Research - Institute of Biophysics - Bionanoscience Research Unit - Optical Micromanipulation Research Group

Lóránd KELEMEN
senior research associate

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Pál ORMOS scientific adviser
Lajos KESZTHELYI professor emeritus
András BUZÁS scientific administrator
Gaszton VIZSNYICZAI junior research associate
Tamás FEKETE Ph.D. student
Zoltán IMRE technical assistant
Izabella EGRI laboratory assistant

OPTICAL MICROMANIPULATION

Our research group is studying how light can be utilized to actuate solid objects or liquid on the micrometer scale. The most intensive study is carried out on micromanipulation methods using artificial microstructures in holographic optical tweezers (HOT) but we also use light to dynamically control the flow pattern of liquids in a microfluidic channel. HOT provides an efficient tool to study various processes with relevance in physics, biology or microfluidics. For these goals we use artificial microstructures either by themselves or acting as intermediate objects to hold or probe our target objects. The microtools are made by the ultrashort-pulsed laser-based two-photon polymerization (TPP) technique in our home-made setup; the tools are in the micrometer size range with sub-micrometer feature size. The actuation of the complex-shaped microstructures takes advantage of that HOT can generate multiple trapping focal points and thereby grab these structures by their various parts and move them with 6 degrees of freedom. We are continuously broadening the applicability of the tools by functionalizing their surfaces with various molecules such as simple functional groups, proteins, antibodies or even inorganic nanoparticles.



Left: Drawing and brightfield images of free-floating trappable microrotors, Galajda and Ormos, 2001. Right: Surface-integrated, light driven micromotor and light-guide, Kelemen et al. 2006


We use Spatial Light Modulator (SLM) to accelerate and simplify the TPP process by creating several independent polymerizing laser beams. This way we can either polymerize identical objects in a parallel manner in large quantity or one structure at a time with a coordinated motion of several beams. We can test and use the microstructures in our home-built and continuously upgraded holographic optical trapping system.


Applications

We have demonstrated that the impulse of light can be converted to controlled mechanical movement by carefully designed artificial parts in the micrometer scale. First, we created helical-shaped free-floating microrotors that were trapped and rotated by optical tweezers [Galajda and Ormos 2001]. This system provided a torque of about 2*10-17 Nm. Then we prepared an integrated micromotor system consisting of a wheel of 10µm diameter [Kelemen et al. 2006], held by a supporting structure and a properly aligned light guide, all polymerized on the same supporting glass slide. The light, that was coupled into the light guide and emerged from it near the wheel structure, rotated the wheel with about 2 Hz, providing a torque of about 6*10-18 Nm.



Scheme of a micromanipulator structure illustrating the spatially separated probe and handle parts.



Left: SEM image of a two-photon polymerized mobile light guide. Palima et al. 2012. Right: Brightfield (top) and fluorescence (bottom) microscopy images of microbeads selectively excited by a mobile light guide. Palima et al. 2012


One of the main field of applications of the OT-actuated microtools is single cell study. For this we develop tools that exploit the sophisticated ways of motions that optical traps are able to provide. These tools consist of two main parts separated by micrometers: one that interacts with the trapping optical field and one that interacts with the object under study. This concept was first demonstrated by a portable micro-light guide that is capable of aiming fluorescence excitation to almost any point and any direction in the sample space. The precise maneuvering of the tools in 3D allowed for the selective excitation of fluorescent microbeads at various locations. When the same concept is elaborated for fluorescently labeled cells, spatially selective excitation of their surface will be possible from directions normally not achievable. [Palima et al. 2012]



Left: Brightfield images of the rotors used for hydrodynamic studies illustrating their phases. Right: The phase difference of the rotors showing phase-locked states at low torque differences. Di Leonardo et al. 2012


Hydrodynamic interactions, that are intensively studied for many biological systems, such as flagellar motion, have also been studied by polymerized microstructures. A microscopic model for hydrodynamic coupling was built from two helical micro-rotors of opposite sense that were optically trapped and rotated by radiation pressure. The coupling was manifested through the synchronization between the two rotors during which phase-locked states appeared and were maintained throughout small detuning of the torque applied on the rotors. [Di Leonardo et al. 2012]



Left: SEM image of a micromanipulator structure. Right: SEM image of the tip of a micromanipulator coated with 80nm Au nanoparticles.


