Holographic optical tweezers (HOT) are a versatile tool allowing for the generation of complex arrays of multiple optical traps which act as force sensors with piconewton resolution. The combination of HOT and stop-flow microfluidics integrated with a fluorescence microscope gives full spatial, chemical and visual control over the microenvironment. This allows us to investigate the dynamic properties of complex biosystems at the microscale, such as crosslinking in biomimetic actin networks.
Creating Biomimetic in vitro Models
Studying the chemo-mechanical interactions of complex microstructures like cellular or subcellular systems requires special tools, which allow for the precise control of the chemical and physical microenvironment. Chemical control is realized by a stop-flow microfluidic device. Nanoliter volumes of different liquids can be exchanged and mixed in a defined way. The physical control is gained by implementing HOT in our setup. The HOT-technique enables simultaneous trapping of multiple microbeads in arbitrary geometries. Trapped microbeads can be moved independently by dynamically changing the holograms and thereby forces up to several piconewtons can be exerted. Using static holograms the beads act as force sensors which are imaged by a highspeed camera.
We apply this integrated setup to create biomimetic models of the actin cortex which is a thin, quasi two-dimensional protein-network located directly underneath the cell membrane of eukaryotic cells. It plays a key role in maintaining and modifying the cells shape and controlling mechanical cell response to external stress. This dynamic regulation of the actin cortex is governed by a vast number of proteins and ions. Due to the inherent complexity the impact of an individual component can not be easily dissected in vivo. With our setup it becomes possible to investigate the forces induced by a certain bundling agent separately in a controlled chemical environment. We demonstrate these capabilities by analyzing the magnesium-induced crosslinking dynamics of an in vitro actin network.
Holographic Optical Tweezers as Micromanipulators and Force Sensors
The HOT-technique utilizes a computer controlled diffractive optical element which modifies the wavefront of the trapping laser via variation of the phase by a spatial light modulator (SLM).
Thus, a single optical trap can be split up into multiple traps. One to hundreds of optical traps can be generated in arbitrary patterns. Each trap can be manipulated independently with sub-nanometer precision even in three dimensions.
To completely fill the SLMs chip, the trapping laser beam is expanded by a telescope (T1 in fig. 2). The telescope T2 in figure 2 places the SLM in the conjugate plane to the back focal plane of the objective. Computer-generated holograms allow for spatial positioning of optical tweezers with nanometer resolution. For the network experiments, seven optical traps were arranged in a hexagonal structure with an edge length of eight micrometers (fig. 2).
Microfluidics for Controlled Microenvironments
The microfluidic system consists of a thin layer (40 µm) of micropatterned poly(dimethylsiloxane) between two cover slips. The PDMS structure is produced by standard soft lithography techniques. The overall height of the flow cell, including the glass slides on top and bottom, is about 400 µm, thus providing excellent optical properties and good transparency required for imaging (fig. 1). The microfluidic system consists of five channels for feed-in of reactants and one experimental channel, the microreactor (MR). This channel is wider than the others to balance pressure peaks during fluid exchange. Additionally, one channel is connected to the outside of the flowcell to compensate for pressure differences in the system. Polyethylene tubings connect the channels to microliter syringes actuated with micrometer screws which facilitate the defined injection and retraction of solutions. Therefore, liquid amounts in the order of nanoliters can be handled precisely. Moreover, air bubbles are used as switchable elements to keep different liquids separated, thus avoiding uncontrolled mixing of reactants.
Optical Setup for Highspeed and Fluorescence Imaging
The system is based on a commercial platform and consists of two microscopes: an upright microscope and an inverted microscope for HOT generation and imaging. The setup has threefold imaging capability and five separated optical paths. The upright microscope visualizes fluidic events in low magnification (2.5x air objective, fig. 2). A 630 nm LED serves as narrow band bright field illumination source. The inverted microscope (63x water immersion objective, NA 1.2) contains three beam paths: a fluorescence microscopy system, using computer controlled laser excitation at 532 nm, imaged at 570-590 nm on a CCD camera (fig. 2b), a bright field path at 630 nm, imaged on the high-speed camera at frame rates up to 10 kHz and the trapping beam path for HOT at 1064 nm (5 W), modulated by the SLM with 512 x 512 pixels. All beam paths can be used simultaneously in order to have maximum control of the experimental settings. The hardware components are addressed by virtual instruments programmed in LabView.
Constructing and Probing Biomimetic Protein Networks
Biomimetic actin networks are constructed on a scaffold of functionalized and optically trapped microbeads. Therefore, the bead-solution is added to the pressure compensation channel. Applying the corresponding hologram, the phase-profile of the incoming Gaussian beam is altered in a way that seven beads can be trapped in a hexagonal pattern. These trapped beads are moved to the MR. The remaining bead-solution is removed and exchanged by a solution of polymerized and fluorescently labeled actin filaments. Due to electrostatic interaction, actin filaments bind to the functionalized microbeads. This scaffold is moved back to the MR followed by injection of a buffer, containing magnesium ions, in the pressure compensation channel. Magnesium ions diffuse into the MR, bundle filaments and thus start to contract the network. Contraction pulls the trapped beads out of their individual foci. The bundling forces exerted on the network can be calculated by the displacement of the beads.
In a similar approach, we study the un-bundling of actin filaments. Therefore, we attach two single actin filaments to three optically trapped beads. These filaments are bundled subsequently by magnesium ions (fig. 4). By dynamically changing the holograms we move the optical traps and thus exerting forces which separate the bundled filaments.
To access the forces quantitatively, optical traps are calibrated. Highspeed-brightfield-imaging provides the possibility to calibrate the optical traps via power-spectral-density calibration or via Boltzmann statistics. In the network-experiment the displacements of all trapped beads were measured simultaneously at 3 kHz resulting in a sufficient temporal resolution for the calibration. Calculated forces are shown exemplarily for three microbeads in figure 3, marked red, yellow and green.
Conclusions and Outlook
The combination of microfluidics with HOT and a versatile optical setup provides an excellent system for biological experiments: The demanding tasks of controlling the chemical environment, exerting and measuring forces in the piconewton regime along with parallel observation in brightfield and fluorescence are accomplished. Hence we can create defined chemical environments, realized by special microfluidic flow cells. With the HOT-technique force generation and measurement with high spatial and temporal resolution are conducted on a biomimetic model of the actin cortex.
Having investigated the influence of divalent magnesium ions on actin networks, we will focus our future research on the effects of actin related proteins. The integrated setup offers a promising novel tool to gain deeper insight, not only into the dynamic regulation of the actin cortex, but also into further complex biological model systems.
 Uhrig K. et al.: Lab on a chip 9, 661-668 (2009)
Dipl.-Phys. Martin Streichfuss, Max Planck Insitute for Metals Research and University of Heidelberg, Germany
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