CLSM-based 4D biofilm reference for quantitative coupling of biofilm structure and sensor signals

Systematic development and experimental evaluation of empirical, numerical and data-driven models linking biofilm structure to sensor signals are often limited by the lack of biological reference data that can be directly assigned to the measurements. Here, we present a platform that couples a 4D biofilm reference obtained by confocal laser scanning microscopy (CLSM) with time-resolved sensor data. Thermomechanical stabilization, axial cover-glass referencing and sub-voxel reconstruction provide stable axial registration over large fields of view. For each fluorescence marker, the approach yields pixel- and time-resolved maps of interfacial height h(x,y,t), fluorescence intensity I(x,y,t) and axial transition width w(x,y,t). These data provide quantitative reference variables for systematically evaluating sensor descriptors and forward and inverse structure-signal models. Electrochemical impedance spectroscopy (EIS) on planar interdigitated electrodes is used as an illustrative application. The example shows how frequency-dependent signal descriptors can be compared and how identified structure-signal relationships can be tested for transferability. The framework is not restricted to EIS and can be applied to other temporally coupled sensing modalities. Independently of sensor coupling, the 4D reference also supports analyses of biofilm growth, heterogeneity and marker-resolved structural differences.