Ensemble and Single Particle Fluorescence Characterization of Dye-Labeled Cellulose Nanocrystals.

Cellulose nanocrystals (CNCs) have been covalently labeled with both fluorescein and rhodamine and studied by a combination of UV-vis absorption spectroscopy and ensemble and single molecule fluorescence spectroscopy. For all samples, the fluorescence anisotropy and lifetimes were consistent with effects expected for covalently bound dye molecules. Low dye loading levels (∼0.1 dye/particle) were estimated for the fluorescein-labeled CNC which coupled with the strong pH dependence make this a less suitable fluorophore for most applications. Rhodamine-labeled CNCs were prepared from both sulfated and carboxylated CNCs and had loading levels that varied from 0.25 to ∼15 dye molecules/CNC. For the sulfated samples, the absorption due to (nonfluorescent) dimeric dye increased with dye loading; in contrast, the carboxylated sample, which had the highest rhodamine content, had a low dimer yield. Single particle fluorescence studies for two of the rhodamine-labeled CNCs demonstrated that individual particles are readily detected by their stepwise blinking/bleaching behavior and by polarization effects. Overall, the results indicate the importance of understanding the effects of loading on dye photophysics to select an optimal dye concentration to maximize sensitivity while minimizing the effect of the dye on the CNC behavior. The results also demonstrate that CNCs with relatively low dye loadings (e.g., ∼1 dye/particle) are readily detectable by fluorescence and should be adequate for use in fluorescence-based biological assays or to probe the distribution of CNCs in composite materials.

Correlating Cellulose Nanocrystal Particle Size and Surface Area.

Cellulose nanocrystals (CNCs) are negatively charged nanorods that present challenges for characterization of particle size distribution and surface area-two of the common parameters for characterizing nanomaterials. CNC size distributions have been measured by two microscopy methods: atomic force microscopy (AFM) and transmission electron microscopy (TEM). The agreement between the two methods is good for length measurements, after taking into consideration tip-convolution effects for AFM. However, TEM widths are almost twice as large as AFM heights-an effect that we hypothesize is due to counting of a larger fraction of laterally associated CNCs in the TEM images. Overall, the difficulty of selecting individual particles for analysis and possible bias due to selection of a specific particle size during sample deposition are the main limitations associated with the microscopy measurements. The microscopy results were compared to Z-average data from dynamic light scattering, which is a useful method for routine analysis and for examining trends in size as a function of sample treatment. Measurements as a function of sonication energy were used to provide information on the presence of aggregates in the sample. Magic-angle-spinning solid-state NMR was employed to estimate the surface area of CNCs based on the ratio of integrated spectral intensities of resonances stemming from C4 sites at the crystallite surfaces and from all C4 sites. Our approach was adapted from the application of solid-state NMR to characterize larger cellulose microfibers and appears to provide a useful estimate that overcomes the limitations of using the BET method for measuring surface areas of highly aggregated nanomaterials. The solid-state NMR results show that the lateral dimension of the CNCs is consistent with that of elementary cellulose crystallites.

In Situ Confocal Raman Microscopy of Hydrated Early Stages of Bacterial Biofilm Formation on Various Surfaces in a Flow Cell.

Bacterial biofilms are precursors to biofouling by other microorganisms. Understanding their initiation may allow us to design better ways to inhibit them, and thus to inhibit subsequent biofouling. In this study, the ability of confocal Raman microscopy to follow the initiation of biofouling by a marine bacterium, Pseudoalteromonas sp. NCIMB 2021 (NCIMB 2021), in a flow cell, using optical and confocal Raman microscopy, was investigated. The base of the flow cell comprised a cover glass. The cell was inoculated and the bacteria attached to, and grew on, the cover glass. Bright field images and Raman spectra were collected directly from the hydrated biofilms over several days. Although macroscopically the laser had no effect on the biofilm, within the first 24 h cells migrated away from the position of the laser beam. In the absence of flow, a buildup of extracellular substances occurred at the base of the biofilm. When different coatings were applied to cover glasses before they were assembled into the flow cells, the growth rate, structure, and composition of the resulting biofilm was affected. In particular, the ratio of Resonance Raman peaks from cytochrome c (CC) in the extracellular polymeric substances, to the Raman phenylalanine (Phe) peak from protein in the bacteria, depended on both the nature of the surface and the age of the biofilm. The ratios were highest for 24 h colonies on a hydrophobic surface. Absorption of a surfactant with an ethyleneoxy chain into the hydrophobic coating created a surface similar to that given with a simple PEG coating, where bacteria grew in colonies away from the surface rather than along the surface, and CC:Phe ratios were initially low but increased at least fivefold in the first 48 h.