biology

A beginner’s guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications

Hydrogel materials show a number of properties which make them interesting candidates to be utilized to mimic the extracellular matrix (ECM). Therefore, these materials are attractive for use in biological applications such as tissue engineering, cell culture 3D bioprinting and more.

Are you planning to use hydrogels for the first time in your research?

Then have a look at the insightful article “A beginner’s guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications” by Francisco Drusso Martinez-Garcia, Tony Fischer, Alexander Hayn, Claudia Tanja Mierke, Janette Kay Burgess and Martin Conrad Harmsen.

In their article the authors describe and evaluate the different technologies that are most commonly used to assess hydrogel microarchitecture.

Francisco Drusso Martinez-Garcia et al. explain the working principle of the various methods and also discuss the merits and limitations of each of them in view of their usefulness for the characterization of hydrogels.

They introduce and explore the pros and cons of the following methods: Scanning Electron Microscopy (SEM), Cryogenic Scanning Electron Microscopy (Cryo-SEM), Environmental Scanning Electron Microscopy (ESEM), Micro-Computed Tomography (µ-CT), Confocal Laser Scanning Microscopy (CLSM), Second Harmonic Generation and Atomic Force Microscopy (AFM).*

Atomic force microscopy (AFM) can be used to investigate the hydrogel surface topology as well as a hydrogel’s mechanical properties. The latter can be achieved through mathematical modelling of force-distance curves.

When using the AFM to characterize the elasticity of a hydrogel sample it is essential to take the stiffness of the investigated material into account when choosing what kind of AFM probe to use for these experiments.

If an AFM cantilever used for probing a soft sample is too stiff (if the force constant/spring constant is too high) this might result in a poor signal-to-noise ratio.

If a soft AFM probe (an AFM probe with an AFM cantilever with a low force constant) is chosen to investigate a soft material this should lead to a better signal-to-noise ratio. On the other hand, if an AFM cantilever is too soft (if the force constant is too low) then it might not be stiff enough to indent the investigated material.

Another critical factor is the shape and the size of the AFM tip.

Spheroidal AFM probes might stick to the material, resulting in artefacts, disrupted force–distance curves, or even damaged AFM cantilevers. If the AFM tip is much smaller than the pore size of the hydrogel, it might get stuck in the fibrous network microarchitecture.

On the other hand, if the spherical AFM tip, e.g. as in colloidal AFM probes (a sphere glued to end of a tipless AFM cantilever), is too large, the weight of the sphere can have a negative influence on the spring characteristics of the AFM cantilever.

All these factors and more as described in the cited article have to be carefully weighed before deciding on the settings of the atomic force microscope and choosing an AFM probe for the investigation of a specific hydrogel.

NanoWorld tipless ArrowTL2 cantilever arrays with polystyrene beads glued to them were used by the authors of this beginner’s guide to achieve the AFM data presented in the article.*

