Protein tracking in living plant cells has become routine with the emergence of reporter genes encoding fluorescent tags. Unfortunately, this imaging strategy is not applicable to glycans because they are not directly encoded by the genome. Indeed, complex glycans result from sequential additions and/or removals of monosaccharides by the glycosyltransferases and glycosidases of the cell's biosynthetic machinery. Currently, the imaging of cell wall polymers mainly relies on the use of antibodies or dyes that exhibit variable specificities. However, as immunolocalization typically requires sample fixation, it does not provide access to the dynamics of living cells. The development of click chemistry in plant cell wall biology offers an alternative for live-cell labeling. It consists of the incorporation of a carbohydrate containing a bio-orthogonal chemical reporter into the target polysaccharide using the endogenous biosynthetic machinery of the cell. Once synthesized and deposited in the cell wall, the polysaccharide containing the analog monosaccharide is covalently coupled to an exogenous fluorescent probe. Here, we developed a metabolic click labeling approach which allows the imaging of cell wall polysaccharides in living and elongating cells without affecting cell viability. The protocol was established using the pollen tube, a useful model to follow cell wall dynamics due to its fast and tip-polarized growth, but was also successfully tested on Arabidopsis root cells and root hairs. This method offers the possibility of imaging metabolically incorporated sugars of viable and elongating cells, allowing the study of the long-term dynamics of labeled extracellular polysaccharides.

Purpose: The aim of this study is to describe visual outcomes and epithelial remodeling following the implantation of asymmetric intracorneal ring segments (ICRSs) of variable thickness and base width for the management of duck-type keratoconus. Methods: A prospective observational study of patients with duck-type keratoconus was conducted. All patients received one ICRS AJL PRO + implant (AJL Ophthalmic). We analyzed demographic and clinical data, anterior segment optical coherence tomography (AS-OCT) data and Scheimpflug camera images obtained with a Placido disc MS-39 (CSO, Firenze, Italy) one and six months after surgery to determine keratometric and aberrometric outcomes and epithelial remodeling. Results: We studied 33 keratoconic eyes. ICRS implantation significantly improved both corrected distance visual acuity (CDVA) and uncorrected distance visual acuity at six months, as assessed with the logMAR (minimum angle of resolution) system, from 0.32 ± 0.19 to 0.12 ± 0.12 (p < 0.001) and from 0.75 ± 0.38 to 0.37 ± 0.24 (p < 0.001), respectively. Overall, 87% of implanted eyes gained ≥ 1 line of CDVA, and 3% of patients (n = 1) lost one line of CDVA; 55% of eyes attained a manifest refraction spherical equivalent between +1.50 and −1.50 D. Epithelial remodeling was greater at the wider and thicker end (+11.33 µm ± 12.95; p < 0.001 relative to the initial value) than at the narrower and thinner end (+2.24 µm ± 5.67; p = 0.01). Coma aberration was significantly reduced from 1.62 ± 0.81 µm to 0.99 ± 0.59 µm (p < 0.001). Conclusions: AJL-PRO + ICRS implantation for duck-type keratoconus improves refractive, topographic, aberrometric and visual parameters and induces progressive epithelial thickening along the segment.