In cellular research, timing is everything. The ability to observe biological processes as they happen has opened a new chapter in the study of physiology and pathology.
Thanks to the combination of flow cytometry and real-time fluorescence, researchers can now track, quantify, and analyse dynamic cellular events with unprecedented temporal and spatial resolution.
Far from being just an extension of traditional cytometry, this technology has become a strategic tool for understanding how cells respond to stimuli, interact with others, and adapt to their environment
Real-time fluorescence is a technique that uses fluorescent probes sensitive to specific changes inside or on the surface of the cell. These probes emit light at a defined wavelength when excited by a laser, and this emission is detected and recorded in real time.
In flow cytometry, this means that each cell passing through the stream can be analysed at the very moment the event of interest occurs, for example:
Intracellular calcium fluxes following cell activation.
Alterations in mitochondrial membrane potential during apoptosis.
Production of reactive oxygen species (ROS) under cellular stress.
For a more comprehensive technical overview, you can consult the Molecular Probes® handbook on fluorescent probes.
High-precision temporal resolution
Detects changes occurring within milliseconds – impossible with static methods.
Closer-to-physiology analysis
Avoids the use of fixatives or destructive processes that alter cell biology.
More information per experiment
Dynamic signals can be correlated with phenotypic and functional markers measured simultaneously.
Versatility of applications
From basic cell biology research to advanced therapeutic evaluation, such as CAR-T cells or immune modulators.
Functional immunology: monitoring the activation of T and NK lymphocytes in response to antigens or target cells.
Cellular pharmacology: analysing the impact of a drug on intracellular signalling in real time.
Translational oncology: monitoring the efficacy of CAR-T cells against tumour cells during direct interaction.
Neuroscience: studying changes in calcium signalling in neurons and glial cells.
Implementing real-time fluorescence in a flow cytometry workflow requires:
Stable, fast-kinetic markers: to capture fleeting changes without signal loss.
Optimised instrumentation: lasers and detectors with high acquisition speed.
Advanced temporal analysis software: able to manage and visualise data such as kinetic curves and multiparametric graphs.
Real-time fluorescence applied to flow cytometry allows us not only to see the what and the how much, but also the when and how cellular processes occur.
This temporal dimension adds immeasurable value for:
Designing more specific drugs.
Understanding therapeutic resistance mechanisms.
Developing more effective immunotherapies.
With the integration of artificial intelligence and high-throughput multiparametric analysis, the future points to systems capable of predicting cellular behaviour based on dynamic signals.
Tracking cellular events dynamically through real-time fluorescence and flow cytometry represents a paradigm shift in biomedical research. It allows us to observe cell life without pauses or cuts, just as it occurs in its natural context.
To learn more about how to implement this technology in your laboratory, visit our advanced flow cytometry solutions.