Measuring Sodium Without Relying on Fluorescence Intensity
Quantifying intracellular sodium in intact brain tissue is technically difficult. Intensity based measurements with chemical indicators can be affected by differences in dye loading, local dye concentration, bleaching, and optical conditions.
In their 2026 Nature Communications study, Meyer et al.1 therefore used fluorescence lifetime imaging (FLIM) of the sodium sensitive indicator ING 2. The fluorescence lifetime of the dye changes with sodium concentration and is largely independent of the amount of indicator present. By calibrating this relationship, the recorded lifetime values could be converted into quantitative sodium concentrations. Multiphoton excitation at 840 nm enabled measurements within acute mouse brain slices and in the cortex in vivo. The lifetime data were recorded using time correlated single photon counting, providing spatially resolved measurements from astrocyte somata and processes.
Astrocytes Do Not Share a Uniform Sodium Baseline
In hippocampal tissue slices, the mean somatic sodium concentration was approximately 14 mM. This average, however, concealed a broad distribution ranging from about 2 to 46 mM. The distribution was best described by two populations centered near 9 and 17 mM. This indicates that astrocytes cannot be treated as a single functionally uniform population based on their sodium homeostasis.
Sodium Gradients Within Individual Astrocytes
The differences were also present within individual cells. Astrocyte processes contained higher sodium concentrations than the corresponding somata, and sodium levels increased with distance from the cell body. Processes located 15 to 25 µm from the soma showed substantially higher average concentrations than processes located closer to it. Individual branches of the same astrocyte could also differ considerably.
These observations are particularly relevant because astrocyte processes interact directly with synapses and contain high densities of sodium dependent glutamate transporters. Local sodium concentrations may therefore reflect the specific transport demands of the surrounding neural network.
Connecting Sodium Levels to Astrocyte Function
The researchers combined dynamic FLIM measurements with pharmacological experiments, molecular staining, and biophysical modeling.
Astrocytes with higher baseline sodium concentrations showed stronger sodium changes when the activity of the Na⁺/K⁺ ATPase was altered. They also displayed a stronger response to elevated extracellular potassium, suggesting a greater capacity for potassium uptake through the pump. RNAscope and immunohistochemistry revealed different spatial expression patterns of the β1 and β2 subunits of the Na⁺/K⁺ ATPase. The modeling results showed that differences in pump composition, pump expression, and sodium influx could reproduce both the cellular and subcellular sodium distributions observed experimentally.
These findings support a model in which astrocytes and their processes are locally adapted to different homeostatic requirements. Sodium concentration is therefore not simply a stable background parameter. It is linked to the capacity of individual astrocytes to regulate ions and neurotransmitters in their immediate environment.
Why Quantitative FLIM Was Essential
By calibrating the fluorescence lifetime of ING 2 against sodium concentration, the researchers could convert lifetime values into quantitative intracellular Na⁺ measurements. The TCSPC based acquisition provided the photon resolved lifetime data required for analyzing cells and subcellular regions across tissue slice and in vivo experiments. To ensure reliable lifetime fitting, only regions containing more than 2,000 photons and at least five photons per pixel and frame were included in the analysis. These criteria were especially important for weakly fluorescent astrocyte processes.
Instrumentation Used in This Study by PicoQuant
PicoQuant’s LSM Upgrade Kit adds time resolved detection, TCSPC acquisition, and dedicated analysis capabilities to an existing laser scanning microscope. It provides a modular route to quantitative FLIM while retaining the microscope’s established scanning optics and excitation platform. In this study, the upgrade enabled multiphoton fluorescence lifetime measurements of the sodium sensitive indicator ING 2 in acute brain slices and in vivo.
The configuration used in the study included:
- PMA Hybrid Photomultiplier Detector: The spectrally separated ING 2 and SR101 fluorescence signals were detected with single photon sensitivity. A cooled PMA Hybrid 40 detector was used for the in vivo ING 2 measurements.
- MultiHarp 150 Multichannel Time Tagging & TCSPC Unit: The TCSPC unit recorded photon arrival times together with the microscope scanning signals, enabling pixel resolved fluorescence lifetime acquisition in tissue slices and in vivo.
- SymPhoTime 64: The fluorescence lifetime imaging and correlation software was used for FLIM acquisition and analysis. Amplitude weighted fluorescence lifetimes were calculated using rapid reconvolution and converted into intracellular sodium concentrations through a calibrated response function.
In this configuration, the existing multiphoton microscope provided excitation and spatial scanning, while the LSM Upgrade Kit added photon detection, timing, and analysis workflow required for quantitative sodium imaging. This allowed the researchers to extend an established microscope platform to functional FLIM measurements without replacing the core imaging system.
Quantitative FLIM for Functional Neuroscience
By resolving sodium concentrations across large cell populations and within individual astrocyte processes, the researchers identified a previously hidden level of functional organization in the brain.
The results show that quantitative FLIM can move beyond the visualization of cellular structure. When combined with an appropriate indicator and a calibrated TCSPC workflow, fluorescence lifetime becomes a direct readout of intracellular physiology.
1Reference: Jan Meyer, Viola Bornemann, Alok Bhattarai, Sara Eitelmann, Petr Unichenko, Simone Durry, Karl W. Kafitz, Nicholas Chalmers, Jianfeng Fan, Ruth Beckervordersandforth, Christian Henneberger, Ghanim Ullah & Christine R. Rose. Cellular and subcellular heterogeneity of astrocytic Na⁺ homeostasis tuning astrocytes into functionally distinct subgroups in the mouse brain. Nature Communications 17, 4515 (2026). DOI: 10.1038/s41467-026-73435-z.
Explore the PicoQuant LSM Upgrade Kit and learn how TCSPC based FLIM can be integrated into existing laser scanning microscopes.
