For AFM imaging of a clean silicon wafer immerse in a 300 μM solution of peptide in 10 mM MOPS (pH 7.4) and incubated overnight at room temperature was analyzed. Topographic, amplitude and phase AFM images were recorded using tapping mode AFM on a Cypher Instrument (Asylum Research). All the AFM images were flattened with a first order line-wise correction fit. AFM tips used were super-sharp silicon probes (Nanosensors; resonant frequency ~330 kHz, tip radius of curvature, 5 nm, force constant 42 N/m). Images were processed using proprietary software, SPIP 6.0.2.
Cypher instrument
Manufactured by Oxford Instruments
The Cypher instrument is a high-performance atomic force microscope (AFM) designed for nanoscale imaging, characterization, and analysis. It provides precise control and measurement of surface topography and properties at the nanometer scale.
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6 protocols using cypher instrument
1
AFM Imaging of Peptide-Coated Silicon Wafer
2
Visualizing WT MT1-MMP Ectodomain on Mica
3
Atomic Force Microscopy of Peptides
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AFM images were obtained for a drop of peptide solution (8 µL, 100 µM) placed on a clean silicon wafer with the buffer excess removed by blotting. All measurements were carried out on a Cypher instrument (Asylum research) in a tapping mode using super-sharp PPP-NCHR type cantilevers (Nanosensors™: resonant frequency ~330 kHz, tip radius of curvature <5 nm, force constant 42 N m−1). Images were flattened via line-by-line subtraction of first-order fits to the background, using SPIP software, version 6.0.2.
4
Atomic Force Microscopy of Cochleates
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50 µl precipitated pellet was harvested from the cochleate sample and diluted with 200 µl purified water. 50 µl of this sample was applied to the surface of freshly cleaved mica and, after 1 minute incbation, washed gently with purified water and dried in a stream of N 2 gas of high purity. Cochleates were imaged with a Cypher instrument (Asylum Research, Santa Barbara, CA) with 0.4 to 1 Hz line-scanning rate in air. A silicon cantilever (OMCL AC-160TS, Olympus, Japan) was used in non-contact mode, oscillated at its resonance frequency (300-320 kHz, typically). Force spectra were taken in contact mode at defined locations of previously scanned areas and at constant 1 µm/s velocity. The cantilevers (stiffness typically around 35 N/m) were calibrated by the thermal method. 27 Temperature during the measurements was 29±1°C.
5
Atomic Force Microscopy of Biological Samples
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Atomic force microscope (AFM) images were recorded with a Cypher instrument (Asylum Research, Santa Barbara, CA) by scanning the samples in fluid with a gold-coated silicon nitride cantilever (Olympus Biolever, A lever, typical spring constant: 30 pN/nm). 100 µl sample was applied on a cleaned borosilicate glass coverslip and incubated in a vapor chamber at 23±1 ˚C. Non-contact-mode images were recorded at a linescan rate of 0.5-1 Hz. All measurements were carried out at 28±1 °C. Images were analyzed by using the built-in algorithms of the AFM driving software (IgorPro, WaveMetrics, Inc., Lake Oswego, OR).
6
Atomic Force Microscopy of Cochleates
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For AFM imaging 10 µl of the control or dispersed cochleate sample was diluted with 50 µl 100 mM TRIS (pH 7.4) then applied to the substrate and, after 1 minute incubation, washed gently with MilliQ water and dried in a stream of N 2 gas. For control cochleates freshly cleaved mica sheets were used as substrate. In the case of dispersed cochleates we used mica sheets pre-coated with 100 μl 0.01% poly-L-lysine for 20 min. Cochleates were imaged with a Cypher instrument (Asylum Research, Santa Barbara, CA) with 0.5 to 2.5 Hz line-scanning rate in air at 29±1°C. A silicon cantilever (OMCL AC-160TS, Olympus, Japan) was used in non-contact mode, oscillated at its resonance frequency (300-320 kHz, typically). Images with 512 x 512 pixel dimensions were collected and analyzed with the built-in algorithms of the AFM driver software.
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