Tag: biology

High-speed AFM height spectroscopy reveals microsecond-dynamics of unlabeled biomolecules

In their recent publication “High-speed AFM height spectroscopy reveals μs-dynamics of unlabeled biomolecules” in Nature Communications George R. Heath and Simon Sheuring develop and apply HS-AFM height spectroscopy (HS-AFM-HS, a technique inspired by fluorescence spectroscopy), a technique whereby the AFM tip is held at a fixed x–y position and  the height fluctuations under the tip in z-direction with Angstrom spatial and 10µs temporal resolution are monitored.

They demonstrate “how this technique can be used to simultaneously measure surface concentrations, diffusion rates and oligomer sizes of highly mobile annexin-V molecules during membrane-binding and self-assembly at model membranes and derive its kinetic and energetic terms. Additionally, HS-AFM-HS at specific positions in the annexin lattice where the freedom of movement is restricted to rotation allowed determination of the interaction free energies of protein-protein contacts.”* The applicability of this technique is wide and is discussed at the end of the publication.

NanoWorld Ultra-Short Cantilevers (USC) for Fast-/High-Speed AFM  ( USC-F1.2-k0.15 ) were used.

Congratulations to the authors to this publication which pushes the speed limits of AFM even further!

Increasing the temporal resolution of HS-AFM by reducing the dimensionality of data acquisition. a HS-AFM image of a DOPC/DOPS (8:2) membrane in the presence of annexin-V and NP-EGTA-caged Ca2+. Blue arrows illustrate the slow- (vertical) and the fast-scan axis (horizontal). Images can be captured at up to 10–20 frames s−1. b HS-AFM movie frames of A5 membrane-binding, self-assembly and formation of p6 2D-crystals upon UV-illumination induced Ca2+-release. c Average height/time trace of the membrane area in b. d Averaged HS-AFM image of an A5 p6-lattice overlaid with the subsequent line scanning kymograph, obtained by scanning repeatedly the central x-direction line as illustrated by the blue arrow with a maximum rate of 1000–2000 lines s−1. e Line scanning kymograph across one protomer of the non-p6 trimer, marked by * in d and e at a rate of 417 lines s−1 (2.4 ms per line). f Histogram of state dwell-times of the molecule in e. g HS-AFM image of an A5 p6-lattice partially covering a DOPC/DOPS (8:2) SLB surface during self-assembly. HS-AFM height spectroscopy (HS-AFM-HS) is performed following halting the x- and y-piezos to capture height information at a fixed position at the center of the image (illustrated by the target). h Schematic showing the principle of HS-AFM-HS. The AFM tip is oscillated in z at a fixed x,y-position, detecting single molecule dynamics such as diffusion under the tip. i Height/time trace obtained by HS-AFM-HS with the tip positioned at the center of image (g). The height/time trace allows determination of the local A5 concentration analyzing the time fraction of the occurrence of height peaks. j Dwell-time analysis of each height peak of diffusing A5 from 60 s height/time data and subsequent fitting of the distribution to multiple Gaussians (possible molecular aggregates corresponding to the fits with distinct dwell-times (τD) are shown above the graph). All scale bars: 20 nm, NanoWorld Ultra-Short Cantilevers (USC) for Fast-/High-Speed AFM ( USC-F1.2-k0.15 ) were used.
Figure 1 from “High-speed AFM height spectroscopy reveals μs-dynamics of unlabeled biomolecules”: Increasing the temporal resolution of HS-AFM by reducing the dimensionality of data acquisition. a HS-AFM image of a DOPC/DOPS (8:2) membrane in the presence of annexin-V and NP-EGTA-caged Ca2+. Blue arrows illustrate the slow- (vertical) and the fast-scan axis (horizontal). Images can be captured at up to 10–20 frames s−1. b HS-AFM movie frames of A5 membrane-binding, self-assembly and formation of p6 2D-crystals upon UV-illumination induced Ca2+-release. c Average height/time trace of the membrane area in b. d Averaged HS-AFM image of an A5 p6-lattice overlaid with the subsequent line scanning kymograph, obtained by scanning repeatedly the central x-direction line as illustrated by the blue arrow with a maximum rate of 1000–2000 lines s−1. e Line scanning kymograph across one protomer of the non-p6 trimer, marked by * in d and e at a rate of 417 lines s−1 (2.4 ms per line). f Histogram of state dwell-times of the molecule in e. g HS-AFM image of an A5 p6-lattice partially covering a DOPC/DOPS (8:2) SLB surface during self-assembly. HS-AFM height spectroscopy (HS-AFM-HS) is performed following halting the x- and y-piezos to capture height information at a fixed position at the center of the image (illustrated by the target). h Schematic showing the principle of HS-AFM-HS. The AFM tip is oscillated in z at a fixed x,y-position, detecting single molecule dynamics such as diffusion under the tip. i Height/time trace obtained by HS-AFM-HS with the tip positioned at the center of image (g). The height/time trace allows determination of the local A5 concentration analyzing the time fraction of the occurrence of height peaks. j Dwell-time analysis of each height peak of diffusing A5 from 60 s height/time data and subsequent fitting of the distribution to multiple Gaussians (possible molecular aggregates corresponding to the fits with distinct dwell-times (τD) are shown above the graph). All scale bars: 20 nm

*George R. Heath & Simon Scheuring
High-speed AFM height spectroscopy reveals μs-dynamics of unlabeled biomolecules
Nature Communicationsvolume 9, Article number: 4983 (2018)
DOI: https://doi.org/10.1038/s41467-018-07512-3

Please follow this external link for the full article: https://rdcu.be/bdaKU

Open Access The article “High-speed AFM height spectroscopy reveals μ s-dynamics of unlabeled biomolecules” by George R. Heath & 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 http://creativecommons.org/licenses/by/4.0/.

