Photonic Force Microscopy (PFM)

Photonic Force Nanospectroscopy, based on Optical Trapping Interferometry (OTI), consists of a custom-made optical microscope for sample visualization, Optical Tweezers (OT), an ultra-fast 3D position detection scheme [1] and a fluorescence light path for single photon detection. In OT, a tightly focused laser beam generates enough forces on a small object, like a micron-sized sphere, a nanoparticle, a micro-organism, or a living cell, so that it remains trapped in the focus. The object can then be dragged through its fluid medium, manipulated in 3D and precisely positioned. When the optical trapping forces are kept minimal, the trapped object acts as a probe, which, driven by Brownian motion, scans its local environment in a volume defined by the optical trap. Any additional forces that act on the probe will alter the position fluctuations of the probe. The principle of data acquisition is illustrated below:

The Brownian motion of a bead in an otical trap is followed by a high reolution interferometer, and its 3D motion is calculated within the trapping volume. Any dviation from a free Brownian motion carries information of interaction of the bead with its surrounding, which could be a vscous medium, a polymer network, an iterface etc.

 

This tool, with its temporal and spatial resolution (1 µs, and 2°A) is very powerful to study hydrodynamics at short time and nanometer legth-scales, to explore molecular recogniton, biomechanics, viscosity inside and outside a cell etc. Herebelow we give few illustration of the applications of PFM.

 

Fundamental aspects of Brownian motion in viscous and viscoelastic fluids

We are systematically validating the use of a Brownian particle to probe fluids by experimentally verifying existing and newly developed theories based on the Langevin equation. Thereby, we were able to track with OTI a single micrometer-sized sphere in water on time scales sufficiently short to detect hydrodynamic contributions and measure the spectrum of the thermal noise driving Brownian motion [1-3]. In addition, we examined in the greatest detail the influence of the harmonic trapping potential confining the particle’s motion [4], as well as the influence of a hard surface on the mobility of the Brownian sphere [5]. We have also developed a method to directly measure the viscosity of a fluid [6] and also modelled Brownian motion in a viscoelastic model fluid [7].

 

In bulk environment we study the fundamental aspects of Brownian motion in a viscoelastic fluid, microrheology in complex fluids. We did important contribution to Brownian motion close to hard surfaces.

PFM is an excellent tool to study single molecule mechanics, receptor-ligand interactions, mechanical response of soft, biological molecule for which the conventional AFM represents a too large load.

The latest achievement of our group is showing correlations in Brownian fluc­tuations in viscous media. In the classical “over­damped” regime, one would expect the fluctua­tions to be uncorrelated with one another and hence to display a white power spectral density. Our exceptional PFM device allowed us to observe correlated fluctuations due to the colloid’s hydrodynamic self-interaction and the resulting power spectral density is not white but “coloured”. The computer simulations showed that the observed resonance in the power spectral density depends on a particle’s shape and surface properties opening possibilities for biophysical applications [4]. The work is performed in collaboration with Profs. Thoas Franosch and Giuseppe Foffi.

Monitoring molecular recognition

Thanks’ to the automation of OTI experiments, we are able to routinely perform single molecule force measurements of ligand–receptor interactions. Biotin-functionalized microspheres, manipulated with the optical trap, and a streptavidin-functionalized surface were used to measure the effect of different pulling forces, in the range of tens of pN, on the lifetime of individual streptavidin-biotin complexes [1].


A) A microsphere covered with specific ligands is trapped and pulled towards or away from a surface containing the corresponding receptor. B) 2D position histograms of the sphere in the X-Z, Y-Z, and X-Y planes at different distances from the surface. Changes in thermal position fluctuations along the X, Y, and Z axes during the course of sphere-surface interaction and eventual receptor-ligand binding are clearly visible [1]. 

 

Mechanical properties of molecular nanomachines

We demonstrated the power of thermal fluctuation analysis in 3D by characterizing nanomechanical properties of single motor proteins. The flexibility of the motility-determining part of the molecular motor kinesin was studied, while it was bound to a microtubule and exposed to minimal external forces in the range of a few pN. The stiffness of kinesin during stretching and compression with respect to its backbone axis were measured [1].


