Monomeric collagen (1μg/ml) was mixed with monomeric DDR2-V5-His or dimeric DDR2-Fc proteins (1 μg/ml in DDR2 concentration) in ice cold PBS and incubated at 4 °C for 4 hrs. As a control, the recombinant DDR2 ECD proteins (without collagen) were also incubated in PBS under similar conditions. After 4 hrs of incubation, the samples were aliquoted onto chilled and freshly cleaved mica substrates, incubated for 5 min, washed and air dried and subjected to AFM imaging using the Multimode AFM (Digital Instruments, Santa Barbara, CA) as described earlier[18 (link)]. AFM imaging was performed in tapping mode in ambient air using NSC15 cantilevers (Micromasch, Estonia) with a nominal spring constant of 40 Nm−1. Both height and amplitude images were recorded at 512 lines per scan direction. Topographic heights of DDR2 proteins and collagen filaments were measured from AFM images, by the section analysis feature of the Nanoscope software. At least n= 50 particles were analyzed using three identical replicates for each sample type (except for DDR2-V5-His bound to collagen for which n= 22 due to the low number of binding events). Student’s unpaired two-tailed t-test was used to determine statistically significant differences in height measurements across samples. A p<0.05 was considered significant.
Multimode afm
Manufactured by Digital Instruments
Sourced in United States
The Multimode AFM is a versatile scanning probe microscope capable of imaging and characterizing a wide range of samples at the nanoscale. It utilizes atomic force microscopy (AFM) technology to provide high-resolution topographical and material property information.
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Lab products found in correlation
Nanoscope 3 controller, by Digital Instruments (5 mentions)
Nanoscope iiia controller, by Digital Instruments (2 mentions)
Fastscan d probes, by Bruker (2 mentions)
Tr400psa silicon nitride probes, by Olympus (2 mentions)
Fast scan bio afm, by Bruker (2 mentions)
Zeta plus analyzer, by Brookhaven Instruments (1 mentions)
Nanoscope 4 controller, by Digital Instruments (1 mentions)
Mpp 11100, by Bruker (1 mentions)
Snl 10 silicon tip, by Bruker (1 mentions)
Axiovert 200m epifluorescence microscope, by Zeiss (1 mentions)
Tap300al g cantilever, by Budget Sensors (1 mentions)
Escalab 250, by Thermo Fisher Scientific (1 mentions)
Nicolet 6700 spectrometer, by Thermo Fisher Scientific (1 mentions)
Hr800 spectrometer, by Horiba (1 mentions)
12 protocols using multimode afm
1
Characterization of DDR2-Collagen Interaction
2
Atomic Force Microscopy of Protein Adsorption
3
Synthesis of Prussian Blue Nanoparticles
4
Tapping Mode AFM Imaging
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AFM images were obtained in tapping mode under buffer using a Digital Instruments Multimode AFM with a Nanoscope (R) III controller. Bruker SNL-10 silicon tip on a nitride lever with a spring constant of ≈ 0.24 N/m were used for imaging, with a drive frequency of ≈ 9–10 kHz. AFM buffer consisted of the same buffer used for annealing unless otherwise noted.
5
Atomic Force Microscopy Imaging of Biomolecules
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1-5 μl of transcription product was mixed with 40 μl AFM dilution buffer (12.5 mM Mg(OAc)2, 40mM KCl, 40mM NaCl, Tris-Borate pH 7.8) directly on the surface of a freshly-cleaved mica puck. Mixing is performed by vigorously pumping a 200 μl pipette tip ten times, before removing and discarding the fluid. The mica was washed with a solution of 60 mM NiCl2. Most AFM images were collected using a Multimode AFM (Digital Instruments) with a Nanoscope IIIA controller and a J-scanner. Olympus TR400PSA silicon nitride probes with a spring constant of ~.08 N/m were used for imaging, with a drive frequency of ~6-9 kHz. AFM in Supplementary Figs. 10 and 11 were collected with a Bruker Fastscan Bio AFM (Bruker) under buffer using FastScan-D probes (Bruker).
