Flow Cytometry Innovations: Imaging, Mass, and Spectral Techniques
Author: Mike Blundell | Reviewer: Chloe Fenton
The latest innovations in flow cytometry include imaging flow cytometry, mass cytometry, and spectral flow cytometry. These advanced technologies enable higher-parameter analysis, improved sensitivity, and more detailed insights into cell structure and function. These innovations have expanded the capabilities of flow cytometry across imaging, high-parameter analysis, and multicolor detection.
Flow cytometry has become more accessible to researchers through a reduction in the complexity of instrument set up, true automation, increased sensitivity, and more user-friendly software. The increasing number of fluorophores and antibodies available and improved protocols have also made this technique accessible to an increasing number of researchers. Although multicolor flow cytometry using fluorescent markers is still one of the most powerful tools in research, there are some new innovations.
Imaging Flow Cytometry
Imaging flow cytometry allows capture of images of the particles as they pass through the laser using a CCD camera. Multiple (spectrally different) images can be captured simultaneously, allowing composites to be made and analysis of antigen location to be determined.
Mass Cytometry
Another innovation is mass cytometry. The introduction of multiple laser containing flow cytometers capable of detecting 27 parameters in addition to forward and side scatter, e.g., the ZE5 Cell Analyzer from Bio-Rad, has vastly increased the complexity of fluorescent flow cytometry experiments. Mass cytometry, however, has the capability to detect in 135 channels, allowing multiplex panels to be built, and currently over 40 markers can be measured per cell.
Mass cytometry relies on labeling the samples with antibodies bound to metal isotopes, which can then be measured by analyzing the time each isotope takes to pass through an electric field towards the detector. The larger the isotope the longer it takes. Sample acquisition is slower with mass cytometry and, as the cells are vaporized, only analysis can be performed. However, there are fewer problems with spillover and compensation. Analysis of the sample can, however, be time-consuming and problematic as it requires specialized software due to the number of parameters that can be collected on one cell.
Spectral Flow Cytometry
Spectral flow cytometry captures the full emission spectrum of fluorophores, enabling improved separation of signals and more flexibility in multicolor panel design.
Comparison of Flow Cytometry Innovations
| Technology | Parameters | Strengths | Limitations | Use Case |
|---|---|---|---|---|
| Imaging Flow Cytometry | Moderate | Captures spatial and morphological data | Lower throughput than standard flow | Subcellular localization studies |
| Mass Cytometry | Very high (100+) | Minimal signal overlap, high multiplexing | Cells destroyed during analysis | High-dimensional immune profiling |
| Spectral Flow Cytometry | High | Improved fluorophore separation | Requires advanced analysis tools | Complex multicolor panel design |
Frequently Asked Questions
What is imaging flow cytometry used for?
Imaging flow cytometry is used to capture images of cells during analysis, allowing researchers to study cell morphology and the localization of specific markers.
What is mass cytometry?
Mass cytometry is a technique that uses metal isotope-labeled antibodies to enable very high-parameter analysis, reducing signal overlap compared to fluorescence-based methods.
Which flow cytometry method allows the highest multiplexing?
Mass cytometry currently allows the highest multiplexing capability, with the ability to measure more than 40 markers per cell and detect over 100 parameters.
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