AFM topography imaging

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/.

Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes

In the last decades enormous advances have been made in characterizing the atomic and molecular structure of respiratory chain supercomplexes. *

However, it still remains a challenge to stitch this refined spatial atomistic description with functional information provided by biochemical studies of isolated protein material. Development of functional assays that detect respiratory chain complexes in their native membrane environment contribute to address the open questions related to the role played by their association and interactions. *

In the article “Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes” Daniel G. Cava, Julia Alvarez-Malmagro, Paolo Natale, Sandra López-Calcerrada, Iván López-Montero, Cristina Ugalde, Jose Maria Abad, Marcos Pita, Antonio L. De Lacey and Marisela Vélez present a characterization assay in which a functionalized gold electrode is modified with mitochondrial membrane fragments that allows monitoring electrochemically the activity of different respiratory chain complexes immersed in the mitochondrial membrane. *

Daniel G. Cava et al. measure the intensity of the reducing current of the electron mediator CoQ1 at the electrode surface and its variation upon addition of the corresponding enzymatic substrates. The activities of Complex I, Complex II and Complex III were monitored by the way in which they reduce the current, reflecting the amount of quinone reduced by the complexes in the presence of their substrates. *

The authors detect that CoQ1H2 produced by Complex I remains partially trapped within the membrane and is more easily oxidized by Complex III or the electrode than the quinone reduced by Complex II. *

Atomic Force Microscopy (AFM) was used to image the topography of the membrane modified electrode. NanoWorld Pyrex-Nitride Silicon-Nitride AFM probes (PNP-DB, diving board shaped cantilevers, the short AFM cantilever with a typical force constant of 0.48 N/m and 67 kHz resonance frequency) were used. *

The surfaces analysed were the electrodes. The two surfaces imaged are the same previously polished electrodes used for electrochemical measurements. The microscope sample holder was adapted in-home to support the electrodes. Two surfaces were analysed: the polished gold functionalized with 4-aminothiophenol and the electrode after incubation with mitochondria subparticles prepared similarly to the electrodes used for the electrochemical measurements.*

Fig. 2 from Daniel G. Cava et al 2024 “Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes”
QCM and AFM characterization of modified gold. Panel A shows the frequency (left, black) and dissipation (right red) changes detected on a gold covered quartz crystal previously modified with a 4-ATP after injection in the chamber of the mitochondrial fragments at the time point indicated by the thick arrow. Panel B show AFM images of the surface topography of a modified gold electrode before (left) and after (right)incubation with the mitochondrial membrane. The inset below shows the height profile of the lines indicated in the images.

*Daniel G. Cava, Julia Alvarez-Malmagro, Paolo Natale, Sandra López-Calcerrada, Iván López-Montero, Cristina Ugalde, Jose Maria Abad, Marcos Pita, Antonio L. De Lacey and Marisela Vélez
Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes
Electrochimica Acta, Volume 484, 20 April 2024, 144042
DOI: https://doi.org/10.1016/j.electacta.2024.144042

The article “Electrochemical detection of quinone reduced by Complex I Complex II and Complex III in full mitochondrial membranes” by Daniel G. Cava, Julia Alvarez-Malmagro, Paolo Natale, Sandra López-Calcerrada, Iván López-Montero, Cristina Ugalde, Jose Maria Abad, Marcos Pita, Antonio L. De Lacey and Marisela Vélez 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/.

C-Axis Textured, 2–3 μm Thick Al0.75Sc0.25N Films Grown on Chemically Formed TiN/Ti Seeding Layers for MEMS Applications

In the search for lead-free, Si-microfabrication-compatible piezoelectric materials, thin films of scandium-doped aluminum nitride (Al,Sc)N are of great interest for use in actuators, energy harvesting, and micro-electromechanical-systems (MEMS).*

While the piezoelectric response of AlN increases upon doping with Sc, difficulties are encountered during film preparation because, as bulk solids with completely different structures and large differences in cation radii, ScN (rock salt, cubic) and AlN (wurtzite, hexagonal) are immiscible. *

