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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.
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.
Ando,
A high-speed atomic force microscope for studying biological macromolecules.
2001, Pubmed
Ando,
A high-speed atomic force microscope for studying biological macromolecules.
2001,
Pubmed
Bednenko,
Nucleocytoplasmic transport: navigating the channel.
2003,
Pubmed
Binnig,
Atomic force microscope.
1986,
Pubmed
Duman,
Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.
2010,
Pubmed
Ebner,
A new, simple method for linking of antibodies to atomic force microscopy tips.
2007,
Pubmed
Ebner,
Localization of single avidin-biotin interactions using simultaneous topography and molecular recognition imaging.
2005,
Pubmed
Ebner,
Comparison of different aminofunctionalization strategies for attachment of single antibodies to AFM cantilevers.
2007,
Pubmed
Fantner,
Components for high speed atomic force microscopy.
2006,
Pubmed
Fantner,
Kinetics of antimicrobial peptide activity measured on individual bacterial cells using high-speed atomic force microscopy.
2010,
Pubmed
Florin,
Adhesion forces between individual ligand-receptor pairs.
1994,
Pubmed
Hinshaw,
Architecture and design of the nuclear pore complex.
1992,
Pubmed
,
Xenbase
Hinterdorfer,
Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.
1996,
Pubmed
Ilk,
Molecular characterization of the S-layer gene, sbpA, of Bacillus sphaericus CCM 2177 and production of a functional S-layer fusion protein with the ability to recrystallize in a defined orientation while presenting the fused allergen.
2002,
Pubmed
Kaftan,
From chloroplasts to photosystems: in situ scanning force microscopy on intact thylakoid membranes.
2002,
Pubmed
Kamruzzahan,
Imaging morphological details and pathological differences of red blood cells using tapping-mode AFM.
2004,
Pubmed
Kienberger,
Visualization of single receptor molecules bound to human rhinovirus under physiological conditions.
2005,
Pubmed
Kienberger,
Monitoring RNA release from human rhinovirus by dynamic force microscopy.
2004,
Pubmed
Kobayashi,
Real-time imaging of DNA-streptavidin complex formation in solution using a high-speed atomic force microscope.
2007,
Pubmed
Leitner,
Single-molecule AFM characterization of individual chemically tagged DNA tetrahedra.
2011,
Pubmed
Madl,
A combined optical and atomic force microscope for live cell investigations.
2006,
Pubmed
Miyagi,
Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy.
2008,
Pubmed
Müller,
Imaging purple membranes in aqueous solutions at sub-nanometer resolution by atomic force microscopy.
1995,
Pubmed
Picco,
High-speed AFM of human chromosomes in liquid.
2008,
Pubmed
Rout,
The yeast nuclear pore complex: composition, architecture, and transport mechanism.
2000,
Pubmed
Schitter,
Fast contact-mode atomic force microscopy on biological specimen by model-based control.
2004,
Pubmed
Sleytr,
Two-dimensional protein crystals (S-layers): fundamentals and applications.
1994,
Pubmed
Sleytr,
Crystalline surface layers on bacteria.
1983,
Pubmed
Sleytr,
S-layers as a tool kit for nanobiotechnological applications.
2007,
Pubmed
Stewart,
Molecular mechanism of translocation through nuclear pore complexes during nuclear protein import.
2001,
Pubmed
Stroh,
Simultaneous topography and recognition imaging using force microscopy.
2004,
Pubmed
Tang,
High-affinity tags fused to s-layer proteins probed by atomic force microscopy.
2008,
Pubmed
Tang,
Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore.
2007,
Pubmed
Tang,
Recognition imaging and highly ordered molecular templating of bacterial S-layer nanoarrays containing affinity-tags.
2008,
Pubmed
Viani,
Probing protein-protein interactions in real time.
2000,
Pubmed
Wielert-Badt,
Single molecule recognition of protein binding epitopes in brush border membranes by force microscopy.
2002,
Pubmed
Yamashita,
Tip-sample distance control using photothermal actuation of a small cantilever for high-speed atomic force microscopy.
2007,
Pubmed