Effective gamma-ray sterilization and characterization of conductive polypyrrole biomaterials

“Conductive polymers, including polypyrrole (PPy), have been extensively explored to fabricate electrically conductive biomaterials for bioelectrodes and tissue engineering scaffolds. For their in vivo uses, a sterilization method without severe impairment of original material properties and performance is necessary. Gamma-ray radiation has been commonly applied for sterilization of medical products because of its simple and uniform sterilization without heat generation.[…]”*

In the article “Effective gamma-ray sterilization and characterization of conductive polypyrrole biomaterials” by Semin Kim et. al cited here, the authors describe the first study on gamma-ray sterilization of PPy bioelectrodes and its effects on their characteristics.

The surface topography and roughness of the PPy and γ-PPy electrodes were analyzed by atomic force microscopy. The experiments were performed using a NanoWorld Pointprobe® NCHR AFM probe. All images were acquired at a 0.3 Hz scan rate in tapping mode.

Figure 2 from “Effective gamma-ray sterilization and characterization of conductive polypyrrole biomaterials”: (a) Atomic force micrographs of PPy and γ-PPy samples irradiated with different doses of gamma-ray. (b) Average roughness (root mean square) of PPy and γ-PPy samples. NanoWorld Pointprobe NCHR AFM probes were used for the imaging.
Figure 2 from “Effective gamma-ray sterilization and characterization of conductive polypyrrole biomaterials”: (a) Atomic force micrographs of PPy and γ-PPy samples irradiated with different doses of gamma-ray. (b) Average roughness (root mean square) of PPy and γ-PPy samples.

*Semin Kim, Jin-Oh Jeong, Sanghun Lee, Jong-Seok Park, Hui-Jeong Gwon, Sung In Jeong, John George Hardy, Youn-Mook Lim, Jae Young Lee
Effective gamma-ray sterilization and characterization of conductive polypyrrole biomaterials
Nature Scientific Reports, volume 8, Article number: 3721 (2018)
DOI: https://doi.org/10.1038/s41598-018-22066-6

Please follow this external link for the full article: https://rdcu.be/bariF

Open Access: The article “Effective gamma-ray sterilization and characterization of conductive polypyrrole biomaterials” by Semin Kim et. al 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/.

 

Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

We have a month with “R” again and the shellfish season has started in the Northern Hemisphere. So we’d like to share the Nature Communications article by Petrone et. al “Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins” with you.
A NanoWorld Pointprobe® NCSTR AFM probe was used for the AFM images in this paper. This AFM probe is designed to give extra stability and accuracy during soft tapping mode imaging in order to produce higher quality AFM images while minimizing sample damage.

Supplementary Figure 16 from Petrone et. al "Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins": Atomic Force Microscopy (AFM) of mussel adhesive proteins on mica. AFM images of dry Pvfp-3α and Pvfp-5β adsorbed from 0.02 mg ml-1 solution in 5% acetic acid and 0.25 MO3 on mica. After 20 min adsorption, the mica surfaces were washed with protein -free buffer, and the AFM images show the homogenous distribution of the resulting adsorbed proteins. The height profiles for both proteins are shown in the graphs below, corresponding to the dotted red and blue lines in the respective AFM images (see black arrows).
Supplementary Figure 16 from Petrone et. al “Mussel adhesion is dictated by time-regulated
secretion and molecular conformation of mussel adhesive proteins”:
Atomic Force Microscopy (AFM) of mussel adhesive proteins on mica. AFM images of dry Pvfp-3α and Pvfp-5β adsorbed from 0.02 mg ml-1 solution in 5% acetic acid and 0.25 MO3 on mica. After 20 min adsorption, the mica surfaces were washed with protein -free buffer, and the AFM images show the homogenous distribution of the resulting adsorbed proteins. The height profiles for both proteins are shown in the graphs below, corresponding to the dotted red and blue lines in the respective AFM images (see black arrows).

Luigi Petrone, Akshita Kumar, Clarinda N. Sutanto, Navinkumar J. Patil, Srinivasaraghavan Kannan, Alagappan Palaniappan, Shahrouz Amini, Bruno Zappone, Chandra Verma, Ali Miserez
Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins
Nature Communications volume 6, Article number: 8737 (2015)
DOI https://doi.org/10.1038/ncomms9737

Please follow this external link for the full article: https://rdcu.be/5vcI

The article by Petrone, L.et al. “Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins” is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/