Efficiency of fluorescent labeling on fines
Fines were labeled with RBITC after separation from the paper pulp (Fig. 1).
Optical microscopy and CLSM of functionalized labeled fines were performed to distinguish between autofluorescence of lignocellulosic fibers (Fig. 2a) and the fluorescence signal emitting at 595 nm from RBITC (Fig. 2b) induced by covalent attachment of RBITC. Further, the images revealed that the fluorescent labelling is homogenous throughout the whole surface of the fines. The zeta potential of fines and labeled fines further proved the successful labelling of the fines revaling a zeta potential close to 0 mV while the unmodified fines featured a zeta potential of − 30 mV.
Influence of fluorescent labeling on sheet properties
Relevant physical and mechanical properties were compared between paper sheets containing no additional (blank), unlabeled (BSK1 and BSK2) and fluorescent fines (FFBSK1 and FFBSK2). The blank, BSK1 and BSK2 exhibited the same brightness, while FFBSK2 appeared visually slightly more intense in color than FFBSK1. This is caused by the higher concentration of labeled fines in those sheets. Nevertheless, labeled fines neither affected the zeta potential, nor the isoelectric point of the handsheets (Table S1). The water retention values of the pulps (Fig. 3a) increased from BSK1 to BSK2, which relates to the higher concentration of fines in the latter which retain more water (Mayr et al. 2017b). FFBSK1 and FFBSK2 showed slightly higher ability to retain water compared to BSK1 and BSK2, regardless of the concentration. While FFBSK2 was in the confidence interval of BSK2, FFBSK1 was beyond the one of BSK1. The increased water retention value (WRV) indicates more pronounced swelling that corresponds to the higher density of the paper sheets (Fig. 3b). Consequently, the higher density is reflected in the mechanical properties (Fig. 3c) and air permeability (Fig. 3d) as well. Nevertheless, the differences between unlabeled and labeled fines in handsheet density, mechanical properties and air permeability were not significant, when taking the corresponding confidence intervals (95% level of confidence) into account.
Furthermore, sheet formation was investigated by beta radiography. The results revealed no significant change in mass distribution, which indicates that flocculation does not seem to be impaired (Fig. S1). Therefore, the labeled fines should represent the properties of unlabeled ones well and may interact with the paper network in a similar way. Studying these interactions shall therefore give new insights into the role of fines within the network and their technological impact.
Localization of fines within the paper matrix
All samples were analyzed by confocal laser scanning microscopy (CLSM) and multiphoton microscopy (MPM). Both techniques should allow for the localization of fibers and fines within the x-,y- and z-direction of the handsheets.
With CLSM, both, labeled fines and the pulp fibers, were excited at 405 nm and the emission was recorded between 420 and 500 nm, corresponding to autofluorescence. The detected autofluorescence (grey areas in Fig. 4) clearly reflected the fiber network of the studied paper samples. In these measurements, any differences for paper sheets containing 1% and 2% fines (i.e. BSK1 and BSK2, Fig. 4a) were not observed. Excitation at 532 nm in turn, just led to noisy signals for sheets made from BSK1 and BSK2 (Fig. S2). In contrast, FFBSK1 and FFBSK2 labeled fines gave a clear response after excitation at 532 nm and allowed for their visualization inside the paper. This further proves that the fluorescent labeled fines can be easily differentiated from the non-labeled cellulosic matrix. A considerable increase of labeled segments is evident in Fig. 4b, c, which is consistent with the increased amount of labeled fines. Obviously, the labeled fines are well distributed within the fiber network. Based on such measurements also 3D investigations and analysis of cross sections are possible with resolution limited by the light refraction in z-direction. Such investigations could enable the characterisation of the segments within the fiber joints. At this point, CLSM reaches its resolution limits for paper sheets.
The application of MPM extends the investigations of fluorophores as labeling technique in paper sheets reducing the blurring by exciting the fluorophore with two photons at the same time, while suppressing the out-of-focus light. The reduction of out-of-focus light enables deeper insight into the cellulosic structure (second harmonic generation) while increasing simultaneously the resolution. The second harmonic generation from cellulose was measured between 400 and 410 nm (green) superimposed by the fluorescence signal of the labeled fines between 589 and 625 nm (red). BSK1 and BSK2 that only contained non-labeled fines (Fig. 5a) clearly revealed a fiber network, while the fluorescence signal results in noise, similar to the one in Fig. 4a. Superimposing the second harmonic generation with the fluorescence signal of FFBSK1 and FFBSK2 showed particles with a specific structure. Larger particles (white arrows) and smaller particles (yellow arrows) are visible (Figs. 5, S3).
Herein, the larger fiber segments (primary fines) seem to participate in the network just like pulp fibers do, in general, due to their size. Smaller particles (secondary fines) on the other hand seem to be more concentrated in specific spots. Due to their size, they are not limited to the same extent from moving within the structure. Therefore, they are allowed to migrate within the network, most probably following the retreating water during dewatering into the smallest interstices between fibers, which later on form fiber–fiber joints. These small particles are attached to a cellulose fiber and the fiber–fiber joint, as evident in the bottom of the image that is wrapped by fluorescent fines, tagged with orange arrows in Fig. 5c. This accumulation in the fiber–fiber joints supports the idea of Retulainen (Retulainen et al. 1993), who proposed less free loops (unbonded fiber segments) at higher fines concentration. Higher fines concentrations also promote accumulation, especially fibrillar parts. Hence, secondary fines have a much bigger impact on paper properties than primary fines (Bäckström et al. 2008; Chen et al. 2009).