Advances in High-Speed Atomic Force Microscopy

High-speed atomic force microscopy (HS-AFM) is a scanning probe technique capable of recording processes at the nanometre scale in real time. By sequentially increasing the speed of individual microscope components, images of surfaces can be recorded at up to several images per second. We present a HS-AFM platform composed of custom¿built measurement head, controller and software, scanners and amplifiers that is shared with the community in an open¿hardware fashion. A new scanner design combined with an advanced control system is shown. The simple addition of a secondary actuator to widely available tube scanners increases the scan speed by over an order of magnitude while allowing for a 130 ¿m × 130 ¿m wide field of view, which is not possible with traditional high¿speed scanner designs. Controllers beyond standard proportional-integral controllers are capable of significantly increasing imaging speed by anticipating resonances. Such filters are cumbersome to design with conventional methods. It is shown how convex optimization can be used to design optimal controllers with guaranteed stability for atomic force microscopy in an automated fashion. By integrating two lasers into the small spot¿size optics of an AFM readout head we are able to use the first laser for detecting the deflection of the smallest, and thus fastest currently available high¿speed cantilevers, while using the second for photo¿thermal actuation. Using this instrument, we demonstrate multi¿frequency atomic force microscopy (MF-AFM) at previously not accessible frequencies of more than 20 MHz. By employing the driving laser not for resonant excitation as is usual in dynamic AFM, a new imaging mode, photothermal off-resonance tapping (PORT) is presented. By repeatedly thermally bending the cantilever below it¿s resonant frequency, the surface is probed at a rapid rate. The resulting force is extracted from the deflection of the cantilever in time¿ domain at real time and used for feedback and image generation. The dynamic and static force contributions in both PORT and state of the art high-speed amplitude modulation atomic force microscopy (AM-AFM) are measured and analyzed in detail. It is shown that by decoupling the driving frequency from the resonant frequency the dynamic tip¿sample impact forces can be drastically reduced when compared to resonance based AFM modes. SAS-6 is a centriolar scaffolding protein with a crucial role in the duplication of centrioles, which are the main microtubule organizing organelle of eukaryotic cells. Defects in centriole duplication are associated with cancer and microencephaly. To understand these defects, is therefore important to understand the kinetics of SAS-6. In¿vitro, SAS-6 polymerizes into rings of between eight and ten monomers. Using the new PORT mode we are able to study the dynamic assembly of SAS-6. It is shown how SAS-6 rings can not only assemble by canonical one-by-one addition, but can form as a fusion of larger, already assembled fragments. Finally, it is shown how PORT can be used to observe fast processes of and on living cells. The adhesion and detachment of thrombocyte cells is studied. Membrane disruptive effects are shown on gram¿negative as well as gram¿positive bacteria.