Depth measurement of molecular permeation using inclined confocal microscopy

by Kenji Kikuchi, Shunsuke Shigeta, Takuji Ishikawa

We report a new technique for the high time-resolved depth measurement of molecular concentration distribution in a permeable hydrogel film with micro-depth precision. We developed an inclined observation technique in a laser-induced fluorescence (LIF) system, based on confocal microscopy, which measures the concentration distribution in the depth direction at less than micrometre intervals. The focal plane of confocal microscopy was tilted to enable simultaneous depth scanning in the microscopic field of view inside the permeable substrate. Our system achieved real-time and non-contact depth measurement of concentration distribution in the permeable hydrogel film. Simultaneous depth concentration measurement was realised with < 1 μm/pixel resolution over a maximum depth range of 570 μm, depending on the tilt angle of the stage and optical conditions. Our system measured the concentration of fluorescence materials based on the fluorescence intensities at several depth positions with a minimum concentration resolution of 1.3 nmol/L. Applying the proposed system to real-time concentration imaging, we successfully visualised unsteady concentration transport phenomena, and estimated the mass transport coefficient through the liquid-hydrogel interface. Our findings are useful for investigating the mass transport of physical, biological, and medical phenomena in permeable substrates.