6
Atomic Force Microscopy Analysis of Corneocytes
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Corneocytes from patients were analyzed with AFM, as previously described.21 (link) Briefly, in each case the seventh tape strip was subjected to AFM measurements carried out with a Multimode AFM equipped with the Nanoscope III controller and software version 5.30sr3 (Digital Instruments, Santa Barbara, Calif). Silicon-nitride tips on V-shaped gold-coated cantilevers were used (0.01 N/m, MLCT; VEECO, Mannheim, Germany). Imaging was performed at ambient temperature with forces of less than 1 nN and 1 to 3 scan lines per second (1-3 Hz) with 512 × 512 pixel resolution. For texture analysis, subcellular scan areas of 20 μm2 were recorded. Ten random images were analyzed from each sample.
Topographic cell-surface data were analyzed with the nAnostic method, applying custom-built proprietary algorithms (Serend-ip GmbH, Munster, Germany). The principle of this method has been described elsewhere.22 (link) Briefly, each nanostructure protruding from the mean surface level was morphometrically evaluated. These objects were then filtered by size and shape through computer vision. At this stage, only structures of positive local deviational volume smaller than 500 nm in height and with an area of less than 1 μm2 were considered. The DTI score is the count of identified objects per image (a mean value from 10 randomly recorded images).
Topographic cell-surface data were analyzed with the nAnostic method, applying custom-built proprietary algorithms (Serend-ip GmbH, Munster, Germany). The principle of this method has been described elsewhere.22 (link) Briefly, each nanostructure protruding from the mean surface level was morphometrically evaluated. These objects were then filtered by size and shape through computer vision. At this stage, only structures of positive local deviational volume smaller than 500 nm in height and with an area of less than 1 μm2 were considered. The DTI score is the count of identified objects per image (a mean value from 10 randomly recorded images).
7
Characterization of Aβ Oligomer Morphology
8
Atomic Force Microscopy Imaging of Biomolecules
9
Quantitative Topography of Chemically Fixed Cells by AFM
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Contact mode Atomic Force Microscopy (AFM) on cultivated cells was performed as described before [63 (link)]. In this study, cells were chemically stabilized by glutardialdehyde fixation (1% final concentration). Briefly, AFM measurements were carried out in PBS-buffered solution (pH 7.4) using a Multimode AFM equipped with Nanoscope III controller and software version 5.30 sr3 (Digital Instruments, Santa Barbara, CA, USA). Silicon-nitride tips on V-shaped gold-coated cantilevers were used (0.01 N/m, MLCT, VEECO, Mannheim, Germany). Imaging was performed at ambient temperature with forces <1 nN at 1–3 scan lines per second (1–3 Hz) with 512*512 pixels resolution. For texture analysis, subcellular scan areas of 10μm2 are recorded. Topographical data of the cell surfaces were analyzed using the nAnostic™-method applying custom-built, proprietary algorithms (Serend-ip GmbH, Münster, Germany). The method principle has been described before [64 ]. Briefly, each nanostructure protruding from the mean surface level is morphometrically evaluated. Then, they are filtered by their size and shape through computer vision; here, only structures of positive local deviational volume (LDV), smaller than 103 nm in height and an area smaller than 106 nm were considered. Values are given for the average depth of such objects (per image) and the sum of their deviational volumes (LDVs).
10
Eu-psa-10 Particle Characterization by AFM
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The measurements were acquired using a Digital Instruments Multimode AFM with Nanoscope III controller. AFM images were acquired in Tapping mode with all parameters including set-point, scan rate and feedback gains adjusted to optimize image quality and minimize the force between the probe and sample. The Eu-psa-10 particles present in the ultrasonicated ethanolic suspensions were collected and dispersed by drop casting the colloidal suspensions onto a glass flat substrate for characterization under AFM.
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