The FluorescenceModule extends real-time deformability cytometry (RT-DC) by enabling synchronized fluorescence detection within the microfluidic measurement channel.
Molecular marker identification and intrinsic mechanical characterization are recorded simultaneously for every single cell — without altering the hydrodynamic measurement conditions.
Fluorescence-based flow cytometry is widely used to identify cell populations via established surface markers. Real-time deformability cytometry enables label-free quantification of intrinsic mechanical properties such as deformation, size, and morphology.
The FluorescenceModule integrates both approaches into a single measurement region, allowing synchronized acquisition of fluorescence intensity and mechanical parameters at high throughput.
This enables:
Identification of defined cell populations within heterogeneous samples
Direct correlation of fluorescence markers with mechanical phenotype
Functional immune profiling without physical pre-sorting
Translational mechanophenotyping of blood-derived cells
A confined excitation light sheet is projected across the microfluidic channel. Cells pass through a narrow illumination curtain, enabling one-dimensional fluorescence imaging along the flow direction.
The fluorescence signal is detected by a dedicated detector array and synchronized with the RT-DC measurement.
Key characteristics:
Up to three excitation lasers (typically 488 nm, 561 nm, 640 nm)
Up to three independent detection channels
Real-time fluorescence analysis (>100 cells per second)
Minimal bleaching outside the excitation zone
Temporal resolution down to 0.5 µs for 1D fluorescence imaging
This optical configuration preserves the mechanical measurement integrity while adding molecular specificity.
Optical configuration of the FluorescenceModule. Excitation lasers are shaped into a confined light sheet across the microfluidic channel. Emitted fluorescence is detected by a multi-channel detector array and synchronized with mechanical measurements in RT-DC.
Published in: Rosendahl et al., Nature Methods (2018) → View publication
Simultaneous fluorescence detection allows identification of cell types based on established surface markers while mechanical parameters are extracted in parallel.
For each detected event, deformation, size, brightness and fluorescence intensity are stored and analyzed in real time.
This enables:
Immune cell profiling without marker-based cell sorting
Stem cell characterization with mechanical readout
Comparative analysis of mechanical biomarkers across defined subpopulations
Simultaneous fluorescence detection and mechanical phenotyping. Surface-marker-defined cell populations (e.g., CD3⁺ T-cells, CD34⁺ stem cells, CD14⁺ monocytes) are identified in parallel with deformation and size measurements, enabling direct correlation of molecular identity and mechanical phenotype.
Published in: Rosendahl et al., Nature Methods (2018) Supplementary Figure 5 → View publication
Because fluorescence intensity is recorded as a temporal profile while cells traverse the excitation sheet, the shape of the detected peak reflects the spatial distribution of labeled structures within the cell.
This allows discrimination between:
Membrane-localized signals
Cytoplasmic labeling
Nuclear or chromatin-associated fluorescence
For example, fluorescently labeled histones produce distinct signal patterns depending on cell-cycle stage, enabling correlation of intracellular organization with mechanical phenotype.
The method provides subcellular insight in flow without full 2D fluorescence microscopy.
One-dimensional fluorescence imaging in flow. As cells traverse the confined light sheet, the temporal fluorescence signal reflects the spatial distribution of labeled intracellular structures. Narrow or broadened peaks correspond to distinct subcellular localization patterns.
Published in: Rosendahl et al., Nature Methods (2018) Supplementary Figure 8 → View publication
Fluorescence detection is fully integrated into the Shape-In acquisition software.
The system enables:
Definition of measurement parameters and acquisition conditions
Real-time gating of fluorescence-defined subpopulations
Simultaneous storage of image data, deformation parameters and fluorescence signals
Direct export of synchronized mechanical and molecular datasets
Subpopulations can be identified and analyzed during acquisition without interrupting the measurement workflow.
The FluorescenceModule is configurable according to experimental requirements.
Configuration options include:
1–3 excitation lasers (customer selectable)
1–3 detection channels
Laser safety integration compliant with EN 60825-1 (Class 1 system)
Upgrade option for existing AcCellerator systems
Installation, calibration and user training included
The FluorescenceModule is commercially available as an add-on for the AcCellerator platform or as part of new system configurations.
The price for the highest technical configuration (3 Lasers, 3 Detection channels) is 148 000 €.
(price only for business customers from academia within Europe, excl. VAT)
