Atomic Force Microscopy Samrat Dutta, Ph.D. Samrat.Dutta@nau.edu Experience, interest: Surface chemistry, microscopy, integrate, biomolecule/biocelluar interaction,solution surface interface, talk about AFM

Outline AFM background AFM modes and techniques (imaging, force spectroscopy) Surface treatments and functionalization Potential applications Mode and techniques: Multiple modes, talk about imaging and force spectroscopy mode, audience might be interested. Surface treatment: Integral part of microscopy, surface chemistry technique. Potential application: W.L Gore. and associate interests.

AFM Basic Working Principle AFM: AFM working qualitatively explain by assuming an example of blind person walking on street, blind person walks with a stick and adjust his walking path by sensing obstackles. AFM: Similarly, AFM tip raster scan a surface with a cantilever with a sharp tip at its end, adjust its movement in X,Y,Z upon encountering features on surface, deflects laser reflected from the cantilever to the photo-sensor, this deflection is recorded and gives information of surface. Key features: of AFM at NAU Scan range: 100 µm lateral (~5nm res) 12 µm vertical (Å res) Force Resolution: 18 pN Physiological environments Key features

AFM Imaging Force Applications Actin paracrystals Force Tapping (Topography): Biological samples Tapping (Topography, phase): Material characterization Contact (Force): Hard surfaces, Molecular interactions, mechanical properties Applications Contact mode: AFM tip physically applies force on surface represented by repulsive region in the lennard-jones curve. Interaction with feature changes force, producing image. Not suitable bio-sample. Tapping mode: Raster scans by tapping the surface represented by attractive region in Lennard-jones curve. Feature dampens oscillation producing image.

Imaging modalities Topography Phase Chemical mapping Unfixed rat brain: mechanical heterogeneity, surfacesciencewestern.com Topography Phase Chemical mapping AFM can also be used to measure other characteristics of surface while scanning in imaging mode. Chemical mapping of PTCDA molecule: Arrow showing Hydrogen bonds, ACS.org

AFM molecular force spectroscopy On surface Above surface Retracted Force Rupture force AFM can also be operated in force spectroscopy mode: where it can measure forces of interaction between two molecules. In this mode: AFM cantilever is constrained to move only in Z-direction w.r.t to the surface, causing any molecular bond formed between the molecule attach to the AFM-tip and the molecule attached surface, when AFM tip is on the surface, to rupture when the AFM tip is retracted back. We calculate the dissociation rate between antiplatellte aggregation ligand: alpha2B beta 3. Tip sample separation Example result: Ligand (cHarGD): αIIbβ3 interaction energy landscape cHarGD:αIIbβ3: toff ~26 + 15 seconds; cHarGD ≡ eptifibatide

AFM Lateral Force Spectroscopy Fiber mechanics W. Liu. Science 2006

AFM Indentation Spectroscopy Example Result: Soft matter and cell-mechanics

AFM cell force spectroscopy Example: Cell-substrate interactions

Surface chemistry and functionalization Type Advantage Disadvantage Application Amino (APS, APTES) Simple protocol Non-specificity DNA, protein, and ligand attachment on surface Thiol Specific conjugation Disulfide bond formation, de-protonation needed Lipid Mimic membranous systems Multi-step protocol F-actin polymerization PEG Surface passivation, finger-print of interaction Diverse molecular weight

Potential applications Polymer and material characterization Chemical profiling of substrates Mechanical testing of polymers Dynamics of molecular interactions Cell mechanics Cell-substrate interaction