Figure 6. from Francisco Drusso Martinez-Garcia et al. 2022: Atomic force microscopy. (A) Equipment. (B) Schematic of an AFM setup with a four-quadrant photodiode (1), in which the four-quadrant photodiode (1) receives a laser (2) reflected from a cantilever (3), in this case positioned over a hydrogel (4) mounted in a piezo stage (5). For example, the height differences in a sample (4) are measured by adjusting the stage using piezo elements (5) to counter the cantilever bending on a nanometer scale. (C) The AFM can then generate a surface heightmap of the hydrogels such as a GelMA hydrogel (shown). AFM can also be used to determine the mechanical properties of hydrogels. (D) Schematic of the AFM technique to determine the elastic moduli of hydrogels with a tipless cantilever (1), spheroidal probe (2, red), hydrogel (3), and stiff substrate (4). As the cantilever represents a spring with a known spring constant, the cantilever bending due to elastic counterforces exerted by the soft material is correlated with the piezo stage height (4). (E) The so-called force–distance curves are recorded. Data from a collagen type-I hydrogel (3.0 g/L) are shown. (F) Young’s moduli of a 1.5 g/L and 3.0 g/L collagen type-I hydrogel. Outliers indicated by ◆. AFM equipment detailed in Appendix A of the cited article. NanoWorld tipless ArrowTL2 cantilever arrays with polystyrene beads glued to them were used by the authors of this beginner’s guide to achieve the AFM data presented in the article. — Figure 6. from Francisco Drusso Martinez-Garcia et al. 2022:
Atomic force microscopy. (A) Equipment. (B) Schematic of an AFM setup with a four-quadrant photodiode (1), in which the four-quadrant photodiode (1) receives a laser (2) reflected from a cantilever (3), in this case positioned over a hydrogel (4) mounted in a piezo stage (5). For example, the height differences in a sample (4) are measured by adjusting the stage using piezo elements (5) to counter the cantilever bending on a nanometer scale. (C) The AFM can then generate a surface heightmap of the hydrogels such as a GelMA hydrogel (shown). AFM can also be used to determine the mechanical properties of hydrogels. (D) Schematic of the AFM technique to determine the elastic moduli of hydrogels with a tipless cantilever (1), spheroidal probe (2, red), hydrogel (3), and stiff substrate (4). As the cantilever represents a spring with a known spring constant, the cantilever bending due to elastic counterforces exerted by the soft material is correlated with the piezo stage height (4). (E) The so-called force–distance curves are recorded. Data from a collagen type-I hydrogel (3.0 g/L) are shown. (F) Young’s moduli of a 1.5 g/L and 3.0 g/L collagen type-I hydrogel. Outliers indicated by ◆. AFM equipment detailed in Appendix A of the cited article.

*Francisco Drusso Martinez-Garcia, Tony Fischer, Alexander Hayn, Claudia Tanja Mierke, Janette Kay Burgess and Martin Conrad Harmsen
A Beginner’s Guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications
Gels 2022, 8(9), 535
DOI: https://doi.org/10.3390/gels8090535

The article “A Beginner’s Guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications” by Francisco Drusso Martinez-Garcia, Tony Fischer, Alexander Hayn, Claudia Tanja Mierke, Janette Kay Burgess and Martin Conrad Harmsen is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

Correlation between plant cell wall stiffening and root extension arrest phenotype in combined abiotic stress of Fe and Al

The plasticity and growth of plant cell walls (CWs) is still not sufficiently understood on its molecular level. *

Atomic Force Microscopy (AFM) has been shown to be a powerful tool to measure the stiffness of plant tissues. *

In the article “Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al” Harinderbir Kaur, Jean-Marie Teulon, Christian Godon, Thierry Desnos, Shu-wen W. Chen and Jean-Luc Pellequer describe the use of atomic force microscopy (AFM) to observe elastic responses of the root transition zone of 4-day-old Arabidopsis thaliana wild-type and almt1-mutant seedlings grown under Fe or Al stresses. *

In order to evaluate the relationship between root extension and root cell wall elasticity, the authors used Atomic Force Microscopy to perform vertical indentations on surfaces of living plant roots. *

NanoWorld Pyrex-Nitride silicon-nitride PNP-TR AFM probes with triangular AFM cantilevers were used for the nanoindentation experiments with atomic force microscopy. (PNP-TR AFM cantilever beam 2 (CB2) with a typical force constant of 0.08 N/m and a typical resonant frequency of 17 kHz, typical AFM tip radius 10 nm, macroscopic half cone angles 35°). *

Force-distance (F-D) curves were measured using the Atomic Force Microscope and the PNP-TR AFM tips. *

Because of the heterogeneity of seedling CW surfaces, Harinderbir Kaur et al. used the recently developed trimechanics-3PCS framework for interpreting force-distance curves. The trimechanics-3PCS framework allows the extraction of both stiffness and elasticity along the depth of indentation and permits the investigation of the variation of stiffness with varied depth for biomaterials of heterogeneous elasticity responding to an external force. *

