Single-cell sorting is a powerful and indispensable technology that enables the isolation and analysis of individual cells from heterogeneous populations. By dissecting cellular heterogeneity at the single-cell level, researchers can unravel complex biological processes, identify rare cell types, and better understand disease mechanisms and developmental pathways. Over the past two decades, single-cell sorting technologies have undergone transformative advances, becoming essential tools in immunology, oncology, stem cell biology, neurobiology, and many other fields.

This article provides an in-depth review of the core technologies underlying single-cell sorting, explores their diverse applications, and highlights recent advances pushing the boundaries of single-cell science. The discussion integrates fundamental principles with cutting-edge innovations to offer a comprehensive understanding suitable for researchers and professionals in biomedical sciences.

1.1. The Challenge of Cellular Heterogeneity

Biological tissues and fluids are composed of heterogeneous mixtures of cells with distinct molecular profiles and functional states. Traditional bulk assays average signals across millions of cells, obscuring critical information about rare subpopulations or dynamic cellular responses. Single-cell sorting allows separation of individual cells based on specific phenotypic or molecular characteristics, enabling downstream analyses such as genomic, transcriptomic, proteomic, or functional studies.

1.2. Basic Workflow

Typical single-cell sorting involves three main steps:

• Labeling: Cells are stained or tagged with markers (e.g., fluorescent antibodies) to identify features of interest.
• Detection: Cells flow past optical or electronic sensors to measure the labeled parameters.
• Sorting: Cells matching predefined criteria are physically isolated into collection vessels for further study.

2.1. Fluorescence-Activated Cell Sorting (FACS)

Fluorescence-Activated Cell Sorting is the most widely used technology for high-throughput single-cell sorting. Developed in the 1970s, FACS instruments use hydrodynamic focusing to align cells into a single stream and interrogate each cell by laser-induced fluorescence.

• Key Features:
• Multiparameter detection (fluorescence channels, forward and side scatter)
• Sorting via electrostatic deflection of droplets containing single cells
• Sorting rates up to tens of thousands of cells per second
• Scientific Foundation:
• FACS exploits fluorescence-labeled antibodies targeting cell surface markers or intracellular molecules, enabling the discrimination of cell subtypes based on expression patterns. The technology also uses scatter properties to infer size and granularity.
• Advantages:
• High throughput and speed
• Multiparametric capabilities
• Sorting purity and recovery rates can exceed 95%
• Limitations:
• Requires fluorescent labeling and viable cells
• Instrument complexity and cost
• Potential cell stress due to high pressure and electrostatic deflection

2.2. Magnetic-Activated Cell Sorting (MACS)

Magnetic-Activated Cell Sorting uses magnetic beads conjugated to antibodies that specifically bind target cells.

• Mechanism:
• Cells bound to magnetic beads are retained in a magnetic field while unlabeled cells flow through.
• Applications:
• Rapid enrichment or depletion of cell populations
• Compatible with large sample volumes
• Advantages:
• Gentle separation preserving cell viability
• Scalable and simple
• Limitations:
• Typically low purity compared to FACS
• Limited multiparametric capability

2.3. Microfluidic-Based Sorting

Microfluidic technologies use precisely engineered channels and valves to manipulate and sort single cells in miniaturized devices.

• Techniques Include:
• Droplet microfluidics: encapsulating single cells in droplets for sorting by fluorescence or other signals
• Acoustic sorting: using surface acoustic waves to direct cells
• Dielectrophoretic sorting: applying electric fields to move cells based on dielectric properties
• Advantages:
• Low reagent consumption
• High precision and gentle handling
• Integration with downstream molecular analyses (e.g., single-cell RNA-seq)
• Limitations:
• Lower throughput compared to FACS
• Complexity in device fabrication

2.4. Optical Tweezers and Laser Capture Microdissection

While less common for high-throughput sorting, these techniques allow isolation of single cells or small groups from tissue sections or culture.

• Optical Tweezers: Use focused laser beams to trap and move individual cells.
• Laser Capture Microdissection (LCM): Uses laser pulses to excise cells of interest from tissue slices.
• Applications: Targeted isolation from complex tissues.

3.1. Immunology and Hematology

• Identification and isolation of immune cell subsets such as T cell subsets, B cells, dendritic cells.
• Studying clonal expansion, activation states, and antigen specificity.
• Single-cell sorting underpins flow cytometry–based diagnostics and vaccine research.

Example: Sorting of antigen-specific T cells using tetramer staining for downstream functional assays.

3.2. Cancer Research

• Isolation of rare circulating tumor cells (CTCs) from blood for molecular characterization.
• Sorting cancer stem cells based on surface markers.
• Understanding intratumoral heterogeneity and drug resistance.

Example: Enrichment of CD44^high/CD24^low breast cancer stem-like cells to study metastasis.

3.3. Stem Cell Biology and Regenerative Medicine

• Isolation of embryonic stem cells, induced pluripotent stem cells (iPSCs), and adult stem cells.
• Sorting for differentiation state markers to purify progenitor populations.
• Quality control of cell products in cell therapy manufacturing.

3.4. Neuroscience

• Sorting neuronal subtypes based on molecular markers.
• Isolation of glial cells, microglia, or oligodendrocytes for transcriptomic studies.

3.5. Microbiology

• Sorting individual bacteria or microbial cells based on fluorescent probes.
• Single-cell genomics of unculturable microbes.

4.1. Integration with Multi-Omics

Single-cell sorting is increasingly integrated with multi-omics workflows that combine transcriptomics, epigenomics, proteomics, and metabolomics from the same cell.

• Technologies such as CITE-seq use antibody-derived tags enabling simultaneous protein and RNA quantification.
• Sorted single cells feed into next-generation sequencing or mass spectrometry.

4.2. High-Dimensional Cytometry and Spectral Flow Cytometry

• Conventional FACS instruments are limited by spectral overlap in fluorescent dyes.
• Spectral flow cytometry uses full emission spectra and computational unmixing, enabling >40 simultaneous markers.

4.3. Automated and High-Throughput Platforms

• Robotic cell sorters automate sample processing, improving reproducibility.
• Microfluidic droplet sorters can isolate thousands of cells per minute with reduced reagent volumes.

4.4. Label-Free Sorting Techniques

• Techniques based on intrinsic cell properties such as size, deformability, or autofluorescence enable sorting without labels.
• Advances include real-time deformability cytometry and digital holographic microscopy.

5.1. Minimizing Cellular Stress and Viability Loss

High pressure, laser exposure, and electrostatic forces during sorting can induce stress or apoptosis. Innovations aim to reduce these impacts to preserve cell function.

5.2. Sorting Rare and Fragile Cell Types

Enhanced sensitivity and gentle sorting methods are needed to isolate extremely rare populations such as circulating tumor cells or fragile neuronal cells.

5.3. Integration with In Situ Technologies

Combining sorting with spatial transcriptomics and imaging techniques allows correlation of molecular profiles with tissue architecture.

5.4. Single-Cell Sorting in Clinical Diagnostics

Single-cell sorting is becoming integral to precision medicine, e.g., isolating minimal residual disease cells in leukemia, or CTCs for personalized oncology.

Single-cell sorting is a cornerstone technology that continues to evolve with innovations in optics, microfluidics, and molecular biology. Its capacity to dissect cellular heterogeneity has transformed biomedical research and opened avenues for diagnostic and therapeutic advances. As technologies become more accessible, multiparametric, and integrated with omics platforms, single-cell sorting will further illuminate the cellular complexity underlying health and disease.