Tag Archives: cell biology

Simultaneous co-localized super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for the life sciences

Correlating data from different microscopy techniques holds the potential to discover new facets of signalling events in cellular biology.*

In the article “Simultaneous co-localized super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for the life sciences” Ana I. Gómez-Varela, Dimitar R. Stamov, Adelaide Miranda, Rosana Alves, Cláudia Barata-Antunes, Daphné Dambournet, David G. Drubin, Sandra Paiva and Pieter A. A. De Beule report for the first time a hardware set-up capable of achieving simultaneous co-localized imaging of spatially correlated far-field super-resolution fluorescence microscopy and atomic force microscopy, a feat only obtained until now by fluorescence microscopy set-ups with spatial resolution restricted by the Abbe diffraction limit.*

The authors detail system integration and demonstrate system performance using sub-resolution fluorescent beads and applied to a test sample consisting of human bone osteosarcoma epithelial cells, with plasma membrane transporter 1 (MCT1) tagged with an enhanced green fluorescent protein (EGFP) at the N-terminal.*

The simultaneous operation of AFM and super-resolution fluorescence microscopy technique provides a powerful observational tool on the nanoscale, albeit data acquisition is typically obstructed by a series of integration problems. The authors of the above-mentioned article believe that the combination of SR-SIM with AFM presents one of the most promising schemes enabling simultaneous co-localized imaging, allowing the recording of nanomechanical data and cellular dynamics visualization at the same time.*

For measurements on cells in liquid NANOSENSORS™ uniqprobe qp-BioAC-CI AFM probes ( CB1 ) with a nominal resonance frequency of 90 kHz (in air), spring constant of 0.3 Nm−1, partial gold coating on the detector side, and quartz-like circular symmetric hyperbolic (double-concaved) tips with ROC of 30 nm were used. The corresponding AFM areas for the cell images were acquired with a Z-cantilever velocity of 250 μms−1 at a max Z-length of 1.5 μm, resulting in an acquisition time (based on the number of pixels) for Figs. 2, 3, 4 of ca. 13, 8 and 15 min respectively.*

Figure 4 a and b from Ana I. Gómez-Varela et al “Simultaneous co-localized super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for the life sciences :  Simultaneous SR-SIM/AFM acquisition. The AFM measurements were carried out on fixed U2OS cells in medium/buffer with (a) and without N-SIM illumination (b). For convenience and enhanced feature/noise contrast, both AFM topography images in the SR-SIM/AFM overlays are displayed with an edge detection algorithm using a pixel difference operator in X. The topography images from Petri dish surface on three positions (labelled in the figures) were planefit (1st order polynomial function) to compensate for tilts in the sample surface, and subjected to surface roughness analysis Please have a look at the full article to view the full figure. https://rdcu.be/b4Iot
Figure 4 a and b from Ana I. Gómez-Varela et al “Simultaneous co-localized super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for the life sciences : Simultaneous SR-SIM/AFM acquisition. The AFM measurements were carried out on fixed U2OS cells in medium/buffer with (a) and without N-SIM illumination (b). For convenience and enhanced feature/noise contrast, both AFM topography images in the SR-SIM/AFM overlays are displayed with an edge detection algorithm using a pixel difference operator in X. The topography images from Petri dish surface on three positions (labelled in the figures) were planefit (1st order polynomial function) to compensate for tilts in the sample surface, and subjected to surface roughness analysis. Please have a look at the full article to view the full figure. https://rdcu.be/b4Iot

*Ana I. Gómez-Varela, Dimitar R. Stamov, Adelaide Miranda, Rosana Alves, Cláudia Barata-Antunes, Daphné Dambournet, David G. Drubin, Sandra Paiva and Pieter A. A. De Beule
Simultaneous co-localized super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for the life sciences
Nature Scientific Reports volume 10, Article number: 1122 (2020)
DOI: https://doi.org/10.1038/s41598-020-57885-z

Please follow this external link to read the full article https://rdcu.be/b4Iot

Open Access: The article “Simultaneous co-localized super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for the life sciences” by Ana I. Gómez-Varela, Dimitar R. Stamov, Adelaide Miranda, Rosana Alves, Cláudia Barata-Antunes, Daphné Dambournet, David G. Drubin, Sandra Paiva and Pieter A. A. De Beule 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/.

Nonlinear Biomechanical Characteristics of Deep Deformation of Native RBC Membranes in Normal State and under Modifier Action

The mechanical properties and structural organization of membranes determine the functional state of red blood cells (RBCs). Deformability is one of the key physiological and biophysical indicators of RBC. Changes of the mechanical characteristics of cell membranes can lead to a decrease in the rate of capillary blood flow and to development of stagnant phenomena in the microcirculation, and it can also reduce the amount of oxygen delivered to the tissues.*

In the article “Nonlinear Biomechanical Characteristics of Deep Deformation of Native RBC Membranes in Normal State and under Modifier Action” Elena Kozlova, Aleksandr Chernysh, Ekaterina Manchenko, Viktoria Sergunova and Viktor Moroz describe how they evaluated the ability of membranes of native human red blood cells (RBCs) to bend into the cell to a depth comparable in size with physiological deformations using the methods of atomic force microscopy ( AFM ) and atomic force spectroscopy ( AFS ).*

