Collagen fibrils are a widely accepted standard sample for measurements of biological and soft material properties. Like many biological samples, these fibrils soften and swell once rehydrated and can sway if disturbed with an AFM probe. This third sample, provided by our collaborators at Niigata University in Japan, allows us to demonstrate SICM’s capability to image a sample whose nanoscale topography would otherwise be difficult to acquire with other microscopy techniques.
The collagen fibril was cut and spin-cast on a petri dish and imaged with a Park NX10 system in PBS solution with SICM. Due to the height variation of collagen fibrils, we applied ARS mode only. The XEP software was used to run all the tests. As shown in Figure 5, the protein bundles as well as each individual fibril can be clearly identified in the 10 μm sized images at a 256 x 256 pixel resolution. The thinnest fibril SICM can distinguish (indicated by the black arrow) is only about 90 nm in width. Based on the fact that there is no real contact or force between the end of the pipette and the sample surface, this resolution is quite impressive.
Figure 5: (a) Topography and (b) contrast enhanced topography images of collagen sample imaged in PBS solution with SICM ARS mode. Individual fibril can be clearly identified and the thinnest one observed is about 90nm in width, as pointed by the black arrow. Scan size 10 μm ×10 μm. Image size 256 px × 256 px.
Experimental
The Park NX10 SICM is based on the Park NX10 AFM platform. The hardware is virtually the same with the exception of an SICM head replacing the standard AFM head on the system. Instead of using an AFM tip to sense the interaction force between the tip and the sample then image the sample topography, the SICM head uses a either a glass pipette with an inner diameter ranging from 80-100 nm or one made of quartz with an inner diameter of 30-50 nm. The pipette is filled with electrolyte solution and connected with an Ag/AgCl electrode while another electrode is connected with the sample in liquid (Figure 1a). A closed circuit forms and with an applied bias between the two electrodes, the ionic current flows from the pipette to the sample (Figure 1b). When the pipette gets closer to the sample, the current decreases and the current-displacement relationship (Figure 1c) is tracked by the system’s fast and accurate feedback loop. When the pipette truly touches the sample surface, the current will drop to zero. The degree of current decrease can be used to back calculate the surface topography. The pipette scans the surface at a given current set-point (normally 99%), which means it always keeps a few hundred nm away from the surface. Quite similar to Scanning Tunneling Microscopy (STM), which takes advantage of the tunneling current to characterize the materials surface, SICM keeps track of the current change to provide not just non-contact, but no-force imaging. In addition, it completely gets rid of cantilever tuning which can add complexity to non-contact in-liquid AFM imaging. This technique not only provides remarkably stable imaging and quantitative data, but also makes the observation of extra soft or sensitive biological materials, such as live cells, at nanoscale possible.
Figure 1: The snapshots showing the (a) SICM hardware setup, (b) circuit mechanism and (c) current-distance relationship between the pipette end and the sample surface.