10.1021/acs.bioconjchem.5b00048.s001 Jay W. Grate Jay W. Grate Kai-For Mo Kai-For Mo Yongsoon Shin Yongsoon Shin Andreas Vasdekis Andreas Vasdekis Marvin G. Warner Marvin G. Warner Ryan T. Kelly Ryan T. Kelly Galya Orr Galya Orr Dehong Hu Dehong Hu Karl J. Dehoff Karl J. Dehoff Fred J. Brockman Fred J. Brockman Michael J. Wilkins Michael J. Wilkins Alexa Fluor-Labeled Fluorescent Cellulose Nanocrystals for Bioimaging Solid Cellulose in Spatially Structured Microenvironments American Chemical Society 2015 bind Alexa Fluor dyes oxidizing vicinal diols Reductive amination reactions Alexa Fluor dye Spatially Structured MicroenvironmentsMethods pore network microfluidic structures cellulose deposits Fluorescent cellulose nanocrystals model cellulose materials Alexa Fluor 546 covalently conjugate Alexa Fluor dyes PDMS Alexa Fluor dyes reductive step molecule fluorescence microscopy linker section cellulose nanocrystals 2015-03-18 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Alexa_Fluor_Labeled_Fluorescent_Cellulose_Nanocrystals_for_Bioimaging_Solid_Cellulose_in_Spatially_Structured_Microenvironments/2185126 Methods to covalently conjugate Alexa Fluor dyes to cellulose nanocrystals, at limiting amounts that retain the overall structure of the nanocrystals as model cellulose materials, were developed using two approaches. In the first, aldehyde groups are created on the cellulose surfaces by reaction with limiting amounts of sodium periodate, a reaction well-known for oxidizing vicinal diols to create dialdehyde structures. Reductive amination reactions were then applied to bind Alexa Fluor dyes with terminal amino-groups on the linker section. In the absence of the reductive step, dye washes out of the nanocrystal suspension, whereas with the reductive step, a colored product is obtained with the characteristic spectral bands of the conjugated dye. In the second approach, Alexa Fluor dyes were modified to contain chloro-substituted triazine ring at the end of the linker section. These modified dyes then were reacted with cellulose nanocrystals in acetonitrile at elevated temperature, again isolating material with the characteristic spectral bands of the Alexa Fluor dye. Reactions with Alexa Fluor 546 are given as detailed examples, labeling on the order of 1% of the total glucopyranose rings of the cellulose nanocrystals at dye loadings of ca. 5 μg/mg cellulose. Fluorescent cellulose nanocrystals were deposited in pore network microfluidic structures (PDMS) and proof-of-principle bioimaging experiments showed that the spatial localization of the solid cellulose deposits could be determined, and their disappearance under the action of Celluclast enzymes or microbes could be observed over time. In addition, single molecule fluorescence microscopy was demonstrated as a method to follow the disappearance of solid cellulose deposits over time, following the decrease in the number of single blinking dye molecules with time instead of fluorescent intensity.