Materials and methods
A deflection signal that appears when usual wavelengths of fluorescence excitation light are shone onto a soft AFM lever has been observed and described. This coupling signal varies (i) in shape as a function of the presence of a reflective gold layer and as a function of the wavelength used, and (ii) in intensity as a function of the power of the light when a gold coating is present. However, this signal appeared to be, in the absence of gold reflective coating, regular and of small amplitude (~50–100pN), with the same order SLx-2119 as the imposed excitation, and with a controlled duration that varies with the duration of the excitation. It was concluded from controls that this signal cannot be totally removed, but minimized by using the proper type of commercially available AFM levers.
A coupling signal existing between AFM and fluorescence microscopy, manifesting as a bending of the AFM cantilever upon shinning excitation light, has been described. It has been characterized in terms of duration, periodicity and intensity, as a function of excitation wavelength and power, for four different types of soft cantilevers that are commonly used in biological applications of AFM force mode. Three different geometries and rigidities (<100pN/nm) were examined. For commercial uncoated levers, this signal is of moderate intensity (~50–100pN), reproducible, and can hence be used as an intrinsic timer for real-time use of AFM and epifluorescence. The mechanical stimulation experiments are paving the way to specific stimulations where a given molecule will be oriented, at a known density, grafted on the bead modified lever, to represent a model APC. Physical/mechanical parameters of the contact (duration, frequency, force, rigidity of the lever i.e. apparent APC rigidity ) could then be varied in order to decipher the transfer function of given pathways encoding the information received at the membrane and transmitted to the cytosol, exploiting the fluorescence recording in real-time or relevant markers. Such a simple technique may open the use of AFM to dissect mechanochemistry at single cell level, by allowing forces and their consequences to be easily correlated.
Fundings: Prise de Risques CNRS, ANR JCJC “DissecTion” (ANR-09-JCJC-0091), PhysCancer “H+-cancer” (to PHP). Labex INFORM (ANR-11-LABX-0054) and A*MIDEX project (ANR-11-IDEX-0001-02), funded by the “Investissements d’Avenir” French Government program managed by the French National Research Agency (ANR) (to Inserm U1067 Lab and as PhD grant to AS). GDR MIV (as a master grant to SO).
Providing material or technical help: P. Robert (U1067, Marseille), Y. Hamon and H.-T. He (CIML, Marseille, France). A. Dumêtre (UMD3, Marseille, France) [J774 cells], K. Hahn [Rac–PA plasmid], F. Bedu and H. Dallaporta (CINAM, Marseille) [ion beam cutting]. F. Eghiaian, A. Rigato and F. Rico (U1006, Marseille) [chemical gold removal recipes and discussions]. P. Dumas (CINAM, Marseille) [discussions]. R. Fabre (CIML, Marseille) [preliminary experiments].
Companies: JPK Instruments (Berlin, Germany) for continuous support and generous help. Zeiss France for support.
Since the invention of atomic force microscopy (AFM) , increasing its imaging rate has been one of the major technical challenges for enhancing the usefulness of AFM. The materialization of high-speed AFM (HS-AFM) required various developments and breakthroughs, as described below, while it was expected to bring a great impact on various fields of science and technology. Biological science in particular would most efficaciously receive a great deal of benefit from the materialization. This is because the direct observation of biological molecules in dynamic action at high spatiotemporal resolution certainly facilitates our detailed understanding of their functional mechanism. Driven by this motivation, Paul Hansma\’s group and Ando\’s group independently embarked on the development of HS-AFM more than two decades ago. In the early stage, efforts were focused on the developments of a fast scanner [2–9], small cantilevers with a high resonant frequency in water and a small spring constant [3–7], an optical beam deflection (OBD) detector that detects the deflection of a small cantilever [3,5], and a fast amplitude detector that quickly converts the deflection signal of the oscillating cantilever to its amplitude signal [3,8]. By assembling these (or some of these) devices, prototypic HS-AFM instruments employing the tapping mode were built around 2000 and shown to be able to capture images of protein molecules at a much higher rate than before [3,9]. However, it was evident that the feedback bandwidth was still insufficient and therefore the imaging rate was not high enough or the protein molecules were damaged when imaged too fast. Then, Ando\’s group has further endeavored to enhance the capacity of HS-AFM by developing various techniques such as an active damping technique that suppresses the Z-scanner\’s unwanted vibrations as well as enhances its response speed , a new scheme for the proportional-integral-derivative (PID) control (referred to dynamic PID control) that can make high-speed imaging compatible with low-invasiveness to fragile molecules and weak intermolecular interactions , a technique to compensate for drift of the cantilever excitation power  and a fast phase detector . Moreover, Ando\’s group has developed smaller cantilevers in collaboration with Olympus, a more robust fast scanner and a lower-noise, fast amplitude detector . Finally, around 2008, HS-AFM of practical use was established that achieved a feedback bandwidth of ~100kHz and therefore could capture images of protein molecules at sub-100 ms temporal resolution, without disturbing their function .
Materials and methods