AFM in Bionanotechnology

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Atomic force microscope in bionanotchnology: a summary

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ATOMIC FORCE MICROSCOPY IN BIONANOTECHNOLOGY : 

ATOMIC FORCE MICROSCOPY IN BIONANOTECHNOLOGY Gerald Kada*, Ferry Kienberger*, and Peter Hinterdorfer† *Agilent Technologies, Mooslackengasse 17, 1190 Vienna, Austria † University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz, Austria. VEERAPANDIAN MURUGAN 200840152 Kyungwon university College of Bionanotechnology

Overview : 

Overview Introduction Principle Methods Results and Discussion Conclusion

Introduction : 

Introduction The atomic force microscope (AFM) or scanning force microscope (SFM) is a very high-resolution type of scanning probe microscope Used for imaging surfaces ranging from micro- to nanometer scales Binnig, Quate and Gerber invented the first AFM in 1986 Visualizing, characterizing surface textures and shapes.

Introduction : 

Introduction Provides sub nanometer resolution for imaging biological species like proteins and living cells. Molecular recognition forces provide function and structure of biomolecular assemblies. Topographic imaging of an identification of biomolecules based on fluorescence labeling and high-resolution is possible on combination with fluorescence microscopy

Principle : 

Principle The AFM uses a sharp probe connected to a cantilever spring The probe is scanned across the surface of the sample Interaction between the tip and the sample causes the cantilever to deflect The interaction is translated into a three-dimensional image

Principle : 

Principle Small modulations of the cantilever tip using piezoscanner Feedback: moves sample up/down to maintain constant force or modulation amplitude. Feedback ON: constant force mode Feedback OFF: constant height mode

Principle : 

Principle

Methods : 

Methods High-resolution imaging of biological species (a) A sample is probed by an ultra sharp stylus mounted on a cantilever, which scans over the surface. A reflected laser beam reports deflections of the cantilever to a split photodiode. Photoelectric circuitry then converts the deflections into height information recorded as a digital image. (b) Sketch of a ligand tethered to an AFM tip and probing receptors embedded in a cell membrane.

Slide 9: 

High-resolution topographical imaging of biomolecular assemblies. (a) 3 kbp (base pairs) pDNA on a mica surface imaged in Ni2+ buffer solution. Scale bar 150 nm. (b) Left: crystalline arrangement of human rhinovirus on a lipid bilayer containing receptors. Inset: Fourier spectrum and average lattice. Right: dense packing of virus particles with regular patterns of small protrusions ~0.5 nm high and ~3 nm in diameter. (c) Topographical image of purple membrane to which a single antibody is bound (left) and a three-dimensional representation (right).

Slide 10: 

Higher harmonics imaging of membrane proteins Higher harmonics imaging of bacterial S-layers in buffer solution. (a) Simultaneously recorded topography (left) and second harmonics (right) images. Substructures within the unit cell can be clearly resolved (resolution ~0.5 nm). (b) Frequency spectra recorded on position 1 and 2 of (a). Higher harmonics contributions reflect the different properties of the tip protein (position 2) and tip-hole (position 1) interactions. (c) Average of 55 unit cells and sketch of the lattice structure. (Reprinted with permission from34. © 2007 The American Physical Society.)

Methods : 

Methods Single molecule force measurements AFM tip functionalization Molecular recognition force spectroscopy Single molecule force measurements on living cells Unfolding of membrane proteins

AFM tips : 

AFM tips Sharpness of the scanning tip achieve the imaging resolution The need for sharp tips is normally explained in terms of tip convolution

Slide 13: 

The diagram illustrates this problem; as the tip scans over the specimen, the sides of the tip make contact before the apex, and the microscope begins to respond to the feature. This is what we may call tip convolution.

Results and discussion : 

Results and discussion AFM has evolved into an imaging method that yields fine structural details on live, biological samples in their physiological environment. High lateral resolution and sensitive force detection capabilities leads to an option of measuring inter and intramolecular forces of biomolecules on the single molecule level Exploring kinetic and structural details of interactions and molecular recognition processes combination of AFM and fluorescence microscopy enables a more detailed characterization of cellular processes.

Results and discussion : 

Results and discussion Topography and recognition imaging Combined AFM and optical microscopy Simultaneous AFM and fluorescence imaging Fluorescence guided force spectroscopy

Conclusion : 

Conclusion The field of scanning probe microscopy, atomic force microscopy (AFM) is extensively used in a wide range of disciplines such as life science, solid-state physics, and materials science The AFM has evolved into an imaging method that yields structural details of biological samples such as proteins, nucleic acids, membranes, and cells in their native environment Sharpness of the scanning tip

Conclusion : 

Conclusion AFM is now a companion technique to X-ray crystallography and electron microscopy (EM) for the determination of protein structures Unique technique for providing sub nanometer resolution at a reasonable signal-to-noise ratio under physiological conditions