Cryo-SEM Imaging and Analysis of Frozen-Hydrated PEG-AA Microgels

Liang, Jenn‐Tai; Teng, Feiyue; Libera, Matthew; Chou, Tsengming

doi:10.1017/s1431927616010333

Cited by 3 publications

(4 citation statements)

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Section: Instrumentation Advancesmentioning

confidence: 99%

“…We have shown that the sublimation rate of water in the buffer is significantly higher than that of water in a microgel. 2,43,44 Hence, the microgels protrude from the buffer after the sublimation treatment. Note that extensive sublimation can change the surface composition of the buffer, since only the water will sublimate while salts are left behind.…”

Section: X-ray Spectroscopy From Polyelectrolyte Microgels In the Cry...mentioning

confidence: 99%

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Analytical Cryo-Scanning Electron Microscopy of Hydrated Polymers and Microgels

et al. 2021

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Metrics & MoreArticle Recommendations CONSPECTUS: Despite the fact that scanning electron microscopes (SEM) coupled with energy-dispersive X-ray microanalysis (EDS) has been commercially available for more than a halfcentury, SEM/EDS continues to develop and open new opportunities to study the morphology of advanced materials. This is particularly true in applications to hydrated soft matter. Developments in field-emission electron sources that enable lowvoltage imaging of uncoated polymers, silicon-drift detectors that enable high-efficiency collection of X-rays characteristic of light elements, and cryogenic methods to effectively cryo-fix hydrated samples have opened new opportunities to apply techniques relatively well established in hard-materials applications to challenging new problems involving synthetic polymers. We have applied cryo-SEM imaging and spatially resolved EDS to collect new information characterizing polyelectrolyte microgels. These are charged gel particles with dimensions in the range of 0.1−100 μm. Perhaps most notable is the fact that the high hydration levels the samples are mostly waterallow robust calibration curves to be generated using frozen-hydrated buffers with known salt and/or hydrocarbon compositions. Such calibration curves enable quantitative composition measurements in the low-concentration extremes associated with high-swelling hydrogels. We use an experimentally derived carbon calibration curve to determine the microgel swell ratio, Q. The swell ratio, arguably, is the single most important gel characteristic because it is directly related to the mesh size of the networked polymer, which in turn determines many of the gel's mechanical and transport properties. While Q can be experimentally measured in macroscopic gels based on weight measurements in the dry and hydrated states, it is very difficult to measure in a microgel, and the fact that EDS in a cryo-SEM can determine Q from a single X-ray spectrum is significant. Furthermore, because of the electrostatic charge distributed along the polymer chains, the presence and concentration of counterions play a critical role in polyelectrolyte systems. While conceptually understood for decades, experimental measurements of counter-ion concentrations have been largely limited to a relatively small set of materials that involve macroscopic samples. By developing calibration curves from frozen-hydrated buffer of known ionic strength, we measure the concentration of Na counter-ions in microgels of poly(acrylic acid) (PAA) with a limit of detection of ∼0.014 M. Such measurements may help resolve some longstanding questions in polyelectrolyte science concerning counter-ion condensation. Even in the absence of a calibration curve, we show that spatially resolved X-ray spectroscopy can map the spatial distribution of a cationic oligopeptide complexed within a hydrated PAA microgel because of the nitrogen fingerprint that, albeit at very low concentration, is unique to the peptide. We look specifically at the case of a microgel with ...

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Section: Instrumentation Advancesmentioning

confidence: 99%

Section: X-ray Spectroscopy From Polyelectrolyte Microgels In the Cry...mentioning

confidence: 99%

Analytical Cryo-Scanning Electron Microscopy of Hydrated Polymers and Microgels

et al. 2021

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“…The specimens were 200 µm in thickness. They were frozen by either plunging in liquid nitrogen at 1 atm or high-pressure freezing using a Leica HPM-100 HPF [3][4]. In another set of experiments, the effect of sublimation was studied with a colloidal suspension of poly(acrylic acid) (PAA) microgels in sodium phosphate buffer.…”

