Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method

Abstract

Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories. Here we report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence (CycIF), a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image. Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications.

Highly Multiplexed Single‐Cell Protein Profiling with Large‐Scale Convertible DNA‐Antibody Barcoded Arrays

“…However, this method still does not overcome the limitation of spectrum overlap, and patterning barcode arrays with a pitch < 10 µm in large‐scale is technically challenging. Recently, a few cyclic approaches have been developed to improve the multiplexity, but they have not yet been capable of multiplexed detection of proteins with single‐cell resolution 7, 8, 9…”

Exploiting single-cell quantitative data to map genetic variants having probabilistic effects

“…This requirement also implies using technologies where the throughput of quantitative acquisitions is high. This is the case for flow-cytometry and, although at higher costs, for high-content image analysis [15,16] and digital microfluidics [18,19]. For these practical reasons, it is possible that mouse immunological studies will help making progress in mammalian scPTL mapping.…”

“…Technological developments in high-throughput flow cytometry [13], multiplexed mass-cytometry [14], image content analysis [15][16][17] and droplet-based single-cell transcriptome profiling [18,19] now offer the possibility to estimate empirically the statistical distribution of numerous molecular and cellular single-cell quantitative traits. We therefore propose to scan genomes for variants that modify single-cell traits in a probabilistic manner, which we call single-cell Probabilistic Trait Loci (scPTL).…”

Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry

“…Single-cell chemical profiling could further the current understanding of biological variability and differential susceptibility to disease and treatment, as well as heterogeneity among similar cells (18,19). Most of currently available single-cell techniques require the chemical of interest (usually large molecules, such as DNA, RNA, and proteins) to be labeled, amplified, or electrochemically detectable (4,6,10,12). In addition to these large molecules, small molecules, such as lipids, saccharides, transmitters, and metabolites, play critical roles in cells, including neurons (20).…”

“…single neuron | mass spectrometry | metabolism | patch clamp S ingle-cell genomic, proteomic, and metabolic studies have attracted increasing attention with the development of singlecell technologies, such as flow cytometry (1-3), single-cell PCR (4,5), single-cell RNA sequencing (6)(7)(8), immunofluorescence (9,10), and electrochemistry (11,12). Single-cell analysis has become increasingly important in biology, especially in neuroscience research, because neurons are specialized cells with a huge diversity of shapes, connections, and anatomical, electrophysiological, and biochemical properties (13)(14)(15)(16)(17).…”