High-Speed Atomic Force Microscopy
time, DNA spontaneously unwraps from the histone core, and the core disassociates and leaves the area of observation. Te study of crystal formation
Figure 2: Two Active Pharmaceutical Ingredients (APIs), NK1(1) and CETP(2), were dissolved into four excipients and tested for mixture stability via high-speed AFM. Using a tip-scanning AFM with an automated sample stage, the multiple images were captured quickly and autonomously, enabling the easy collection of large quantities of data from several samples. The tight error bars in the plot of average surface roughness for each sample formulation demonstrates the imaging reliability of high-speed AFM. (Samples provided by researchers at F. Hoffman-La Roche Ltd.)
without user intervention, data management, and batch image analysis also play important roles. In a typical example, a high-speed AFM was used to
determine where an active pharmaceutical ingredient (API) was combined with an inactive excipient to form an amorphous solid with the goal of maximizing the API’s solubility aſter ingestion. Te amorphous formulation was solid at room temperature but would otherwise separate into two phases. Microscopic (~100 nanometers) separation and recrystallization of the API must occur to observe the possible phase separation. Researchers at F. Hoffman–La Roche Ltd. and the University of Basel used the latest generation of high-speed AFMs to detect the indicators of instability on a much smaller size scale and at a point earlier in the development process than was possible in the past (Figure 2), thereby accelerating the formulation development cycle. Te AFM provided data on multiple sites of 100 or more samples per day [5], enabling the researchers to quickly and easily collect statistically significant data sets.
Dynamic Processes Te time-resolved study of dynamic processes is the
application that has provided the greatest impetus to develop faster AFMs. As an example of work in this field, Bruker scientists examined a sample from the group of Yuri L. Lyubchenko at the University of Nebraska Medical Center, with the goal of replicating their methods to loosely bind biomolecules to a substrate for dynamic AFM observation. Lyubchenko et al. use these methods to investigate the structure and dynamics of nucleosomes [6]. In their studies, the samples were prepared by the deposition of nucleosome core particles (NCPs) without the fixation with glutaraldehyde that is typically required for electron microscopy, demonstrating that AFM is gentle enough to image the DNA-NCP complex in its active state (Figure 3). Time-lapse experiments were performed by scanning in liquid over an area of about 800 × 800 nanometers to follow the dynamics of NCPs. Te frames show that, over
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is another important application of AFM involving dynamic processes. AFMs have been used to study polyhydroxybutyrate-co- valerate (PHBV), a biodegradable thermoplastic polymer produced by microbial fermentation [7]. Te formation of the crystalline lamella structure of PHBV may determine the mechanical properties of the bulk material. Te sample crystal- lizes from various nucleation points so quickly that by the time conventional AFMs have taken a single scan, the crystal is already formed. Fast scanning is needed to capture the dynamic processes of crystallization. Using a PBHV sample courtesy of Jamie Hobbs at the University of Sheffield, Bruker
researchers employed a high-bandwidth AFM to take a 1 µm × 1 µm, 256-line image with a frame time of 2.5 seconds. Te resulting movie shows the lamella forming as the crystallization front moves across the frame. It also shows two fronts joining, passing over defects, sometimes incorporating them, and sometimes not. Lower magnification images of this process are shown in Figure 4. Tese results provide important insights into the growth rates and dynamics of the crystalline lamella.
AFM Technology Advances that Affect Increased Bandwidth AFM measures the sample by probing the sample surface
with an ultra-sharp tip that is attached to the end of a cantilever. Interaction forces between the tip and sample can be measured by detecting the displacement of the cantilever, for example via the laser deflection method [8]. When scanning a sample, the AFM employs a feedback loop, which monitors the cantilever displacement and actuates the AFM Z-scanner to maintain a
Figure 3: High-speed, 1-frame-per-second AFM images of DNA strands, obtained using the Dimension FastScan™ system, indicate that force control on loosely bound biomolecules can be achieved at high frame rates. Three of 2,100 AFM frames are shown for DNA-NCP in various active states. (APS mica samples courtesy of Yuri L. Lyubchenko of the University of Nebraska Medical Center.)
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