Carbotrace as a tool for the study of cellulose oxidation and paper aging

Since its invention, paper has skyrocketed technological advances as a key medium for recording thoughts and ideas. However, over time paper ages and oxidation leads to loss of strength and renders sheets of paper brittle. Therefore, the study of paper aging becomes relevant not only for the preservation of historical and artistic values, but also because competition from electronic media and increasing demands from customers force the pulp and paper industry to improve its competitive edge by optimizing production processes and paper quality. In a recent publication from the Departments of Physics and Chemistry as well as Biological, Chemical and Pharmaceutical Sciences at the University of Palermo, Ferrara et al. used fluorescence-lifetime imaging microscopy (FLIM) to study aging of cellulosic material. The researchers leveraged the structure-dependent photo-physical properties of Carbotrace 680 to track aging-induced chemical alterations in cellulose fibers. Paper aging was simulated by treating cellulose fibers with potassium metaperiodate which reacts with cellulose to yield the oxidized derivative known as dialdehyde cellulose in a controlled Malaprade reaction. Paper acidification and carbonyl content were studied with conventional methods such as pH measurement, a redox colorimetric method (TTC assay) and crystallinity analysis using X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) techniques. As an alternative, novel method, steady-state fluorescence and phasor-FLIM analysis of paper oxidation were performed using Carbotrace. Confocal microscopy of cellulose fibers labeled with Carbotrace 680 before and after aging revealed morphological differences in the aged fibers. Strikingly, the fluorescence emission spectrum of Carbotrace 680 showed a blue-shift of the emission maximum and a narrowing of the spectrum in aged paper related to alterations in the chemical environment of the dye. The data indicate that Carbotrace 680 fluorescence is sensitive to paper changes induced by oxidation, and showcase Optotracing as a useful technique for screening physicochemical properties of cellulosic materials. Using an advanced method called Fluorescence Lifetime Imaging (FLIM), it is possible to map aging-induced alterations at the sub-micrometric scale. With FLIM, a distribution of fluorescence lifetimes is observed. Using phasor analysis fluorescence lifetime distributions can be compared, revealing striking differences between fluorescence lifetimes of untreated and aged paper. The high-resolution data show that the paper fibers undergo oxidation in a non-uniform way, with the outer portions of the fiber oxidized at the beginning of the process, followed by a complete oxidation of the fiber also in the inner part within 24 h. The study by Ferrara et al. paves the way for use of novel technologies like FLIM and Optotracing in pulp and paper industry that are critical to improve sustainability while maintaining quality.

Image: Phasor-FLIM images illustrate kinetics of paper oxidation during 30, 60 and 1440 min treatment with metaperiodate showing the change of the Carbotrace 680 lifetime (blue: 0.10 ns, red: 2.94 ns) during the oxidation process (scale bar: 20 µm). Image from Figure 7C by Ferrara et al. (2024) International Journal of Biological Macromolecules 260(2), 129452 (CC BY 4.0).

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