Nanomaterials 2023 , 13 , 2536
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2.3.2. Morphology The morphological analysis encompassed several parameters, including average length weighted in length (length w ), diameter (d), coarseness, and fine percentage (%). These measurements were determined using image analysis performed with a MorFi Compact analyzer (TechPap, Gi è res, France). However, as the resolution of MorFi images was limited and we were unable to accurately measure nanofibers, the aspect ratio of the pretreated samples was estimated using the measurement of the gel point (GP) based on the methodology developed by Varanasi et al. (2013) and Sanchez-Salvador et al. (2021) [37,38]. Furthermore, optical images of the pretreated pulps were taken using a Zeiss Axio Lab.A1 optical microscope and a color microscope camera Zeiss AxioCam ERc 5s (Carl Zeiss Microscopy GmbH, Göttingen, Germany). Subsequently, the optical images were processed using the version 1.53i of ImageJ software package to enhance the clarity of the images for further analysis. 2.4. Characterization of the CNFs Characterization of the CNFs involved the measurement of several properties, includ- ing nanofibrillation yield, transmittance at 600 nm, cationic demand (CD), and aspect ratio of the CNF suspensions, following the methods described in previous studies [6,27]. To determine the nanofibrillation yield, CNF suspension was diluted to 0.1 wt.% and centrifu- gated at 4500 × g for 30 min to isolate nanofibrillated cellulose from non-nanofibrillated components, which settled in the sediment. The weight of the sediment was measured to calculate the nanofibrillation yield. A UV–vis Shimadzu spectrophotometer UV-160A was used to measure the transmittance of the CNF suspensions at two different wavelengths. The anionic character of CNFs correlates with their CD, which was determined via back titration, with polydiallyldimethylammonium chloride (PDADMAC) and polyvinylsulfate potassium salt using a Mütek PCD04 particle charge detector (BTG Instruments, Weßling, Germany). The aspect ratio of the CNFs was determined using a recently developed methodology by Sanchez-Salvador et al. (2021) [38]. This approach modified the traditional GP methodology by dying the fibers with crystal violet, enabling the visualization of the fibril sedimentation and optimizing the sedimentation time to ensure complete settling. An optical microscope was used to obtain images from CNF hydrogels for qualitative support of our results. In this case, the parameters and method used for obtaining these images were consistent with those utilized for the pretreated pulps. Additionally, the optical images underwent processing with the ImageJ software package to seamlessly eliminate the continuous background. This was achieved by applying the “Process › Subtract Background” command from the menu, with the specified parameters including a rolling ball radius of 50 pixels, a light background, and a sliding paraboloid. For the observation of CNFs, a JEOL JEM 1400 PLUS transmission electron mi- croscopy (TEM) device (Tokyo, Japan) was utilized at the Spanish National Centre of Electronic Microscopy (CNME), following the methodology developed by Campano et al. (2020) [39]. The TEM microscope operated at 100 kV accelerating voltage, with an Orius SC200 CCD camera manufactured by Gatan (Pleasanton, CA, USA), featuring a resolution of 2048 × 2048 pixels and a pixel size of 7.4 microns. With the TEM images obtained, projected fractal dimension (D2) was measured. D2 gives an idea of the “tree structure” of the sample, which can influence the mechanical properties of the material [40]. The acquired images were also processed and analyzed with ImageJ software, editing images to achieve good definition and high contrast in CNFs and converting these into 8-bit images. They were binarized through a threshold and corrected with a close filter. Elements in the images were selected individually and copied into a separate file. The fractal analysis was performed with the fractal box count plugin. To reduce processing times, this procedure was automatized through a script [41,42].
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