CyTOF
CyTOF, also known as cytometry by time-of-flight or mass cytometry, is a single-cell analysis technology that combines the hydrodynamic focusing of flow cytometry with inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) to simultaneously measure dozens of cellular parameters using stable metal isotope tags. In this technique, cells are labeled with antibodies conjugated to distinct metal isotopes, typically from the lanthanide series, which are then vaporized into ions and analyzed based on their mass-to-charge ratio, allowing for high-resolution detection without the spectral overlap inherent in fluorescence-based methods.[1] This enables the quantification of up to 50 or more protein markers per cell, far exceeding the 10–20 parameters typical of conventional flow cytometry.[2] The technology was pioneered in the late 2000s by researchers at DVS Sciences, including Scott D. Tanner, Vladimir I. Baranov, and Dmitry R. Bandura, who published the first prototype description in 2009, adapting ICP-MS principles originally commercialized in the 1980s for biological applications. Following its introduction, DVS Sciences was acquired by Fluidigm (renamed Standard BioTools in 2022 and merged with SomaLogic in 2024) in 2014, leading to commercial instruments like the CyTOF2 and Helios systems, which process up to 1,000 cells per second with a detection efficiency of around 30%.[1] [3] [4] Key advancements include the development of metal-chelating polymers for stable antibody conjugation and intercalators like iridium for viability and DNA staining, enhancing data quality and throughput. CyTOF has revolutionized fields such as immunology, oncology, and hematology by facilitating deep phenotyping of immune cell subsets, tumor microenvironments, and signaling pathways in complex tissues.[2] Notable applications include mapping hematopoietic differentiation hierarchies and profiling drug responses in cancer immunotherapy, where its ability to handle rare cell populations (down to 10^6–10^7 total cells analyzed) provides quantitative, high-dimensional insights unattainable with traditional cytometry.[1] Compared to fluorescence cytometry, CyTOF offers superior multiplexing without compensation artifacts, though it requires specialized sample preparation and generates large datasets necessitating advanced computational tools like t-SNE or FlowSOM for analysis.[5] Ongoing developments, including imaging mass cytometry (IMC) for spatial analysis and expanded reagent libraries, continue to broaden its utility in translational research as of 2025.[6]Introduction
Definition and Scope
CyTOF, or Cytometry by Time-Of-Flight, is a proprietary mass cytometry platform developed by DVS Sciences—now integrated into Standard BioTools—that facilitates flow-based analysis of single cells through time-of-flight mass spectrometry. This system represents a specific implementation of mass cytometry technology, trademarked by Standard BioTools Canada Inc., designed for high-throughput, multiplexed detection of cellular components at the individual cell level.[7][8][9] At its core, CyTOF combines the hydrodynamic focusing and cell interrogation principles of traditional flow cytometry with inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) to enable elemental ion detection. Cells are labeled with probes conjugated to distinct stable metal isotopes, which are ionized in a plasma torch and analyzed based on their mass-to-charge ratio, providing quantitative data on biomarker expression without the optical limitations of fluorescence-based assays. This integration allows for precise, real-time measurement of transient ion signals from individual cells passing through the instrument at rates up to thousands per second.[7] The scope of CyTOF is centered on single-cell proteomics and phenotyping, supporting the simultaneous analysis of over 50 protein markers per cell using rare earth metal tags, such as lanthanides, to achieve high-dimensional resolution of cellular heterogeneity. This capability is particularly valuable in immunology, oncology, and translational research, where it enables deep profiling of surface and intracellular targets without spectral overlap or compensation requirements. Notably, "CyTOF" specifically denotes the commercial instrument lineup, including models like the Helios, XT, and XT PRO systems (as of 2024). Recent models, such as the CyTOF XT PRO introduced in 2024, further enhance automation and sample throughput for clinical applications.[10][11][12]Basic Principles
CyTOF, or cytometry by time-of-flight, relies on the conjugation of biological probes, such as antibodies, to stable isotopes of rare-earth lanthanide elements for multiplexed detection of cellular targets. These isotopes, spanning the mass range of 139 to 176 atomic mass units (amu), are attached to antibodies via chelating polymers, enabling specific binding to proteins or other markers on or within cells while minimizing natural background interference due to the rarity of these elements in biological samples.[1] This metal-tagging approach supports simultaneous measurement of dozens of parameters per cell, far exceeding the limitations of traditional fluorescence-based cytometry.