Fluorescence staining and confocal laser scanning microscopy study of hydrogen-bonded poly(vinylpyrrolidone)/poly(acrylic acid) film

Poly(vinylpyrrolidone) (PVPON) and poly(acrylic acid) (PAA) were layer-by-layer (LBL) assembled to prepare the thin films based on hydrogen-bonding complexation. The hydrogen-bonded PVPON/PAA films were incubated in acidic, neutral and basic vapors separately. To study the morphologies after incubation, the films were stained by pH-sensitive fluorescent dyes using chemical and physical ways, and investigated with confocal laser scanning microscope (CLSM). The chemical way (labeling) was covalently linking fluoresceinamine (FAM) to some monomer units of PAA while the physical way was adsorbing rhodamine B (RB) molecules from dilute solution. Atomic force microscope (AFM) was combined with CLSM to find that after incubation in neutral or basic vapor the hydrogen-bonded PVPON/PAA films form porous structure and the pores are through the whole film.

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► Hydrogen-bonded films were stained both by chemical and physical methods.
► The stained films were incubated in different vapor environments.
► The films’ morphologies were studied with confocal laser scan microscopy (CLSM).
► CLSM and AFM were combined together to unveil the film's structure after incubation.