The surface of the SU8 microtools requires further treatment to interact with biological objects. We are adopting established surface treatment protocols to them using small functional groups, macromolecules, proteins, as well as metal nanoparticles. Such coating, for instance, with streptavidin enables their use in single cell manipulation studies [Aekbote et al. 2012]. The indirect optical trapping of the cells through micro-carriers allows their manipulation with 6 degrees of freedom without significant interaction with the intense optical field which is otherwise a major drawback of the OT systems. This method can considerably lengthen the duration of the trapping experiments that can be performed on living cells.



Left: Scheme of the arrangement to change the flow pattern in a microfluidic channel. The flow is generated by electroosmosis. The photoconducting CdS layer is 200 nm thin; it is removed in a 100μm wide stripe. Right: Analyzed images showing the local direction of the flow. Upper panel: lack of illumination results in practically undisturbed flow. Lower panel: illumination of the CdS layer induces highly disturbed flow pattern. Oroszi et al. 2009.


We are using light to induce changes not only locally but also on the hundreds of micrometer scale in microfluidic environments. We demonstrated that liquid flow pattern can be changed by light in a microfluidic channel. The flow was generated by electroosmosis in the transparent channel where its bottom wall was covered by a photoconductor film. Illumination of this surface with visible light resulted in an increase of the conductivity that modified the structure of the electric field inside the channel that in turn changed the electroosmotic flow pattern. This approach was successfully used for directing and mixing the flow [Oroszi et al. 2009].


Future plans

We plan to use metal nanoparticle coating on the microstructures and perform OT-assisted localized metal-enhanced fluorescence and Raman measurements on the single cell level. This work requires on one hand systematic upgrading of our HOT system (spectrograph, camera) and on the other the selective functionalization of the polymerized microstructures where only the probe part is coated with the nanoparticles and the handle part is free from light-scattering particles.

We also plan to use the TPP microtools to study the mechanical properties of single cells. One advantage of using microtools is that they can measure forces in the fN regime, below the limit of AFM systems. Another advantage as compared to the use of trapped microbeads for this purpose is that the trapping field can be placed far from the studied cell which prevents it from being damaged by the beam and also prevents the trapping beam from not being distorted by the vicinity of the cell.

Selected publications

Galajda P, Ormos P Complex micromachines produced and driven by light. Appl. Phys. Lett. 78:249-251. (2001)

Kelemen L., S. Valkai and P. Ormos. Integrated optical motor, Applied Optics, 45:2777-2780, (2006)

Oroszi L, Der A, Kirei H, Rakovics V, Ormos P Manipulation of microfluidic flow pattern by optically controlled electroosmosis. Microfluid. Nanofluid. 6:565-569. (2009)

Rodrigo P. J., L. Kelemen, D. Palima, C.A. Alonzo, P. Ormos, J. Glükstad, Optical microassembly platform for constructing reconfigurable microenvironments for biomedical studies, Opt. Expr. 17:6578-6583, (2009)

Palima D., A. R. Bañas, G. Vizsnyiczai, L. Kelemen, P. Ormos, and J. Glückstad, Wave-guided optical waveguides, Opt. Expr., 20: 2004-2014 (2012)

Aekbote B. L., J. Jacak, G. J. Schütz, E. Csányi, Zs. Szegletes, P. Ormos, L. Kelemen, Aminosilane-based functionalization of two-photon polymerized 3D SU-8 microstructures, Eur. Polym. J. 48:1745–1754 (2012), DOI:10.1016/j.eurpolymj.2012.06.011

Di Leonardo R., A. Buzas, L. Kelemen, G. Vizsnyiczai, L. Oroszi, P. Ormos, Hydrodynamic synchronization of light driven microrotors, Phys. Rev. Lett. 109:034104 - 034108 (2012), DOI: 10.1103/PhysRevLett.109.034104