[7] Labeled cells are aerosolized and injected into an inductively coupled argon plasma, where they are rapidly vaporized and atomized at temperatures of approximately 6000–8000 K, producing a cloud of singly charged atomic ions proportional in number to the abundance of each metal tag.[13] The high temperature ensures complete dissociation of cellular material into free atoms, which are then ionized primarily to the +1 charge state, facilitating downstream mass analysis without molecular fragmentation complicating the signal.[7] The ion cloud is extracted into a time-of-flight (TOF) mass spectrometer, where ions are accelerated by an electric field of voltage V and separated based on their mass-to-charge ratio (m/z) according to their flight times over a fixed path length L. The flight time t for an ion is given by t = \sqrt{ \frac{m L^2}{2 z e V} } where m is the ion mass, z is the ion charge (typically 1 for atomic ions), and e is the elementary charge; lighter ions arrive at the detector faster than heavier ones, generating a time-resolved mass spectrum.[14] In practice, the relationship is calibrated as t = t_0 + A \sqrt{m/z}, with constants t_0 and A determined empirically.[13] CyTOF systems achieve a mass resolution >500 m/z units (full width at half maximum), with values exceeding 900 in standard operation, sufficient to resolve adjacent lanthanide isotopes with minimal overlap, typically less than 1%.[7] This high resolution ensures discrete detection channels for each isotope, eliminating the spectral spillover inherent in fluorophore-based flow cytometry, where overlapping emission spectra require compensation matrices and limit multiplexing to around 20 parameters.[7]History
Early Development
The foundational research leading to cytometry by time-of-flight (CyTOF) began in 1994, when Tsutomu Nomizu and colleagues at Nagoya University explored the application of inductively coupled plasma (ICP) atomic emission spectrometry to analyze biological particles, successfully determining calcium content in individual yeast and mammalian cells by introducing cell suspensions into the plasma for time-resolved measurements.[15] This work established the feasibility of using plasma-based ionization for single-cell elemental analysis, overcoming challenges in sample introduction and detection sensitivity for microscopic biological entities.[15] A major breakthrough occurred in 2007, when Scott Tanner's team at the University of Toronto demonstrated the potential of combining flow cytometry with ICP time-of-flight mass spectrometry (ICP-TOF-MS) for single-cell analysis, using metal isotopes conjugated to antibodies as labels to enable multiplexed biomarker detection without spectral overlap issues inherent in fluorescence methods. This innovation addressed key limitations in traditional cytometry by leveraging the high resolution and dynamic range of mass spectrometry for simultaneous measurement of dozens of parameters per cell. The technique's viability was rigorously validated in a seminal 2009 publication by Dmitry R. Bandura and colleagues, including Tanner, along with Vladimir I. Baranov and Olga I. Ornatsky, in Analytical Chemistry, which described the prototype mass cytometer and demonstrated its capacity for up to 60-plex analysis, with potential for over 100 parameters through stable metal isotope tagging.[7] Central challenges, such as vaporizing and ionizing cells in the ICP without fragmentation while preserving intact ion clouds from single cells, were resolved via optimized nebulization and plasma conditions that achieved detection efficiencies suitable for rare event analysis.[7] Proof-of-concept experiments in this study utilized human leukemia cell lines labeled with lanthanide-tagged antibodies, enabling multiplexed detection of surface and intracellular markers to distinguish cell subtypes with high specificity and minimal background interference.[7] These efforts marked the transition from conceptual elemental mapping to practical immunophenotyping, setting the stage for CyTOF's broader adoption in cellular research.[7]Commercialization and System Iterations
The commercialization of cytometry by time-of-flight (CyTOF) began with DVS Sciences, a Toronto-based company founded in 2004 to develop mass cytometry technology, which unveiled the first CyTOF system in November 2009 as the inaugural commercial platform combining flow cytometry principles with inductively coupled plasma mass spectrometry for multiparametric single-cell analysis.[16][17] In February 2014, Fluidigm Corporation acquired DVS Sciences for approximately $207.5 million in cash and stock, integrating the CyTOF platform into its portfolio of single-cell analysis tools and enabling broader market expansion for high-parameter protein profiling.[9] Following the acquisition, Fluidigm introduced the Helios system in June 2015 as an advanced CyTOF iteration, supporting up to 45 or more parameters per cell through expanded metal isotope conjugation capabilities.[18] The Helios upgrade, often referred to as CyTOF 3.0 in subsequent installations starting around 2017, improved event processing for sustained acquisition rates of approximately 500 events per second, facilitating deeper immune system phenotyping in research settings.[19] Fluidigm continued iterating on the platform, releasing the CyTOF XT in May 2021 as a next-generation system with automated sample barcoding and multiplexing capabilities for up to 50 samples per run, reducing manual handling and enhancing reproducibility in high-throughput studies.