Imaging Flow Cytometry (IFC) combines the best of two worlds: the morphological resolution of microscopy with the statistical robustness of traditional flow cytometry. This advanced technique enables the analysis of individual cells at high throughput while capturing high-quality images to evaluate both cellular phenotypes and functional processes.
In the context of CAR-T therapies and tumor studies, IFC has become an indispensable tool to identify immunological synapses, study the co-localization of granzymes and perforin, and monitor degranulation markers.
The formation of an immunological synapse between a CAR-T cell and a tumor cell is a critical event determining recognition and cytotoxic efficacy. IFC allows researchers to:
Visualize the direct interaction between effector and target cells.
Measure polarization of cytotoxic granules toward the tumor cell.
Quantify adhesion and membrane reorganization events preceding cytotoxic mediator release.
By acquiring thousands of single-cell images, IFC enables statistical evaluation of these interactions. Which is difficult to achieve with conventional confocal microscopy.
A key aspect of CAR-T and NK cell function is the release of granzymes and perforin, essential proteins for inducing apoptosis in target cells. IFC provides the ability to:
Detect granzymes and perforin within cytotoxic granules.
Analyze their co-localization and polarization toward the immunological synapse.
Distinguish activated from non-activated cells based on morphological and functional patterns.
This multiparametric analysis provides a functional readout of cytotoxic activity, complementing conventional flow cytometry, which only measures total protein expression without spatial context.
Markers such as CD107a/LAMP1 allow monitoring of cytotoxic lymphocyte degranulation. IFC offers key advantages:
Identification of effector cell subpopulations actively releasing granules.
Correlation of degranulation with granule polarization and immunological synapse formation.
Analysis of functional heterogeneity of immune responses at the single-cell level.
IFC combines the quantitative robustness of FACS with the spatial resolution of confocal microscopy, offering a unique platform to study complex morphofunctional processes in cellular therapies and tumor microenvironments. Unlike confocal microscopy, IFC allows high-throughput analysis of thousands of cells, and unlike conventional FACS, it provides spatial and co-localization information.
Imaging Flow Cytometry has established itself as a key tool in CAR-T, NK, and tumor microenvironment research. IFC can analyze immunological synapses, granzyme and perforin co-localization, and degranulation markers, offering a robust functional assessment. This approach provides statistically meaningful data on morphofunctional features at the single-cell level. IFC not only complements but also surpasses the limitations of confocal microscopy and traditional FACS analysis. These advantages create new opportunities to optimize cellular therapies and study immune response heterogeneity.