results from patients and healthy controls lead the team to assert that an increase in cellular phenylalanine may be related to mitochondrial/energetic dysfunction and, thus, holds promise as a biomarker for this disease.

Exploring a new tool to track lipid production in genetically modified single yeast cells, Kochan and co-workers (DOI: 10.1039/c8an01477a) apply confocal Raman spectroscopy to both broadly detect and spatially locate cyclopropane fatty acids within cells.

In analysis of microbial communities with clinical and environmental relevance, Weiss and colleagues (DOI: 10.1039/c8an02177e) assessed the feasibility and limitations of surface-enhanced Raman scattering (SERS). Across six different microbial species (an archaeon, two Gram-positive bacteria, three Gram-negative bacteria), the team links the occurrence, intensity, and reproducibility of microbial SERS signals to questions regarding species-specificity of the signals and the role of metabolic activity of individual cells.

To extend the applicability of synchrotron radiation Fourier transform infrared microspectroscopy (SR-microFTIR) to analyses of live cells, the team of Doherty and co-workers (DOI: 10.1039/c8an01566j) reports here the design, fabrication, and application of a cost-effective liquid sample holder. The enhancement allows the team to perform longitudinal analysis of live-cell analysis for up to 24 hours. In one example presented, the new single-cell handling approach allows the team to concurrently induce and monitor biochemical changes.

Couvillion and co-workers (DOI: 10.1039/c8an01574k) outline the challenges faced by single-cell MS-based omics analyses, with particular emphasis on what is needed to realize high throughput single-cell MS.

Duncan and colleagues (DOI: 10.1039/c8an01581c) contribute a minireview, which presents advances over the most recent three years in single-cell MS for metabolomics, highlighting the state-of-the-art and identifying the challenges to move the field forward.

Nemes and Portero (DOI: 10.1039/c8an01999a) present a primary technical contribution of CE-ESI-MS analysis that reports both cationic and anionic metabolites using one-pot microextraction from the aspirate of an 8-cell Xenopus laevis embryo.

Perhaps you will agree that our map reveals wide open space for further innovation, and a multitude of intriguing frontiers for asking and answering important new questions. We thank all of our contributors for sharing their view of the future of single-cell analysis, as well as for sharing original research that underpins the next wave of capability and promise.

Amy E. Herr (University of California, Berkeley, USA), Takehiko Kitamori (University of Tokyo, Japan), Ulf Landegren (Uppsala University, Sweden) and Masood Kamali-Moghaddam (Uppsala University, Sweden).

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