As a true estimation of the elastic properties of RBC membranes can be obtained only by measurement of native cell properties the aim of the experiments was to study nonlinear mechanical characteristics of deep deformation of native RBC membranes in normal state and under the action of modifiers, in vitro to make sure that the result would be the closest to the characteristics of a living biological object.*

NANOSENSORS™ rounded AFM tips of the type SD-R150-T3L450B with a typical tip radius of 150 nm from the NANOSENSORS Special Developments List were used to measure the deformation of the RBC membrane by atomic force spectroscopy ( AFS ).*


Figure 5.2. (c) from “Nonlinear Biomechanical Characteristics of Deep Deformation of Native RBC Membranes in Normal State and under Modifier Action “ by Elena Kozlova et al.:
 Bending of membranes under the action of force F for stiff (1) and soft (2) membranes; F is the force acting on the membrane from the probe, Z is the vertical displacement of the piezoscanner, h is the depth of the membrane bending into RBC, PBS is the phosphate buffer solution, and rd is the bending radius of the membrane.

*Elena Kozlova, Aleksandr Chernysh, Ekaterina Manchenko, Viktoria Sergunova, and Viktor Moroz
Nonlinear Biomechanical Characteristics of Deep Deformation of Native RBC Membranes in Normal State and under Modifier Action
Scanning, Volume 2018, Article ID 1810585, 13 pages
Doi: https://doi.org/10.1155/2018/1810585

Please follow this external link to read the full article: https://www.hindawi.com/journals/scanning/2018/1810585/

Open Access The article « Nonlinear Biomechanical Characteristics of Deep Deformation of Native RBC Membranes in Normal State and under Modifier Action ” by Elena Kozlova, Aleksandr Chernysh, Ekaterina Manchenko, Viktoria Sergunova, and Viktor Moroz 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/.

Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy

The dielectric permittivity of membranes is important for many fundamental electrophysiological functions like selective transport in ion channels, action potential propagation and energy generation.*

In their article “Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy” George Gramse, Andreas Schönhals and Ferry Kienberger investigate the nearfield dipole mobility of protein membranes in a wide frequency range from 3 kHz to 10 GHz.*

They achieved their results by adding the frequency as a second fundamental dimension to quantitative dielectric microscopy thereby demonstrating the possibilities of broadband dielectric microscopy for the investigation of dynamic processes in cell bioelectricity at the individual molecular level. Furthermore, the technique may also shed light on local dynamic processes in related materials science applications like semiconductor research or nano-electronics.*

All AFM measurements were carried out at 25 °C using a NANOSENSORS Platinum Silicide AFM probe ( PtSi-FM ).

Fig. 2 from “Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy” by Gramse et al.: image a) shows the AFM topography and image b) shows the corresponding C′′(z)/C′′dry(z) image obtained in lift mode at z = 10 nm above the last scan line and at a frequency of ω = 10 kHz (inset at 1 MHz). The corresponding topography and C′′(z)/C′′dry(z) profile lines are shown in  image c). Solid lines correspond to profile lines at 10 kHz and the dashed line to 1 MHz. Image d) shows the normalized dielectric spectra on the substrate and protein membrane at constant height z′ = 15 nm and lift mode z = 15 nm. Black solid lines represent fitting with eqn (1) and (2). image e) shows the resulting complex dielectric functions ε′r(f) and ε′′r(f)2 (using the relation ε′′r(f) = −(π/2∂)ε′r/∂ln(2πf)38). All measurements are carried out at 25 °C using conductive and wear-resistant Platinum Silicide AFM probes  (PtSi-FM ) from NANOSENSORS (Germany). Humidity was changed and left to stabilize for 2–3 hours. Imaging conditions were adjusted to maintain the lift distance for the dielectric images identical.

Fig. 2 from “Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy” by Gramse et al.: (a) AFM topography and (b) corresponding C′′(z)/C′′dry(z) image obtained in lift mode at z = 10 nm above the last scan line and at a frequency of ω = 10 kHz (inset at 1 MHz). The corresponding topography and C′′(z)/C′′dry(z) profile lines are shown in (c). Solid lines correspond to profile lines at 10 kHz and the dashed line to 1 MHz. (d) Normalized dielectric spectra on the substrate and protein membrane at constant height z′ = 15 nm and lift mode z = 15 nm. Black solid lines represent fitting with eqn (1) and (2). (e). Resulting complex dielectric functions ε′r(f) and ε′′r(f)2 (using the relation ε′′r(f) = −(π/2∂)ε′r/∂ln(2πf)38).
All measurements are carried out at 25 °C using PtSi-FM tips from NANOSENSORS (Germany). Humidity was changed and left to stabilize for 2–3 hours. Imaging conditions were adjusted to maintain the lift distance for the dielectric images identical.

*Georg Gramse, Andreas Schönhals, Ferry Kienberger
Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy
Nanoscale, 2019, 11, 4303-4309
DOI: 10.1039/C8NR05880F

Please follow this external link for the full article: https://pubs.rsc.org/en/content/articlehtml/2019/nr/c8nr05880f

Open Access The article “Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy” by George Gramse, Andreas Schönhals and Ferry Kienberger is licensed under a Creative Commons Attribution 3.0 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. To view a copy of this license, visit https://creativecommons.org/licenses/by/3.0/