Efficient Labeling of Nanocellulose for High-Resolution Fluorescence Microscopy Applications

Abstract

The visualization of naturally derived cellulose nanofibrils (CNFs) and nanocrystals (CNCs) within nanocomposite materials is key to the development of packaging materials, tissue culture scaffolds, and emulsifying agents, among many other applications. In this work, we develop a versatile and efficient two-step approach based on triazine and azide–alkyne click-chemistry to fluorescently label nanocelluloses with a variety of commercially available dyes. We show that this method can be used to label bacterial cellulose fibrils, plant-derived CNFs, carboxymethylated CNFs, and CNCs with Cy5 and fluorescein derivatives to high degrees of labeling using minimal amounts of dye while preserving their native morphology and crystalline structure. The ability to tune the labeling density with this method allowed us to prepare optimized samples that were used to visualize nanostructural features of cellulose through super-resolution microscopy. The efficiency, cost-effectiveness, and versatility of this method make it ideal for labeling nanocelluloses and imaging them through advanced microscopy techniques for a broad range of applications.

Unraveling the Supramolecular Structure and Nanoscale Dislocations of Bacterial Cellulose Ribbons Using Correlative Super-Resolution Light and Electron Microscopy

“…BC fibrils were fluorescently labeled with sulfo-Cy5 azide dye in a two-step reaction scheme as previously described. 21 Briefly, a triazinyl linker bearing an alkyne group, DTAP, was grafted onto BC through a reaction involving 0.1 wt % suspension of BC fibrils in a 3:1 water/acetone solution containing 10 mM of linker and 0.05 M of sodium hydroxide while mixing at room temperature for 24 h. The unreacted linker was removed through centrifugation (21,100g, 60 s with a Sorvall Legend Micro 21R centrifuge, Thermo Scientific), decanting, and resuspension cycles with water (3×), acetone (3×), PBS (3×), then water (3×). Cellulose fibrils were dispersed using a 0.25-inch point-probe (Branson SLPt, 40% amplitude, or 60 W, for 10 s) every third spindown.…”

“…Glucose, sodium hydroxide (NaOH), glucose oxidase (from Aspergillus niger, type X–S, 100–250 units/mg, 65–85% protein content), catalase (from bovine liver, ≥10,000 units/mg, ≥70% protein content) and cysteamine (98%) were purchased from MilliporeSigma (Oakville, ON, Canada) and used without any further purification. Dichlorotriazinyl propargyl amine (DTAP) was synthesized in-house as previously described. , BC was extracted from foodstuff Nata de Coco (230 g drained weight, New Lamthong Food Industries, Bangkok, Thailand), a fermented desert consisting of a bacterial culture of K. xylinus, using the same procedure described previously .…”

“…Dichlorotriazinyl propargyl amine (DTAP) was synthesized in-house as previously described. , BC was extracted from foodstuff Nata de Coco (230 g drained weight, New Lamthong Food Industries, Bangkok, Thailand), a fermented desert consisting of a bacterial culture of K. xylinus, using the same procedure described previously . All mentions of water refer to distilled 18.2 MΩ water, generated from a Milli-Q Direct purifier system (MilliporeSigma).…”

“…Stochastic optical reconstruction microscopy (STORM) super-resolution images were acquired as previously reported. , Briefly, imaging was performed in widefield, epifluorescence mode on a Leica DMI6000B inverted microscope equipped with a Borealis module (Spectral Applied Research, Richmond Hill, ON, Canada) that couples 405, 488, 561, and 647 nm laser lines from an integrated light engine (Spectral Applied Research, Richmond Hill, ON, Canada) to a 100×/1.47 NA oil-immersion objective. Emitted light passes through a multiline dichroic mirror and is filtered through a 700/75 nm filter before being captured by an Andor iXon Ultra DU-897U EMCCD with a projected pixel size of 96 nm.…”

Nanocellulose: A Fundamental Material for Science and Technology Applications

“…Therefore, the distribution of nanocellulose in cells and tissues should be tracked. Babi et al [132] successfully labeled fluorescently the nanocellulose using triazine-alkyne linker and azide dyes, without affecting the cellulose structure. The labeling was optimized and the labeled nanocellulose was identified using high-resolution fluorescence microscopy.…”