Cypher VRS1250 Video-Rate AFM Movies

Beyond Video-Rate: DNA at 45 fps

Loosely bound DNA was imaged at 1250 lines/s and 160×16 pixels for a frame rate of 45 fps. Dynamics can be observed in the loops in the lower left corner in the first half of the movie. In the second half, we seem more movement in the two strands on the right.

Copper Etching by Ammonium Persulfate at 24 fps

The etching of copper is commercially important in the manufacture of printed circuit boards but is also a common processing step in the deposition of large area graphene on copper. Here, an electropolished copper substrate was imaged in ammonium persulfate solution at a scan size of 130 nm and line scan rate of 1250 lines/s and 160×40 pixels for a frame rate of 24 fps. The video plays back in real-time. The movie shows a transition from unidirectional etching to a spiral etching process, apparently the result of a screw dislocation defect in the copper crystal.

Sample courtesy of Mitsubishi Materials Corporation.

The degradation of a lipid bilayer by an antimicrobial peptide was monitored by video-rate imaging at a scan size of 425 nm and a line scan rate of 1250 lines/second and 160×32 pixels, for a frame rate of 28 fps. The playback rate is 10× the acquisition rate. The lipid patch was imaged continuously for almost 7 minutes during which almost 12,000 image frames were acquired. Observation of similar patches in the absence of the peptide did not result in any degradation of the bilayer (see below).

One question that is sometimes asked about video-rate AFM is whether the dynamics observed are influenced by interactions with the scanning tip. Here we show a control experiment that was conducted prior to the work we showed previously wherein an antimicrobial peptide degraded a lipid bilayer. In the control experiment, a similar DMPC lipid bilayer sample was imaged continuously in plain buffer without the peptide at a scan size of 500 nm and a line scan rate of 1250 lines/second and 160×32 pixels, for a frame rate of 28 fps. The playback rate is 10× the acquisition rate. Note that the bilayer maintains sufficient fluidity that its shape slowly changes slightly over the course of imaging for six minutes, however, no degradation of the bilayer is observed. This helps to confirm that the antimicrobial peptide is responsible for the degradation in the other experiments. The high imaging bandwidth and exceptional stability of the Cypher VRS1250 enable it to image soft biological samples like these without any damage.

One challenge in video-rate AFM measurements is finding a region of the sample where the process is actively occurring. The Cypher VRS1250 features a large 30 µm XY scanner range that provide ample capability to offset to different regions of the sample. Both the scan size and offset can be adjusted in real-time while imaging, allowing the operator to “focus” on wherever dynamics are happening. The example shown here is another experimental run of the lipid degradation experiment. The bilayer was imaged in the presence of the antimicrobial peptide while imaging continuously at 1250 lines/second and 160×32 pixels, for a frame rate of 28 fps. The playback rate is 10× the acquisition rate. The offset and scan size are changed during the four-and-a-half-minute experiment to find areas where bilayer patches are actively degrading.

Crystallization of MoS₂-Binding Peptide at 2.6 s/frame

Researchers at the University of Washington and Pacific Northwest National Labs, led by Professor Jim De Yoreo, studied a MoS₂ binding peptide that self-assembles into 2D crystals. The process was imaged on a Cypher VRS at 2.6 seconds per frame, which allowed the nucleation dynamics to be measured with sufficient time resolution while also clearly resolving nucleated islands as small as 4×9 nm (only ~8 peptide dimers).

What they learned about the nucleation process was surprising. The crystals nucleate in very small islands. Classical nucleation theory predicts that 2D crystals will only grow, on average, if they nucleate above some critical size at which the free energy savings associated with crystallization offsets the energy cost associated with line tension. This apparent contradiction, however, was resolved when the researchers considered that the peptides in this system were crystallizing in one dimension, not two. Even though the rows aggregate to form 2D arrays, the actual crystallization process is happening within each row. For 1D crystals, this energy balance scales differently, allowing the crystals to grow even when they initially nucleate in very small islands.

Data courtesy of Prof. Jim De Yoreo, U. Washington and PNNL

Published in: Chen, J. et al. (2018) Science 362(6419): 1135-1139.

Cetyl Palmitate Adsorbed on HOPG at 13 fps

Cetyl palmitate is a waxy ester molecule that self-assembles in a pattern that templates the underlying HOPG substrate. Here, the sample was imaged at a scan size of 40 nm and line scan rate of 962 lines/second and 208×64 pixels for a frame rate of 13 fps. The playback rate is 2× the acquisition rate. We see a defect in the alignment of the rows at the start of the video. However, the sample remains dynamic and this defects midway through the video.

Insulin Fiber—Stable at 28 fps for 15 Minutes

An insulin fiber was imaged at 1250 lines/second and 160×32 pixels for a frame rate of 28 fps. Over 25,000 image frames were captured over a period of 15 minutes with no intervention by the operator to adjust imaging parameters. Imaging remained stable with no sample degradation and with very little lateral drift, demonstrating remarkable stability even at video-rate imaging speeds.

Tetratetracontane (C₄₄H₉₀)

Tetratetracontane (C₄₄H₉₀) is a long unbranched alkane. It adsorbs on HOPG and forms patches of ordered molecules with a periodicity of 5.8 nm. Though long used as a demonstration of AFM resolution at conventional scan rates, here it was imaged in air at 962 lines/second and 208×512 pixels, for a frame rate of 2 fps. The playback rate is 10× the acquisition rate. The phase channel is shown here for best contrast. These high-speed AFM results demonstrate that the molecules maintain mobility on the HOPG substrate, causing the shape of the patches to change over time.

