Tag: biology AFM probes

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/.

Membrane sculpting by curved DNA origami scaffolds

In the article “Membrane sculpting by curved DNA origami scaffolds” the authors show that “dependent on curvature, membrane affinity and surface density, DNA origami coats can indeed reproduce the activity of membrane-sculpting proteins such as BAR, suggesting exciting perspectives for using them in bottom-up approaches towards minimal biomimetic cellular machineries.”*

The AFM images for this article were taken in high-speed AC mode using NanoWorld Ultra-Short Cantilevers of the USC-F0.3-k0.3 type.

Supplementary Figure 5 b from Membrane sculpting by curved DNA origami scaffolds: Characterization of folded DNA origami nanoscaffolds. ( a ) Assembly of the folded bare origami structures L, Q, H was initially assessed via agarose gel (2%) electrophoresis analysis. Lanes containing marker DNA ladder (1kb) and M13 single - stranded p7249 sc affold (Sc) were also included. ( b ) Structure of folded bare origami L, Q and H was further validated using negative - stain transmission electron microscopy (TEM ; scale bar s : 100 nm ) and atomic force microscopy (AFM ; scale bar s : 200nm ).
Supplementary Figure 5 b from “Membrane sculpting by curved DNA origami scaffolds”:
Characterization of folded DNA origami nanoscaffolds.
b) Structure of folded bare origami L, Q and H was further validated using negative-stain transmission electron microscopy (TEM; scale bars: 100 nm) and atomic force microscopy (AFM; scale bars: 200nm).

*Henri G. Franquelim, Alena Khmelinskaia, Jean-Philippe Sobczak, Hendrik Dietz, Petra Schwille
Membrane sculpting by curved DNA origami scaffolds
Nature Communicationsvolume 9, Article number: 811 (2018)
DOI: https://doi.org/10.1038/s41467-018-03198-9

Please follow this link to the full article: https://rdcu.be/8zZi

Open Access: The article “Membrane sculpting by curved DNA origami scaffolds” by Franquelim 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 http://creativecommons.org/licenses/by/4.0/.

Vertical Light Sheet Enhanced Side-View Imaging for AFM Cell Mechanics Studies

Atomic Force Microscopy is a powerful tool for evaluating cell mechanics.
In the recent article “Vertical Light Sheet Enhanced Side-View Imaging for AFM Cell Mechanics Studies” by Kellie Beicker, E. Timothy O’Brien III, Michael R. Falvo, Richard Superfine published in Nature Scientific Reports, the authors combine sideways imaging and a vertical light sheet illumination system integrated with AFM to achieve their results.

5 µm polystyrene beads attached to NanoWorld Arrow-TL1 tipless AFM probes were used.

igure 5 from Vertical Light Sheet Enhanced Side-View Imaging for AFM Cell Mechanics Studies: Membrane and nuclear displacements observed in response to force-rupture events between the AFM-tip and cell membrane. (a) Retraction portion of force-indentation curve with important points (A-G) identified. A, the point of zero force application to the cell, B-F, force-rupture peaks, and G, after bead releases from cell. (b) A closer examination of peaks E and F with sub-peaks of the E rupture event identified. No point is shown for E1 because this is the frame immediately following Peak E0. Inset indicates regions where displacement is measured between points E and F highlighted in green. These regions were determined through difference imaging using frames taken at E and F. (c) Regions of cell displacements determined through difference imaging highlighted in green for the sub-peaks indicated in (b). Yellow dashed lines indicate outline of AFM mounted bead. Scale bars = 5 um. NanoWorld Arrow-TL1 tipless AFM cantilevers were used.
Figure 5 from Beicker et. al Vertical Light Sheet Enhanced Side-View Imaging for AFM Cell Mechanics Studies: Membrane and nuclear displacements observed in response to force-rupture events between the AFM-tip and cell membrane. (a) Retraction portion of force-indentation curve with important points (A-G) identified. A, the point of zero force application to the cell, B-F, force-rupture peaks, and G, after bead releases from cell. (b) A closer examination of peaks E and F with sub-peaks of the E rupture event identified. No point is shown for E1 because this is the frame immediately following Peak E0. Inset indicates regions where displacement is measured between points E and F highlighted in green. These regions were determined through difference imaging using frames taken at E and F. (c) Regions of cell displacements determined through difference imaging highlighted in green for the sub-peaks indicated in (b). Yellow dashed lines indicate outline of AFM mounted bead. Scale bars = 5 um.

Kellie Beicker, E. Timothy O’Brien III, Michael R. Falvo, Richard Superfine
Vertical Light Sheet Enhanced Side-View Imaging for AFM Cell Mechanics Studies
Nature Scientific Reports,  8, Article number: 1504 (2018)
DOI: https://doi.org/10.1038/s41598-018-19791-3

For the full article please follow this external link: https://rdcu.be/59FM

The article Beicker et. al, Vertical Light Sheet Enhanced Side-View Imaging for AFM Cell Mechanics Studies 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/.