Atomic Force Microscope and its potential use in biology
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- The atomic force microscope (AFM) is a type of scanning probe microscope used to image surfaces at the nanoscale. It measures forces between a sharp probe and the sample surface. - In contact mode, the AFM tip is in contact with the sample surface and repulsive forces are measured. In tapping mode, the tip lightly taps the surface to reduce lateral forces and damage. - The AFM has many applications in biology like imaging living cells, studying protein unfolding, measuring molecular interactions like DNA binding, and more. It can be used to map molecular recognition with single molecule resolution.
Atomic Force Microscope and its potential use in biology
- 1. - Sudhir K Shukla Journal club presentation Atomic Force Microscope and its potential use in biology
- 2. Scanning Probe Microscopy STM: scanning tunneling microscope tunneling of electrons between probe and surface AFM: atomic force microscope measuring of the force on the probe tip OFM: Optical force microscope measuring of the force on the optically trapped particle MFM: magnetic force microscope AFM with magnetical probe y x
- 3. STM: scanning tunneling microscope nA R piezo- element e- e- e- e- e- e- e- e- e- < 1nm tunneling of electrons through air between probe and surface only conducting material probe x-y stage
- 4. STM: scanning tunneling microscope Icontrol piezo-element (changes length at different voltages) nA Itip ∆I R ∆I -> ∆V transfer
- 5. Challenges of the STM 1. Works primarily with conducting materials 2. Vibrational interference 3. Contamination •Physical (dust and other pollutants in the air) •Chemical (chemical reactivity)
- 6. AFM: Atomic Force Microscope • The AFM brings a probe in close proximity to the surface • The force is detected by the deflection of a spring, usually a cantilever (diving board) • Forces between the probe tip and the sample are sensed to control the distance between the the tip and the sample. van der Waals force curve
- 7. AFM probe scans over the surface e.g. living cells, chromatin fibers laser photodiode piezo-element probe AFM: how it works feedback
- 8. AFM: how it works cantilever tip laser cantilever piezo y z x photodiode
- 9. Scanning the Sample Tip brought within nanometers of the sample (van der Waals) Radius of tip limits the accuracy of analysis/ resolution Stiffer cantilevers protect against sample damage because they deflect less in response to a small force
- 10. Scanning Modes 1. Contact (Repulsive force) At short probe-sample distances, the forces are repulsive 2. non-contact (Attractive Force ) • At large probe-sample distances, the forces are attractive • The AFM cantilever can be used to measure both attractive force mode and repulsive forces. 3. Tapping mode (vibrating mode) • Better resolution • Minimal damage to sample van der Waals force curve
- 11. 1. Contact Mode Contact mode operates in the repulsive regime of the van der Waals curve Tip attached to cantilever with low spring constant (lower than effective spring constant binding the atoms of the sample together). In ambient conditions there is also a capillary force exerted by the thin water layer present (2-50 nm thick). van der Waals force curve
- 12. 2. Non-Contact Mode Uses attractive forces to interact surface with tip Operates within the van der Waal radii of the atoms Oscillates cantilever near its resonant frequency (~ 200 kHz) to improve sensitivity Advantages over contact: no lateral forces, non- destructive/no contamination to sample, etc. van der Waals force curve
- 13. 3. Tapping mode Change in amplitude measured Change in phase measured
- 14. Biological Applications 1. Study Unfolding Of Proteins 2. Imagining Of Biomolecules 3. Force Measurements In Real Solvent Environments 4. Antibody-Antigen Binding Studies 5. Ligand-Receptor Binding Studies 6. Binding Forces Of Complimentary DNA Strands 7. Study Surface Frictional Forces 8. Ion Channel Localization
- 15. path of AFM tip AFM tip superhelical DNA plasmid DNA double helix Mg2+ negatively charged mica surface Mg2+ Mg2+Mg2+ Mg2+Mg2+ movement of the AFM tip along the sample
- 16. AFM image of a 6.8 kb superhelical plasmid AFM tip
- 17. Molecular Force Probe •Functionalizing Cantilevers as a live biological substrate • Receptor binding in native environment can be observed Tkk xr x Tk f Boff fB m 0 ln Panorchan, P. et al. Journal of Cell Science, 119. 2006
- 18. Functionalization of AFM Tip
- 19. Imaging live cells: from structure to function
- 20. Single molecule imaging Molecular recognition maps demonstrating that clustering of the yeast sensor Wsc1 (green) is strongly enhanced by hypoosmotic shock Buffered solution Deionized water
- 21. Single molecule imaging Molecular recognition maps documenting the distribution of single Als5p adhesins (red) on a single yeast cell.
- 22. Unfolding studies of spectrin C-terminal Helix C N-terminal Helix B Helix A molecule that contributes to the mechanical properties, especially the elasticity of the cells measurement of its mechanical stability provides information about the physiological function
- 23. Stretching spectrin with an AFM distance force 1 2 3 surface cantilever tip 4 repeated spectrin domain 1. adhesion force between cantilever tip and surface 2. dissociation from the folded state to the intermediate unfolded state 3. dissociation from the intermediate to the total unfolding state
- 24. 0.15 100 30 40 0.05 20 50 0.10 0.20 0.00 unfolding force (pN) probability stretching spectrin with an AFMforce(pN) 0 20 40 60 80 0 40 20 60 80 100 distance (nm) -20
- 25. MFM: magnetic force microscope AFM with magnetic probe e.g. hard disc, tape magnetic tip laser photodiode piezo-element
- 26. Thanks