Multiphoton microscopy is a promising technique to detect spatially and temporally resolved concentration gradients of chemical compounds, e.g., reactants in hydrogel-encapsulated biocatalysts. In contrast to current techniques, the improved spatial and temporal resolution of this method in data acquisition and its ability to measure hydrogel beads facilitates the identification of various kinetic phenomena. To our knowledge, multiphoton microscopy is used here for the first time to examine diffusion, mass transfer, and reaction in immobilized hydrogel systems. In a first step, the phenomena of diffusion and diffusion-coupled mass transfer through the phase interface are investigated in the bead center. Finally, the complete system—consisting of diffusion, mass transfer, and enzymatic reaction—is observed by measuring concentration gradients along the bead radius with temporal and spatial resolution. This metrology enables a subsequent mechanistic model identification, which in turn leads to an enhanced knowledge of reaction kinetics and supports the design of biotechnological processes. This task was only possible due to excellent spatial (25 μm) and temporal (5 s) resolution and the accuracy (±1%) achieved by using a multiphoton microscopy setup.

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