Thermal noise probing of a single kinesin molecule [1]. A) Scheme of the optical trapping force applied to a kinesin molecule when spanned between a probing bead and a microtubule. B) Free energy isosurface calculated from the data of the kinesin-tethered bead exhibiting a shape similar to a section of a spherical shell. C) Stiffness profile along the linker axis through the global minimum of the 3D energy landscape. The bottom panel illustrates possible conformations and behaviours of kinesin for different parts of the energy landscape.

 

Membrane tension study in fish keratocytes

In this study, we incubate fish keratocytes with polystyrene beads (1 μm DIA) in the “liquid cell” of the PFM microscope. Some of the beads settle down on keratocyte lamellipodia. Subsequently, using the optical tweezers, we try to catch one of the beads attached to lamellipodia.  Then, the optical trap being stationary, we move the microscope’s X-Y stage with the “liquid cell” attached to it. This results in pulling of a tether. The tether itself is usually not observable. However, once the laser beam of the optical tweezers is switched off, the bead is pulled back towards the cell.

The product of an average bead displacement from the trapping center (<x>) and the trap constant (k), where k is a characteristic parameter of the optical-tweezers-based trap, allows us to estimate the force with which the membrane pulls the bead back.

A movie showing a tether pulled from a keratocyte. The bead to be pulled is marked by a circle on the preview. At 51 sec of the movie (89 of the data) the laser beam is switched off and the bead returns to the cell.

This research is performed in a close collaboration with Dr. Małgorzata Lekka from the Institute of Nuclear Physics of the Polish Academy of Sciences (Krakow) and with Prof. Jean-Jaques Meister and Dr. Alexander B. Verkhovsky from the Cell Motility group of the Laboratory of Cell Biophysics (LCB)/IPMC/FSB/EPFL, Lausanne.

 

References:
[1] S. Jeney, B. Lukic, C. Guzmán, L. Forró. Probing hydrodynamic fluctuations with a Brownian particle. Nanotechnology, Vol. 6 – Nanoprobes, ed. Harald Fuchs, Wiley-VCH, 2009.
[2] B. Lukić, S. Jeney, C. Tischer, A. J. Kulik, L. Forró, E.-L. Florin. Direct observation of nondiffusive motion of a single Brownian particle. Phys Rev Lett, 95, 160601, 2005.
[3] R. Huang, I. Chavez, K. M. Taute, B. Lukić, S. Jeney, M. G. Raizen, E.-L.Florin.Direct observation of the full transition from ballistic to diffusive Brownian motion in a liquid. Nature Physics, 7, 576, 2011.
[4] T. Franosch, M. Grimm, M. Belushkin, F. Mor, G. Foffi, L. Forró, S. Jeney. Resonances arising from hydrodynamic memory in Brownian motion – The colour of thermal noise. Nature, 478, 85, 2011.
[5] B. Lukić, S. Jeney, Z. Sviben, E.-L. Florin, A. J. Kulik, L. Forró. Motion of a colloidal particle in an optical trap. Phys Rev E, 76, 011112, 2007.
[6] S. Jeney, B. Lukić, J. A. Kraus, T. Franosch, L. Forró. Anisotropic memory effects in confined colloidal diffusion. Phys Rev Lett, 100, 240604, 2008 (invited cover page).
[7] C. Guzmán, H. Flyvbjerg, R. Köszali, C. Ecoffet, L. Forró, S. Jeney. In situ viscometry by optical trapping interferometry. Appl Phys Lett, 93, 184102, 2008.
[8] M. Grimm, S. Jeney, T. Franosch. Brownian motion in a Maxwell fluid. Soft Matter, 7, 2076, 2011.
[9] S. Jeney, E. H. K. Stelzer, H. Grubmüller, E.-L.Florin.Mechanical properties of single motor molecules studied by three-dimensional thermal force probing in optical tweezers. ChemPhysChem, 5, 1150, 2004.