Consequently, (Al,Sc)N is inherently thermodynamically unstable and prone to phase segregation. Film preparation is further complicated by the technological requirement for polar [001] or [00 1̲] out-of-plane texture, which is achieved using a seeding layer.*

In the article “C-Axis Textured, 2–3 μm Thick Al0.75Sc0.25N Films Grown on Chemically Formed TiN/Ti Seeding Layers for MEMS Applications” Asaf Cohen, Hagai Cohen, Sidney R. Cohen, Sergey Khodorov, Yishay Feldman, Anna Kossoy, Ifat Kaplan-Ashiri, Anatoly Frenkel, Ellen Wachtel, Igor Lubomirsky and David Ehre propose a protocol for successfully depositing [001] textured, 2–3 µm thick films of Al0.75Sc0.25N.*

The procedure relies on the fact that sputtered Ti is [001]-textured α-phase (hcp). Diffusion of nitrogen ions into the α-Ti film during reactive sputtering of Al0.75,Sc0.25N likely forms a [111]-oriented TiN intermediate layer. The lattice mismatch of this very thin film with Al0.75Sc0.25N is ~3.7%, providing excellent conditions for epitaxial growth. In contrast to earlier reports, the Al0.75Sc0.25N films prepared in the current study are Al-terminated. Low growth stress (<100 MPa) allows films up to 3 µm thick to be deposited without loss of orientation or decrease in piezoelectric coefficient. *

An advantage of the proposed technique is that it is compatible with a variety of substrates commonly used for actuators or MEMS, as demonstrated here for both Si wafers and D263 borosilicate glass. Additionally, thicker films can potentially lead to increased piezoelectric stress/strain by supporting application of higher voltage, but without increase in the magnitude of the electric field. *

SEM, AFM, EDS, XRD and XPS techniques were used for the film characterization. For the nanoscale topography maps with atomic force microscopy (AFM) NanoWorld Pyrex-Nitride series PNP-TRS silicon nitride AFM probes were used in peak-force tapping^1® mode. *

Figure 3 from Asaf Cohen et al. “C-Axis Textured, 2–3 μm Thick Al0.75Sc0.25N Films Grown on Chemically Formed TiN/Ti Seeding Layers for MEMS Applications”: AFM images of (a) a (100) silicon wafer following cleaning procedures as described in the Materials and Methods section; (b) 50 nm-thick Ti film deposited on the wafer at 300 K; (c) the same film following exposure to N2 plasma at 673 K for 30 min. For the nanoscale topography maps with atomic force microscopy (AFM) NanoWorld Pyrex-Nitride PNP-TRS AFM probes were used in peak-force tapping mode1. * — Figure 3 from Asaf Cohen et al. “C-Axis Textured, 2–3 μm Thick Al0.75Sc0.25N Films Grown on Chemically Formed TiN/Ti Seeding Layers for MEMS Applications”: AFM images of (a) a (100) silicon wafer following cleaning procedures as described in the Materials and Methods section; (b) 50 nm-thick Ti film deposited on the wafer at 300 K; (c) the same film following exposure to N2 plasma at 673 K for 30 min.

*Asaf Cohen, Hagai Cohen, Sidney R. Cohen, Sergey Khodorov, Yishay Feldman, Anna Kossoy, Ifat Kaplan-Ashiri, Anatoly Frenkel, Ellen Wachtel, Igor Lubomirsky and David Ehre
C-Axis Textured, 2–3 μm Thick Al0.75Sc0.25N Films Grown on Chemically Formed TiN/Ti Seeding Layers for MEMS Applications
Sensors 2022, 22, 7041
DOI: https://doi.org/10.3390/s22187041

The article “C-Axis Textured, 2–3 μm Thick Al0.75Sc0.25N Films Grown on Chemically Formed TiN/Ti Seeding Layers for MEMS Applications” by Asaf Cohen, Hagai Cohen, Sidney R. Cohen, Sergey Khodorov, Yishay Feldman, Anna Kossoy, Ifat Kaplan-Ashiri, Anatoly Frenkel, Ellen Wachtel, Igor Lubomirsky and David Ehre 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/.

¹Peak Force Tapping® is a registered trademark of Bruker Corporation.