Imaging Flow Cytometry

Essential Features

• Cells in suspension are hydrodynamically focused into a narrow stream and illuminated by an LED array combined with multiple laser lines. This setup enables imaging at rates of several thousand objects per second.
• A standard excitation laser is used, with the option to expand to multiple additional lasers at different wavelengths. Higher-power laser configurations are available for enhanced signal detection.
• The system can capture images across multiple channels, including several fluorescence channels along with brightfield and darkfield images. Dual detection systems with filters and spectral decomposition are used to increase the number of simultaneously acquired images.
• Images can be collected at different magnifications, typically corresponding to submicron pixel resolutions. This flexibility allows detailed visualization of both whole cells and fine subcellular structures.
• An optional extended depth-of-field setting preserves focus across greater cell thickness. This is useful for quantifying features throughout the entire volume of a cell.
• A high-gain configuration enhances the ability to detect weak fluorescence signals. It is particularly effective for very small or dim targets such as extracellular vesicles or viruses.
• User-friendly acquisition software enables instrument calibration, setup, and real-time monitoring of image data. It also supports gating strategies to selectively capture relevant events and reduce unnecessary data.

Applications of Image Analysis with Imaging Flow Cytometers

• Immunology and cell therapy: Imaging cytometry is used to study immune cell interactions, such as antigen-presenting cell binding, immune synapse formation, and CAR-T cell function. It also enables precise measurement of cell death, protein colocalization, and signaling events at the single-cell level.
• Cell biology and drug response: Researchers apply IFC to track processes like autophagy through LC3 clustering, nuclear localization of transcription factors, and cyclin-dependent cell cycle progression. It is also used to quantify apoptotic indices after drug treatments, providing high-content insights into cellular responses.
• Extracellular vesicles and small particle analysis: The technology allows visualization and quantification of extracellular vesicle uptake by cells. Its high sensitivity enables the detection of small or dim structures that may be missed by conventional cytometry.
• Aquatic microbiology and environmental monitoring: The image-based cytometer supports in situ analysis of plankton, bacteria, fungi, and other microorganisms in aquatic ecosystems. It helps detect ecological changes such as harmful algal blooms by monitoring different compartments simultaneously.
• Genotoxicity and toxicology: For assays like the micronucleus test, IFC offers a more accurate and high-throughput alternative to flow cytometry and microscopy. By imaging intact cells, it reduces false positives, enables counting of multiple micronuclei per cell, and supports automated AI-driven scoring for robust statistics.
• Nuclear translocation studies: Image-based flow cytometry enables precise quantification of nuclear translocation by simultaneously capturing fluorescence images of both the nucleus and the protein of interest within intact cells. This allows researchers to measure localization shifts objectively across thousands of cells, providing statistically robust insights into signaling pathways and transcription factor activation.
• Rare event imaging: Imaging flow cytometry is highly effective for rare event imaging, as it combines high-throughput acquisition with detailed imaging of each individual cell. This enables reliable detection and characterization of uncommon populations, such as circulating tumor cells or stem cell subsets, that may be overlooked using conventional methods.

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