Integration of optical microscopy with high-speed atomic force miscroscopy and development of high speed single molecule force spectroscopy – Opt-Spect-HS-AFM

- Integration of an optical microscopy (OM) path into HS-AFM, allowing bright field and fluorescence microscopy, without loss of HS-AFM performance
- HS-AFM imaging on cells
- Choix du polymère et des nanocristaux de diamant
- Optimisation et reproductibilité du procédé de greffage par voie électrostatique
- Mise au point d’une technique d’auto-assemblage de nanocristaux de diamant

-Successful development of hybrid HS-AFM / OM setup.
-Imaging on cells ~1000 times faster than conventional AFM/OM setups.
-Optimisation de la reproductibilité du dépot de nanocristaux.
-Utilisation des substrats de silicium de 10x10 mm2 avec une solution de nanocristaux de type SYNDIA à une concentration de nanocristaux à 0,005%.
-Immersions contrôlées avec un dip-coater (ultérieurement remplacé par un système de micromanipulation pour les pointes AFM).

-Development of a small cantilever atomic force microscope to perform high-speed single molecule force spectroscopy (HS-SMFS), with a cantilever of 1MHz resonance frequency.
-Greffage électrostatique de nanocristaux sur pointes AFM
-Premiers essais auto-assemblage sur pointe AFM
-Fixation par voie plasma & optimisation du procédé plasma
-Nucléation et croissance par voie plasma : essais sur pointes AFM

A hybrid high-speed atomic force-optical microscope for visualising single membrane proteins on eukaryotic cells
Nature Communications, 2013, 4: 2155, DOI:10.1038/ncomms3155
Adai Colom, Ignacio Casuso, Felix Rico & Simon Scheuring*

High-speed force spectroscopy unfolds titin at the velocity of molecular dynamics simulations
Science, 2013, 342 (6159): 741-743
Felix Rico, Laura Gonzalez, Ignacio Casuso, Manel Puig & Simon Scheuring*

The AFM is unique in its capability of imaging biomolecules at high-resolution and under close-to-native conditions (physiological solution, room temperature, atmospheric pressure), therefore the ideal candidate for characterizing the dynamics of biomolecules. In response to the need for faster imaging rates for monitoring the essential molecular movements a new generation of faster high-speed atomic force microscopes (HS-AFM) have been developed recently. This apparatus features an increased imaging speed by three orders of magnitude compared to previous conventional AFMs, and it is now achieving maximum imaging rates of 50 ms per frame with single molecule resolution.

The INSERM U1006 is one of less than a dozen labs world-wide that operates a functional biological HS-AFM (1, 2). Nonetheless, HS-AFM imaging is a very young technique and still limited to highly purified and controlled biological systems. In order to increase the nativeness of the samples studied and observe biomolecules on life cells, better molecular recognition, tip placement, and reproducibility of HS-AFM methodology are necessary. In this project, we will address these problems and push the HS-AFM studies forward to a higher degree of biological and medical relevancy.

First of all, we are planning to develop a novel AFM tip sharpening procedure and improve the tip quality for reproducible high-resolution high-speed imaging. We have established a concept for fabricating the best possible tips, with a very sharp apex mounted on a globular tip. The sharp apex will assure high-resolution contouring, while the globular tip allows precise control of the electrostatic forces between tip and sample to avoid sample damage. Such tips are ideal for imaging membrane proteins in membranes (3) implicated in many of life’s basic processes such as energy generation, signal transduction, and metabolite transport, and target to medical drugs (4).

Moreover, we are planning to develop an integrated HS-AFM and optical fluorescence microscopy setup to improve the molecular recognition capabilities of the HS-AFM. This integration is to be performed maintaining the performance in speed and resolution of the HS-AFM. The integration of the optical microscopy into the HS-AFM will be accomplished using sophisticated optical components. Such a combined HS-AFM – optical microscopy setup will allow studying the membrane proteins directly on living cells including pathological cell lines.

Additionally, we will develop the use of the HS-AFM as a protein folding analysis tool. Indeed, the short cantilevers used in a HS-AFM setup have low vibrational noise and resonance frequencies higher that 600kHz. The goal is profiting from the high speed of the HS-AFM for characterizing for the first time transient states and vibrations during protein folding (5) and interaction events (6) in the microsecond time range. Protein folding is a question of key importance in fundamental biological research but also cause of numerous diseases (7).

In summary, we will perform technical developments in the HS-AFM that will open the door to the acquisition of novel and so far inaccessible data of the structure, dynamics and assembly of proteins. For this, we will use our broad expertise on membrane biology AFM imaging in the laboratory U1006 INSERM as a baseline from where to build our research and development program on the HS-AFM. We will get additional expertise from the collaboration with the CEA Saclay for the microfabrication of the nano-crystal HS-AFM probes, and from the industrial subcontractor Optique J Fichou for the fabrication of the sophisticated optical components

1.Casuso, I. et al.2009.Biophysical J. 97:1354-1361
2.Casuso, I. et al.2010.Biophysical J. 99:L47-L49
3.Muller, D. J. et al.1999.Biophysical J. 76:1101-1111
4.Drews, J.2000.Science 287:1960-1964
5.Schlierf, M. et al.2004.PNAS. 101:7299-7304
6.Marshall, B. T. et al.2003.Nature 423:190-193
7.Dobson, C. M.2003 Nature 426:884-890