DNA Denaturation by Potassium Hydroxide at 18 fps

The native double-stranded structure of DNA can be disrupted and split into single-stranded DNA in a process known as denaturation. This occurs in nature during DNA replication, transcription and repair. It is also an important step in biochemical techniques like PCR. A variety of methods exist to induce denaturation. One such technique is raising the pH of the solution by the addition of an alkaline compound like potassium hydroxide (KOH). Raising the pH disrupts the hydrogen bonds that stabilize the double-stranded structure. The video here was captured with an Asylum Research Cypher VRS1250 video-rate AFM and shows double-stranded DNA imaged at 962 lines/second and 80x40 pixels with a frame rate of 18 frames/second while KOH solution was slowly perfused into the sample cell. Note when the time stamp on the video reaches ~3m 45s the strand near the middle splits open into single-stranded DNA.

Dissolution of Gypsum Reveals Retreat of Atomic Steps

Understanding the dissolution of the mineral gypsum is important both geologically and commercially. Video-rate AFM using the Asylum Research Cypher VRS1250 was used to monitor gypsum dissolution at the scale of single atomic steps. The video here shows a 550 nm area of a gypsum crystal imaged in solution at 962 lines/second and a frame rate of 12 fps. The kinetics of the dissolution process could be extracted by quantitative analysis of the data.

Assembly of Collagen Fibrils on Mica

Collagen is the most abundant protein found in humans and other animals. It assembles in vivo into hierarchical structures found in numerous tissues. In addition, it is widely used to create substrates and 3D scaffolds in vitro for cell and tissue culture. Hence, understanding the mechanism of collagen self-assembly has both fundamental and practical importance.

The results shown here were obtained on a Cypher VRS video-rate AFM. Briefly, the movie begins with images of bare mica in buffer obtained in tapping mode at a line rate of 400 Hz with 512×256 pixels for a frame rate of 1.5 frames/second (fps). The movie here is played back at 15 fps, i.e. 10× the acquisition rate. A dilute solution of collagen molecules is added. The three steps of assembly are captured:

1. Adsorption of highly mobile collagen molecules on mica within several seconds after injection
2. Nucleation of poorly ordered structures through the spatial fluctuations, interaction and aggregation of adsorbed collagen molecules
3. Growth of fibrils exhibiting the D-band structure by attachment of collagen monomers as well as merging of small nucleated collagen aggregates.

Samples provided courtesy of Jinhui Tao and Jim De Yoreo, Pacific Northwest National Laboratory.

DNase Cleavage of Lambda Digest DNA

DNase1 is an enzyme that nonspecifically cleaves DNA. Here, the Cypher VRS was used to monitor the action of DNase1 on lambda digest DNA. Briefly, the DNA was bound to a mica substrate with MgCl₂ buffer, and DNase was introduced. Images were obtained in tapping mode at a line rate of 625 Hz with 320×64 pixels using an Olympus AC10 probe for a frame rate of 8.7 frames per second (fps). Beside the movie, we’ve selected three key frames taken over an interval of just 2.5 seconds. In the first image (top), several DNA strands are observed. In the second image (center), note the DNase enzyme bound to one of the overlapping strands (circle). In the third and last image (bottom), this strand has been cleaved at the binding point.

Dynamics of CTAB Hemimicelles Captured at 2.4 fps

In this experiment we captured the dynamics of hemicylindrical micelles, formed in a 2 mM CTAB solution, migrating across the surface of HOPG. The micelles are normally quite stable so we perfused in a small amount of isopropyl alcohol to induce dynamics. For these movies we acquired images (200×200 pixels) at 2.4 frames per second. In order to visualize the dynamics more clearly the playback rate is 6× faster than the acquisition rate.

Celgard Polypropylene Membrane Imaged at 2.4 fps in Air

Polycaprolactone Polymer Thin Film Imaged at 1.3 fps in Air

DNA Imaged at 1 fps with Double-Helix Resolution

Denaturation of DNA by Potassium Hydroxide at 9fps

A lambda digest DNA sample was prepared for AFM imaging using a typical preparation where the DNA solution (1 μg/µL λDNA in 10 mM NiCl₂) is allowed to adsorb to a freshly cleaved mica disc. The sample was loaded in the Cypher VRS and prepared for imaging. Then a 100 mM potassium hydroxide solution was introduced, which was expected to denature the DNA (i.e. unravel the DNA double-helix, forming single stranded DNA). Video-rate AFM imaging immediately commenced using an Asylum Research Cypher VRS atomic force microscope, scanning at 625 lines per second with an Olympus AC10 probe at a scan size of 160 nm with 448 points per line and 52 lines, for an effective frame rate of 9.19 frames/second. The video plays back here in real-time (i.e. 1× the acquisition rate).

Initial image frames show the expected intact DNA structure, but finer strands corresponding to single-strand DNA quickly appear, oftentimes appearing to remain connected to the original DNA strand.

Imaged on an Asylum Research Cypher VRS atomic force microscope at 100 Hz with 312×64 pixels using an Olympus AC10 probe for a frame rate of 1.56 fps; playback: 10× acquisition rate (15.6 fps)