Depth measurement of molecular permeation using inclined confocal microscopy.

Kenji Kikuchi,Takuji Ishikawa,Shunsuke Shigeta,Thomas Abraham

doi:10.1371/journal.pone.0214504

Abstract

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.

Highlights

The transport of solvent molecules through a passively permeable material is a fundamental and important dispersion phenomenon in biology, chemistry, and fluid dynamics
We propose a novel approach for the measurement of mass transport in the depth direction via a real-time non-invasive method with micro-scale depth resolution using confocal microscopy with geometrically inclined observation
The fluorescence intensity was produced by a convolution between the intensity distribution of the optical section thickness (OST) and the spatial distribution of the fluorescence molecules

Summary

Introduction

The transport of solvent molecules through a passively permeable material is a fundamental and important dispersion phenomenon in biology, chemistry, and fluid dynamics. Brownian diffusion is governed by Fick’s law, which shows that the concentration flux is proportional to the negative spatial gradient of the concentration and diffusion coefficient of a molecule. In the case of high Peclet number, i.e. the advective velocity exceeds diffusive velocity, and the molecules are rapidly transported by the advective velocity rather than by the diffusive velocity [1, 2]. In the case of low Peclet number, diffusion is more dominant than advection. Concentration, or pH distribution is inhomogeneous, the scalar transport.

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Discussion

Conclusion