A glass slide with a glued seedling (see Figure 1 cited below) was positioned under the AFM cantilever with the help of an AFM optical camera. Due to the large motorized sample stage of the AFM, the glass slide was adjusted in such a way that the AFM cantilever could be positioned perpendicularly at the longitudinal middle of the glued root. The target working area, the transition zone, was 500 µm away from the root apex, almost twice the length of PNP-TR AFM cantilever. *

As shown in the article the presence of single metal species Fe²⁺ or Al³⁺ at 10 μM exerts no noticeable effect on the root growth compared with the control conditions. On the contrary, a mix of both the metal ions produced a strong root-extension arrest concomitant with significant increase of CW stiffness. *

Raising the concentration of either Fe²⁺or Al³⁺ to 20 μM, no root-extension arrest was observed; nevertheless, an increase in root stiffness occurred. In the presence of both the metal ions at 10 μM, root-extension arrest was not observed in the almt1 mutant, which substantially abolishes the ability to exude malate. The authors’ results indicate that the combination of Fe²⁺and Al³⁺ with exuded malate is crucial for both CW stiffening and root-extension arrest. *

It is shown that the elasticity of plant CW is sensitive and can be used to assess abiotic stresses on plant growth and stiffening. *

However, stiffness increase induced by single Fe²⁺ or Al³⁺ is not sufficient for arresting root growth in the described experimental conditions and unexpectedly, the stiffening and the phenotype of seedling roots such as REA are not directly correlated. *

Figure 1 from Harinderbir Kaur et al. 2024 “Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al”:Principle of nanomechanical measurement of seedling roots with atomic force microscopy. A seedling root (R) is deposited on a microscope slide using silicon glue (N, for Nusil). A fastening band of silicon is seen near the tip of the root (T). The thickness of the fastening band must be thin enough to avoid hindering the AFM support (S), but thick enough to withstand the bending of the root tip. The root is placed under the AFM cantilever (C) as observed by the AFM optical camera. The triangular shaped cantilever (200 µm long) was placed 500 µm away from the root tip in the transition zone where nanoindentation measurements proceeded (as shown). The seedling root and the AFM cantilever are placed within a liquid environment (growth solution, see Supplementary file of the cited article). AFM, atomic force microscopy. NanoWorld Pyrex-Nitride silicon-nitride PNP-TR AFM probes with triangular AFM cantilevers were used for the nanoindentation experiments with atomic force microscopy. — Figure 1 from Harinderbir Kaur et al. 2024 “Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al”:
Principle of nanomechanical measurement of seedling roots with atomic force microscopy.
A seedling root (R) is deposited on a microscope slide using silicon glue (N, for Nusil). A fastening band of silicon is seen near the tip of the root (T). The thickness of the fastening band must be thin enough to avoid hindering the AFM support (S), but thick enough to withstand the bending of the root tip. The root is placed under the AFM cantilever (C) as observed by the AFM optical camera. The triangular shaped cantilever (200 µm long) was placed 500 µm away from the root tip in the transition zone where nanoindentation measurements proceeded (as shown). The seedling root and the AFM cantilever are placed within a liquid environment (growth solution, see Supplementary file of the cited article). AFM, atomic force microscopy.

