Observation of wet specimens sensitive to evaporation using scanning electron microscopy

Inoue, Noriyuki; Takashima, Yoshinori; Suga, Mitsuo; Suzuki, Toshiaki; Nemoto, Yoshikazu; Takai, Osamu

doi:10.1093/jmicro/dfy041

Cited by 5 publications

(5 citation statements)

References 31 publications

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“…Hydrogel structure is intrinsically linked to the presence of a hydration shell around the gel polymer, in this case collagen molecules arranged into a network of fibres, and overall degree of hydration, 30 forming distinct layers of bound water according to the degree of energy associated with the binding 41 . Water molecules in closer association to the hydrophilic polymer network of the hydrogel interact more strongly with this network, and so require more energy to become dissociated.…”

Section: Discussionmentioning

confidence: 99%

“…As the structure of hydrogels is so closely linked to the degree of hydration (Figure 2), dehydrated films imaged under SEM do not necessarily reflect the structure of the hydrated gel. Artefacts are induced by, for example, shrinkage and loss of ‘pores’ or void spaces of the gel as the polymer material is made increasingly concentrated by the creeping loss of the gel's water content 30 . This loss of water content, as the bulk volume of the gel, results in a densification of the gel polymer, reducing apparent porosity and potentially generating polymer aggregates that are not present in the hydrated material, and generally disrupting the polymer network relative to how these features exist in the hydrated gel state.…”

Section: Introductionmentioning

confidence: 99%

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Exploring the microstructure of hydrated collagen hydrogels under scanning electron microscopy

Merryweather

Weston

Roe

et al. 2023

Journal of Microscopy

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Collagen hydrogels are a rapidly expanding platform in bioengineering and soft materials engineering for novel applications focused on medical therapeutics, medical devices and biosensors. Observations linking microstructure to material properties and function enables rational design strategies to control this space. Visualisation of the microscale organisation of these soft hydrated materials presents unique technical challenges due to the relationship between hydration and the molecular organisation of a collagen gel. Scanning electron microscopy is a robust tool widely employed to visualise and explore materials on the microscale. However, investigation of collagen gel microstructure is difficult without imparting structural changes during preparation and/or observation. Electrons are poorly propagated within liquid‐phase materials, limiting the ability of electron microscopy to interrogate hydrated gels. Sample preparation techniques to remove water induce artefactual changes in material microstructure particularly in complex materials such as collagen, highlighting a critical need to develop robust material handling protocols for the imaging of collagen hydrogels. Here a collagen hydrogel is fabricated, and the gel state explored under high‐vacuum (10−6 Pa) and low‐vacuum (80–120 Pa) conditions, and in an environmental SEM chamber. Visualisation of collagen fibres is found to be dependent on the degree of sample hydration, with higher imaging chamber pressures and humidity resulting in decreased feature fidelity. Reduction of imaging chamber pressure is used to induce evaporation of gel water content, revealing collagen fibres of significantly larger diameter than observed in samples dehydrated prior to imaging. Rapid freezing and cryogenic handling of the gel material is found to retain a porous 3D structure following sublimation of the gel water content. Comparative analysis of collagen hydrogel materials demonstrates the care needed when preparing hydrogel samples for electron microscopy.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Exploring the microstructure of hydrated collagen hydrogels under scanning electron microscopy

Merryweather

Weston

Roe

et al. 2023

Journal of Microscopy

View full text Add to dashboard Cite

show abstract

“…Such preparation processes may cause topographical, morphological and compositional changes to the cells and fibers of A-PRF membranes. Therefore, future research using environmental SEM, 29 or with modifications to conventional SEM by using the wet cover method 30 will allow the observation of wet specimens and may overcome the limitations of conventional SEM.…”

Section: Discussionmentioning

confidence: 99%

Impact of glutaraldehyde cross-linking on the advanced platelet-rich fibrin membrane: Macroscopic, conventional light microscopic and scanning electron microscopic analysis

Vemanaradhya¹,

Rahman²,

Adi³

et al. 2023

Dent. Med. Probl.

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“…37 Environmental SEM/TEM set-ups can be used to maintain the hydration of the sample, but sample preparation becomes increasingly more complex. 38 While AFM does not warp the sample, this technique has a limited imaging depth restraining analysis to the surface level. 39 Optical microscopy can be used to image soft samples.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Since many ECM-like samples are wet, the high vacuum required for traditional SEM/TEM can lead to warping of the original structure, which is not ideal when attempting to recover accurate spatial details . Environmental SEM/TEM set-ups can be used to maintain the hydration of the sample, but sample preparation becomes increasingly more complex . While AFM does not warp the sample, this technique has a limited imaging depth restraining analysis to the surface level …”

Section: Introductionmentioning

confidence: 99%

Spatially Resolving Size Effects on Diffusivity in Nanoporous Extracellular Matrix-like Materials with Fluorescence Correlation Spectroscopy Super-Resolution Optical Fluctuation Imaging

Chatterjee,

Kramer,

Wellnitz

et al. 2023

J. Phys. Chem. B

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It is well documented that the nanoscale structures within porous microenvironments greatly impact the diffusion dynamics of molecules. However, how the interaction between the environment and molecules influences the diffusion dynamics has not been thoroughly explored. Here, we show that fluorescence correlation spectroscopy super-resolution optical fluctuation imaging (fcsSOFI) can be used to accurately measure the diffusion dynamics of molecules within varying matrices such as nanopatterned surfaces and porous agarose hydrogels. Our data demonstrate the robustness of fcsSOFI, where it is possible not only to quantify the diffusion speeds of molecules in heterogeneous media but also to recover the matrix structure with resolution on the order of 100 nm. Using dextran molecules of varying sizes, we show that the diffusion coefficient is sensitive to the change in the molecular hydrodynamic radius. fcsSOFI images further reveal that smaller dextran molecules can freely move through the small pores of the hydrogel and report the detailed porous structure and local diffusion heterogeneities not captured by the average diffusion coefficient. Conversely, bigger dextran molecules are confined and unable to freely move through the hydrogel, highlighting only the larger pore structures. These findings establish fcsSOFI as a powerful tool to characterize spatial and diffusion information of diverse macromolecules within biorelevant matrices.

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Observation of wet specimens sensitive to evaporation using scanning electron microscopy

Cited by 5 publications

References 31 publications

Exploring the microstructure of hydrated collagen hydrogels under scanning electron microscopy

Exploring the microstructure of hydrated collagen hydrogels under scanning electron microscopy

Impact of glutaraldehyde cross-linking on the advanced platelet-rich fibrin membrane: Macroscopic, conventional light microscopic and scanning electron microscopic analysis

Spatially Resolving Size Effects on Diffusivity in Nanoporous Extracellular Matrix-like Materials with Fluorescence Correlation Spectroscopy Super-Resolution Optical Fluctuation Imaging

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