Wide-area scanner for high-speed atomic force microscopy

Abstract

High-speed atomic force microscopy (HS-AFM) has recently been established. The dynamic processes and structural dynamics of protein molecules in action have been successfully visualized using HS-AFM. However, its maximum scan ranges in the X-and Y-directions have been limited to ∼1 μm and ∼4 μm, respectively, making it infeasible to observe the dynamics of much larger samples, including live cells. Here, we develop a wide-area scanner with a maximum XY scan range of ∼46 × 46 μm 2 by magnifying the displacements of stack piezoelectric actuators using a leverage mechanism. Mechanical vibrations produced by fast displacement of the X-scanner are suppressed by a combination of feed-forward inverse compensation and the use of triangular scan signals with rounded vertices. As a result, the scan speed in the X-direction reaches 6.3 mm/s even for a scan size as large as ∼40 μm. The nonlinearity of the X-and Y-piezoelectric actuators' displacements that arises from their hysteresis is eliminated by polynomial-approximation-based open-loop control. The interference between the X-and Y-scanners is also eliminated by the same technique. The usefulness of this wide-area scanner is demonstrated by video imaging of dynamic processes in live bacterial and eukaryotic cells.

Method of mechanical holding of cantilever chip for tip-scan high-speed atomic force microscope

“…Even for the widearea scanner displaceable up to ∼50 × 50 µm 2 in the X-and Y-directions, this compensation method permits its X-scanner with the first resonant frequency of ∼2 kHz to be displaced at 1 kHz approximately in an isosceles triangle form as a function of time, without producing unwanted vibrations. 18 Rather, the Z-scanner's resonant frequency severely limits the imaging rate because the feedforward compensation technique cannot be applied to the Z-scanner.…”

“…Nonetheless, the lowered resonant frequency of the X-scanner is within a range that the adverse responses of the X-scanner can be removed by using an inverse feedforward compensation method as described previously. 18,26 In fact, this removal of adverse responses was proven, as follows. When the X-scanner was driven with triangle signals of 3.45 kHz (black line in Fig.…”

“…3 The gap space in the scanner is filled with an elastomer having a high loss factor to absorb the mechanical vibration energy. 18 A wedge-shaped stage (hereafter referred to as "w-stage") on which a cantilever chip to be placed is glued onto the top of the Z-scanner. modulus, ∼430 GPa; density, ∼3.2 g/cm 3 ).…”

“…These observations have provided greater insights than ever before into how the proteins function, thus demonstrating the innovative power of this microscopy. Recently, fast widearea scanners displaceable up to 23 × 23 µm 2 or up to ∼50 × 50 µm 2 have been developed 17,18 together with a new vibration damping method for the wide-area scanners, 18 expanding the objects of HS-AFM observation to much larger ones. For example, various dynamic processes occurring in live bacterial and mammalian cells, such as endocytosis, membrane raffling, filopodia growth and bacteriolysis, were visualized within a few seconds.…”

“…For example, various dynamic processes occurring in live bacterial and mammalian cells, such as endocytosis, membrane raffling, filopodia growth and bacteriolysis, were visualized within a few seconds. 18,19 The HS-AFM instrument used in these studies employs the sample stage-scan mode. To achieve the high scan rate and minimize the hydrodynamic effect of fast sample-stage movement on the cantilever behavior, a small sample stage (a glass rod with dimensions of 1-1.5 mm in diameter and 2 mm in height) is attached to the Z-scanner.…”