Leitner M et al. (2012), Increased imaging speed and force sensitivity f...

XB-ART-46475

Micron 2012 Dec 01;4312:1399-407. doi: 10.1016/j.micron.2012.05.007.

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Increased imaging speed and force sensitivity for bio-applications with small cantilevers using a conventional AFM setup.

Leitner M , Fantner GE , Fantner EJ , Ivanova K , Ivanov T , Rangelow I , Ebner A , Rangl M , Tang J , Hinterdorfer P .

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In this study, we demonstrate the increased performance in speed and sensitivity achieved by the use of small AFM cantilevers on a standard AFM system. For this, small rectangular silicon oxynitride cantilevers were utilized to arrive at faster atomic force microscopy (AFM) imaging times and more sensitive molecular recognition force spectroscopy (MRFS) experiments. The cantilevers we used had lengths between 13 and 46 μm, a width of about 11 μm, and a thickness between 150 and 600 nm. They were coated with chromium and gold on the backside for a better laser reflection. We characterized these small cantilevers through their frequency spectrum and with electron microscopy. Due to their small size and high resonance frequency we were able to increase the imaging speed by a factor of 10 without any loss in resolution for images from several μm scansize down to the nanometer scale. This was shown on bacterial surface layers (s-layer) with tapping mode under aqueous, near physiological conditions and on nuclear membranes in contact mode in ambient environment. In addition, we showed that single molecular forces can be measured with an up to 5 times higher force sensitivity in comparison to conventional cantilevers with similar spring constants.

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Species referenced: Xenopus laevis
Genes referenced: avd trim9

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	Fig. 1. Electron microscope images of prototype cantilevers. (A and B) Typical images used for further characterization. The horn shaped and partly hollow tip from the front side. The upper side of the silicon oxynitride cantilever is coated with chromium as adhesive agent and gold for laser reflection. The tip was visualized as cone with a height of about 3 μm and a base radius of about 2 μm. The radius of the curvature is about 5–10 nm for the sharpest cantilevers. (C) The measured resonance frequency for the short 430 nm cantilever (Type C) is about 600 kHz. (D) The measured resonance frequency is about 280 kHz for the long 430 nm (Type D) cantilever. (E) Image of the 430 nm thick cantilevers (Type C and D).
	Fig. 2. The chart in Fig. 2 shows a summary of the results of the different characterized cantilever prototypes. The cantilever prototypes combine low spring constant with a high resonance frequency when compared to common cantilevers. The long 430 nm thick (Type D) and the short 150 nm thick (Type A) cantilevers were used for high speed imaging at nanometer resolution in contact and tapping mode. The long 150 nm thick (Type B) cantilever was used for MRFS on avidin/biotin test system, showing lower thermal fluctuations then conventional cantilevers with similar spring constant. The long and short 600 nm (Type E and F) cantilever was only used for EM imaging and characterization. The difference in the measured and calculated frequencies for Type C and D cantilever is mainly due to meanderings of the cantilever dimensions, the reflex coating, the tip mass and the shape of the tip.
	Fig. 3. Fig. 3 shows a typical force distance cycle with a specific unbinding event using a chemically modified cantilever prototype (Type B). Force distance cycles were recorded on avidin adsorbed to freshly cleaved mica. The inset shows a typical force distance cycle recorded on the same system using a Bruker MSCT C (10 pN/nm) cantilever. Obviously it can be seen that the force noise is much lower using the small prototype cantilever.
	Fig. 4. Fig. 4 shows force noise traces of different cantilevers which are common in molecular recognition force spectroscopy (MRFS). All curves have been extracted from force distance cycles, showing 50 nm ranges of the non-contact region of the retrace at the same loading rate (1 s sweep duration, 600 nm force distance cycles, 2000 data points). (A and B) Force noise traces of the Type B and Type D prototype cantilevers. The 150 nm cantilever with a spring constant of 21 pN/nm showed the lowest thermal force noise. BRUKER Cant. (B) (Fig. 4E, 20 pN/nm), (C) (Fig. 4F, 10 pN/nm) and (D) (Fig. 4G, 30 pN/nm) and the Olympus BioLever (Fig. 4C, 6 pN/nm) and TSP400 (Fig. 4D, 20 pN/nm) cantilever showed higher thermal force fluctuations.
	Fig. 5. The diagram in Fig. 5 summarizes the thermal force fluctuation analysis for common MRFS cantilever and the prototype cantilevers. Type D cantilever showed a thermal fluctuation of about 22 pN which is comparable to Bruker cantilever E (∼28 pN). Type B cantilever showed a thermal fluctuation of about 1.4 pN which is about 5 times lower when compared with Bruker cantilever B, and about 3 times lower when compared with Bruker cantilever C and Olympus BioLever.
	Fig. 6. Bacterial surface layer from Bacillus Sphaericus were imaged with up to ten times higher speed compared to common cantilevers. (A) Overview image recorded in contact mode using Type D cantilever. Imaging speed was about 55 μm/s (∼10 lines/s). (B) Larger magnification image. The typical lattice structure with a constant of 14 nm constant is clearly visible (see inset). Image recorded in tapping mode with nm resolution using the Type C Cantilever.
	Fig. 7. (A) Nuclear pore complexes imaged with Type D cantilever using contact mode at ambient conditions. (B) The imaging speed of 12 lines per second was about ten times higher than in (A) using the same cantilever. (C) Conventional cantilever at a speed of 10 lines/s yielded images with much less resolution.

References [+] :

Ando, A high-speed atomic force microscope for studying biological macromolecules. 2001, Pubmed