Cellulose (2021) 28:5775–5791
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Fig. 2 Schematic illustration of the impregnation process of paper samples with the copolymer dissolved in three different solvents H 2 O, 2-propanol (IPA) and 1-butanol (BuOH)
In brief, (1) a PTFE (Teflon)-dish was filled with the given copolymer solution (H 2 O, IPA or BuOH) and the pre-weighed paper samples were submerged one at a time. A petri dish on top (of the Teflon-dish) was used to prevent large amount of solvent evapo- rating and thus changing the concentration over time. After at least 60 s, the paper samples were pulled out and laid flat on a 3D-printed frame with Teflon yarn (see supporting information for details) for drying overnight under climate-controlled conditions. (2) After equilibration and consecutive weighing of the impregnated paper samples, ambient dried samples were pressed with a defined weight ( * 15 kg) for about four hours to ensure flat samples and a uniform UV-excitation. By illumination of the paper samples with UV-light, the benzophenone groups were excited and thus the cross-linking of the copolymer was induced. The UV source used was a Newport 1000 W Oriel Flood Exposure Source with a wavelength of k = 365 nm. Five samples were illuminated at a time, each with an energy density of E = 16 J cm - 2 , at which approx. 94% of the present benzophenone groups reacted, as reported previously (Toomey et al. 2004; Jocher et al. 2015). (3) After the UV-excitation, extraction with cold H 2 O was carried out, in order to remove any unbound copolymer from the paper samples. For this, the paper samples were extracted in an extraction apparatus for four cycles, which is sufficient for complete removal of unbound copoly- mer, after which the paper samples were dried on the Teflon-sieve under climate-controlled conditions.
After equilibration the dried paper samples were weighed one more time, before they were pressed again to guarantee flat samples for the tensile measurements. Note, for the reference experiments, paper samples were impregnated in H 2 O, IPA or BuOH for at least 60 s and dried afterwards, all other steps were carried out as described above.
Preparation of thin slices for confocal microscopy
To improve the visibility of cellulose fibers under fluorescence microscopic investigation, paper sheets were stained in a 100 l M aqueous Calcofluor White (CW) solution for ten minutes, washed in H 2 O for another 10 min to remove unbound CW and dried overnight. A commercially available polyurethane system consisting of an aliphatic polyisocyanate (Desmodure 3200), a polyol (Albodur 956), and a catalyst (TIB Kat 318) in a ratio of 1:1:5 9 10 –4 was used to embed the samples. After a few vacuum cycles at room temperature to improve the penetration of the embedding medium inside the paper samples, they were cured overnight and consecutively cut into 120 l m slices using a microtome. This technique has been used to analyze the spatial modification of paper and the functionalization of fiber surfaces, where details can be found in recent publi- cations (Bump et al. 2015; Janko et al. 2015; Nau et al. 2019). Despite an increased resolution and contrast that confocal detection provides over standard widefield
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