[20] In April 2022, Fluidigm rebranded to Standard BioTools Inc. following a $250 million strategic capital infusion from investors Casdin Capital and Viking Global Investors, aimed at accelerating innovation in proteomics and mass cytometry technologies.[3] Standard BioTools launched the CyTOF XT Pro in March 2025, featuring enhanced sensitivity via optimized ion optics and quadrupled throughput at up to 2,000 events per second, alongside 21 CFR Part 11 compliance for regulated clinical trial environments.[12][21] Key technological iterations across CyTOF models are summarized below, focusing on parameter capacity and event acquisition rates, which have evolved to support increasingly complex single-cell analyses while maintaining data integrity.| Model | Release Year | Maximum Parameters | Sustained Event Rate (events/sec) |
|---|---|---|---|
| CyTOF 1 | 2009 | ~20 | 300–500 |
| Helios (CyTOF 3.0) | 2015/2017 | 45+ | 500 (peak up to 2,000) |
| CyTOF XT | 2021 | 50+ | 500 |
| CyTOF XT Pro | 2025 | 50+ | 2,000 |
Instrumentation
Core Components
The core components of a CyTOF system form the foundation for single-cell analysis by integrating sample introduction, plasma-based ionization, ion manipulation, and detection within a high-vacuum environment. The process begins with the nebulizer and injector, which aerosolize the cell suspension into fine droplets using a concentric glass nebulizer driven by argon gas flow, enabling the introduction of 200-300 cells per second to ensure single-cell resolution without clustering.[23][7] Central to atomization is the argon plasma torch, configured as an inductively coupled plasma (ICP) source operating at 1-1.5 kW radio-frequency (RF) power from a 40 MHz generator, which sustains a high-temperature plasma (approximately 5,000–10,000 K) to vaporize, atomize, and ionize the desolvated cells within its three concentric quartz tubes.[7][13] Ions generated in the atmospheric-pressure plasma are then extracted through a multi-aperture vacuum interface into the mass analyzer region. Ion selection and separation occur via the quadrupole ion guide and time-of-flight (TOF) analyzer; the RF quadrupole acts as a high-pass filter to remove low-mass interferents (below m/z 80), directing the relevant metal isotope ions toward the dual-stage orthogonal acceleration reflectron TOF analyzer, which sequences ion clouds at 76.8 kHz push frequency for mass-to-charge resolution exceeding 500 (FWHM at m/z 159).[7] The detector, a discrete dynode electron multiplier, captures arriving ions and amplifies the signal for discrete counting, processed through a high-speed digitizer to yield quantitative isotope intensities per cell event.[7] The entire ion flight path operates under a multi-stage vacuum system, with turbo-molecular pumps maintaining pressures around 10^{-6} Torr in the TOF analyzer to minimize ion scattering and ensure precise time-of-flight measurements.[7] Overall system flow proceeds sequentially from sample aerosolization in the nebulizer, desolvation in a heated spray chamber, plasma atomization in the torch, ion extraction and filtering, TOF separation, and final detection, as illustrated in standard schematics of the instrument architecture.[7] This hardware configuration leverages ICP ionization principles to enable metal-tagged antibody detection without spectral overlap.[7]Technical Specifications
CyTOF systems support the simultaneous measurement of up to 50 markers per cell, in addition to dedicated channels for viability assessment and sample duplexing, such as iridium (191Ir/193Ir) DNA intercalators for nucleated cell identification and cisplatin for viability assessment.[12][2] The available detection channels span 135 distinct masses in the range of 75 to 209 atomic mass units (amu), primarily utilizing lanthanide isotopes conjugated to antibodies for multiplexed labeling without spectral overlap.[11] Throughput in CyTOF instruments typically ranges from 300 to 500 cells per second in standard configurations, enabling the analysis of millions of cells per run; the advanced CyTOF XT Pro model achieves up to 2000 events per second through optimized sample uptake rates of 30 µL/min and higher cell concentrations.[24][25] Sensitivity is defined by a detection limit of approximately 100 to 500 metal-tagged antibodies per cell, with signal-to-noise ratios exceeding 100:1 when using isotopes in the optimal mass range of 153 to 176 amu.[26][27] Mass resolution, measured as full width at half maximum (FWHM), is 300 to 600 at m/z ≈ 160, sufficient to resolve adjacent isotopes like ¹⁴³Nd from ¹⁴⁴Nd; in flow cytometry mode, spatial resolution is at the single-cell level (≈10-15 µm). Instruments operate on a power supply of approximately 5 to 7 kW (via a 30 A, 220-240 V AC circuit) and feature a compact benchtop design with a footprint of about 1 m × 1 m and height up to 1.35 m.[13][11] The following table compares key specifications across major CyTOF models:| Model | Maximum Parameters | Event Rate (cells/sec) | Mass Range (amu) | Footprint (width × height) |
|---|---|---|---|---|
| Helios | Up to 50 | 250-500 | 75-209 | ≈1 m × 1.35 m |
| CyTOF XT | Up to 50 | Up to 500 | 75-209 | 0.93 m × 1.35 m |
| CyTOF XT Pro | 50+ | 500-2000 | 75-209 | ≈0.93 m × 1.35 m |