*Harinderbir Kaur, Jean‐Marie Teulon, Christian Godon, Thierry Desnos, Shu‐wen W. Chen and Jean‐Luc Pellequer
Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al
Plant, Cell & Environment 2024; 47:574–584
DOI: https://doi.org/10.1111/pce.14744

The article “Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al” by Harinderbir Kaur, Jean‐Marie Teulon, Christian Godon, Thierry Desnos, Shu‐wen W. Chen and Jean‐Luc Pellequer is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

Intrinsically disordered regions in TRPV2 mediate protein-protein interactions

Transient receptor potential (TRP) ion channels are gated by diverse intra- and extracellular stimuli leading to cation inflow (Na+, Ca2+) regulating many cellular processes and initiating organismic somatosensation. *

Structures of most TRP channels have been solved. However, structural and sequence analysis showed that ~30% of the TRP channel sequences, mainly the N- and C-termini, are intrinsically disordered regions (IDRs). Unfortunately, very little is known about IDR ‘structure’, dynamics and function, though it has been shown that they are essential for native channel function. *

In the article “Intrinsically disordered regions in TRPV2 mediate protein-protein interactions”, Raghavendar R. Sanganna Gari, Grigory Tagiltsev, Ruth A. Pumroy, Yining Jiang, Martin Blackledge, Vera Y. Moiseenkova-Bell and Simon Scheuring imaged TRPV2 channels in membranes using high-speed atomic force microscopy (HS-AFM). *

The dynamic single molecule imaging capability of HS-AFM allowed the authors to visualize IDRs and revealed that N-terminal IDRs were involved in intermolecular interactions. Their work provides evidence about the ‘structure’ of the TRPV2 IDRs, and that the IDRs may mediate protein-protein interactions. *

In total, 1.5 µl of the TRPV2 reconstituted vesicles were deposited on a 1.5-mm2 freshly cleaved mica surface, which was glued with epoxy to the quartz sample stage. After 20–30 min incubation, the sample was gently rinsed with imaging buffer (20 mM Hepes, pH 8.0, 150 mM NaCl) and mounted in the HS-AFM fluid cell. All images in this study were taken using a HS-AFM operated in amplitude modulation mode using optimized scan and feedback parameters and lab-built amplitude detectors and free amplitude stabilizers. *

Short (8 µm) cantilevers (NanoWorld Ultra-Short Cantilevers for High-Speed AFM USC-F1.2-k0.15) with nominal spring constant of 0.15 N/m, resonance frequency of 0.6 MHz, and a quality factor of ∼1.5 in liquid were used. AFM probes were sharpened using oxygen plasma etching to obtain better resolution. *

Fig. 1 from “Intrinsically disordered regions in TRPV2 mediate protein-protein interactions” by Raghavendar R. Sanganna Gari et al. :TRPV2 reconstitution for HS-AFM analysis. b Overview HS-AFM images (Supplementary Movie 1) of TRPV2 (windmill-shaped molecules) in soy polar lipid membranes on mica (dark background areas). False color scale: 0–9 nm. The white oversaturated areas have a height of ~26 nm and represent likely non-ruptured small vesicles. NanoWorld-USC-F1.2-k0.15 AFM probes were used for the HS-AFM — Fig. 1 from “Intrinsically disordered regions in TRPV2 mediate protein-protein interactions” by Raghavendar R. Sanganna Gari et al. :
TRPV2 reconstitution for HS-AFM analysis.
b Overview HS-AFM images (Supplementary Movie 1) of TRPV2 (windmill-shaped molecules) in soy polar lipid membranes on mica (dark background areas). False color scale: 0–9 nm. The white oversaturated areas have a height of ~26 nm and represent likely non-ruptured small vesicles.

*Raghavendar R. Sanganna Gari, Grigory Tagiltsev, Ruth A. Pumroy, Yining Jiang, Martin Blackledge, Vera Y. Moiseenkova-Bell and Simon Scheuring
Intrinsically disordered regions in TRPV2 mediate protein-protein interactions
Communications Biology volume 6, Article number: 966 (2023)
DOI: https://doi.org/10.1038/s42003-023-05343-7

Please follow this external link to read the full article: https://rdcu.be/dnNba

The article “Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response” by Raghavendar R. Sanganna Gari, Grigory Tagiltsev, Ruth A. Pumroy, Yining Jiang, Martin Blackledge, Vera Y. Moiseenkova-Bell and Simon Scheuring is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.