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Freezing and sublimation effects on cryo-SEM imaging and microanalysis

et al. 2019

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“…Here we study the distribution of unlabeled cationic antimicrobials complexed within anionic microgels using EDS in the cryo-SEM. We observe direct evidence for water and counterion release during complexation.Poly(acrylic acid) (PAA) microgels, both as synthesized as well as after complexation loading with the L5 cationic antimicrobial (+6 charge, 2274 g/mol), were soaked in 0.01 M sodium phosphate buffer at pH 7.4 and cryo-fixed by high-pressure freezing (Leica HPM-100 HPF) [4][5]. After freeze fracturing, the specimens were imaged at -135 °C using a Zeiss Auriga Cross-Beam FIB-SEM equipped with a Leica VCT-100 cryo-transfer system.…”

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Cryo-SEM: Direct Evidence of Water and Counterion Release upon Complexation

et al. 2018

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Polyelectrolyte hydrogels are polymeric networks of charged groups, and they retain a high water content. These gels been applied extensively in drug delivery due to their ability to complex with oppositely charged molecules [1][2]. Polyelectrolyte complexation within microgels is traditionally studied by confocal microscopy using fluorescently labeled molecules [3]. However, the labeling changes molecular physicochemical properties and consequently can alter complexation behavior, especially for small molecules. Here we study the distribution of unlabeled cationic antimicrobials complexed within anionic microgels using EDS in the cryo-SEM. We observe direct evidence for water and counterion release during complexation.Poly(acrylic acid) (PAA) microgels, both as synthesized as well as after complexation loading with the L5 cationic antimicrobial (+6 charge, 2274 g/mol), were soaked in 0.01 M sodium phosphate buffer at pH 7.4 and cryo-fixed by high-pressure freezing (Leica HPM-100 HPF) [4][5]. After freeze fracturing, the specimens were imaged at -135 °C using a Zeiss Auriga Cross-Beam FIB-SEM equipped with a Leica VCT-100 cryo-transfer system. In order to generate topographic contrast for imaging, specimens were sublimated at -95 °C for 10 min, 40 min, or 12 h.Figs. 1A and 1B show typical cryo-SEM images of as-synthesized and L5-complexed microgels after 10 min sublimation. The process removes ~1 µm of ice, leaving fractured microgels protruding from the surrounding ice. As-synthesized microgels are homogeneous throughout the cross-section surfaces while L5-complexed microgels exhibit a shell layer with a few microns thickness. To further analyze the difference in composition and antimicrobial distribution within the L5-complexed microgels, carbon, oxygen and nitrogen X-ray line scans were collected across the fracture surface using EDS (Oxford Max-80 SDD detector) with 1 keV incident electrons. Profiles of the C/O intensity ratio reflect hydration levels in these gels [6]. The C/O ratios of 0.1 and 7.1 indicate highly hydrated and highly dehydrated microgel states, respectively. As shown by Fig. 1C, the as-synthesized microgels and the surrounding ice exhibit similar O intensities, indicating a very high water content within the microgel. The slightly higher C intensity reflects the fact that the hydrated microgel contains a small fraction of polymer. In contrast, L5-complexed microgels shows very different C, O and N intensities in their shell (Fig. 1D). The N intensity is unique to amine groups, and the spike in N intensity indicates that the L5 mainly complexes in the shell layer. The C/O ratio in the core is close to that of as-synthesized microgels, indicating the core is still highly hydrated. In contrast, the C/O ratio in the shell is very high and comparable to that of dried microgels. Thus, a significant of water is lost in the complexed shell. Further examination of the N and Na X-ray intensities (Fig. 2) using 2 keV electrons across a fully sublimed sample shows that the Na concentration is reduced i...

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Cryo-SEM Imaging and Analysis of Frozen-Hydrated PEG-AA Microgels

Cited by 3 publications

References 3 publications

Analytical Cryo-Scanning Electron Microscopy of Hydrated Polymers and Microgels

Analytical Cryo-Scanning Electron Microscopy of Hydrated Polymers and Microgels

Freezing and sublimation effects on cryo-SEM imaging and microanalysis

Cryo-SEM: Direct Evidence of Water and Counterion Release upon Complexation

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