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.