Single-Cell Elasticity Measurement with an Optically Actuated Microrobot.

A cell elasticity measurement method is introduced that uses polymer microtools actuated by holographic optical tweezers. The microtools were prepared with two-photon polymerization. Their shape enables the approach of the cells in any lateral direction. In the presented case, endothelial cells grown on vertical polymer walls were probed by the tools in a lateral direction. The use of specially shaped microtools prevents the target cells from photodamage that may arise during optical trapping. The position of the tools was recorded simply with video microscopy and analyzed with image processing methods. We critically compare the resulting Young's modulus values to those in the literature obtained by other methods. The application of optical tweezers extends the force range available for cell indentations measurements down to the fN regime. Our approach demonstrates a feasible alternative to the usual vertical indentation experiments.

Multiview microscopy of single cells through microstructure-based indirect optical manipulation.

Fluorescent observation of cells generally suffers from the limited axial resolution due to the elongated point spread function of the microscope optics. Consequently, three-dimensional imaging results in axial resolution that is several times worse than the transversal. The optical solutions to this problem usually require complicated optics and extreme spatial stability. A straightforward way to eliminate anisotropic resolution is to fuse images recorded from multiple viewing directions achieved mostly by the mechanical rotation of the entire sample. In the presented approach, multiview imaging of single cells is implemented by rotating them around an axis perpendicular to the optical axis by means of holographic optical tweezers. For this, the cells are indirectly trapped and manipulated with special microtools made with two-photon polymerization. The cell is firmly attached to the microtool and is precisely manipulated with 6 degrees of freedom. The total control over the cells' position allows for its multiview fluorescence imaging from arbitrarily selected directions. The image stacks obtained this way are combined into one 3D image array with a multiview image processing pipeline resulting in isotropic optical resolution that approaches the lateral diffraction limit. The presented tool and manipulation scheme can be readily applied in various microscope platforms.

3D Biomimetic Chips for Cancer Cell Migration in Nanometer-Sized Spaces Using "Ship-in-a-Bottle" Femtosecond Laser Processing.

Cancer cells undergo dramatic morphology changes when migrating in confined spaces narrower than their diameter during metastasis, and thus it is necessary to understand the deformation mechanism and associated molecular events in order to study tumor progression. To this end, we propose a new biochip with three-dimensional (3D) polymer nanostructures in a closed glass microfluidic chip. "Ship-in-a-bottle" femtosecond laser processing is an exclusive technique to flexibly create 3D small details in biochips. The wavefront correction by the spatial light modulator significantly improves the fabrication resolution of this technique. The device could then accommodate defect-free 3D biomimetic nanoconfigurations for the evaluation of prostate cancer cell migration in confined spaces. Specifically, polymeric channels with widths of ∼900 nm, which is more than one order of magnitude smaller than the cell size, are integrated by femtosecond laser inside glass channels. The cells are responsive to an in-channel gradient of epidermal growth factor and can migrate a distance greater than 20 µm. After migration, the cells suffer partial cytokinesis, followed by fusion of the divided parts back into single cell bodies.

Nearly Aberration-Free Multiphoton Polymerization into Thick Photoresist Layers.

In the era of lab-on-chip (LOC) devices, two-photon polymerization (TPP) is gaining more and more interest due to its capability of producing micrometer-sized 3D structures. With TPP, one may integrate functional structures into microfluidic systems by polymerizing them directly inside microchannels. When the feature of sub-micrometer size is a requirement, it is necessary to use high numerical aperture (NA) oil-immersion objectives that are optimized to work close to the glass substrate-photoresist interface. Further away from the substrate, that is, a few tens of micrometers into the photoresist, the focused beam undergoes focal spot elongation and focal position shift. These effects may eventually reduce the quality of the polymerized structures; therefore, it is desirable to eliminate them. We introduce a method that can highly improve the quality of structures polymerized tens of micrometers away from the substrate-photoresist interface by an oil-immersion, high NA objective. A spatial light-modulator is used to pre-compensate the phase-front distortion introduced by the interfacial refractive index jump on the strongly converging beam.

Surface-modified complex SU-8 microstructures for indirect optical manipulation of single cells.

We introduce a method that combines two-photon polymerization (TPP) and surface functionalization to enable the indirect optical manipulation of live cells. TPP-made 3D microstructures were coated specifically with a multilayer of the protein streptavidin and non-specifically with IgG antibody using polyethylene glycol diamine as a linker molecule. Protein density on their surfaces was quantified for various coating methods. The streptavidin-coated structures were shown to attach to biotinated cells reproducibly. We performed basic indirect optical micromanipulation tasks with attached structure-cell couples using complex structures and a multi-focus optical trap. The use of such extended manipulators for indirect optical trapping ensures to keep a safe distance between the trapping beams and the sensitive cell and enables their 6 degrees of freedom actuation.

Microfluidic study of the chemotactic response of Escherichia coli to amino acids, signaling molecules and secondary metabolites.

Quorum sensing and chemotaxis both affect bacterial behavior on the population level. Chemotaxis shapes the spatial distribution of cells, while quorum sensing realizes a cell-density dependent gene regulation. An interesting question is if these mechanisms interact on some level: Does quorum sensing, a density dependent process, affect cell density itself via chemotaxis? Since quorum sensing often spans across species, such a feedback mechanism may also exist between multiple species. We constructed a microfluidic platform to study these questions. A flow-free, stable linear chemical gradient is formed in our device within a few minutes that makes it suitable for sensitive testing of chemoeffectors: we showed that the amino acid lysine is a weak chemoattractant for Escherichia coli, while arginine is neutral. We studied the effect of quorum sensing signal molecules of Pseudomonas aeruginosa on E. coli chemotaxis. Our results show that N-(3-oxododecanoyl)-homoserine lactone (oxo-C12-HSL) and N-(butryl)-homoserine lactone (C4-HSL) are attractants. Furthermore, we tested the chemoeffector potential of pyocyanin and pyoverdine, secondary metabolites under a quorum sensing control. Pyocyanin is proved to be a weak attractant while pyoverdine are repellent. We demonstrated the usability of the device in co-culturing experiments, where we showed that various factors released by P. aeruginosa affect the dynamic spatial rearrangement of a neighboring E. coli population, while surface adhesion of the cells is also modulated.

Optically Trapped Surface-Enhanced Raman Probes Prepared by Silver Photoreduction to 3D Microstructures.

3D microstructures partially covered by silver nanoparticles have been developed and tested for surface-enhanced Raman spectroscopy (SERS) in combination with optical tweezers. The microstructures made by two-photon polymerization of SU-8 photoresist were manipulated in a dual beam optical trap. The active area of the structures was covered by a SERS-active silver layer using chemically assisted photoreduction from silver nitrate solutions. Silver layers of different grain size distributions were created by changing the photoreduction parameters and characterized by scanning electron microscopy. The structures were tested by measuring the SERS spectra of emodin and hypericin.

Holographic multi-focus 3D two-photon polymerization with real-time calculated holograms.

Two-photon polymerization enables the fabrication of micron sized structures with submicron resolution. Spatial light modulators (SLM) have already been used to create multiple polymerizing foci in the photoresist by holographic beam shaping, thus enabling the parallel fabrication of multiple microstructures. Here we demonstrate the parallel two-photon polymerization of single 3D microstructures by multiple holographically translated foci. Multiple foci were created by phase holograms, which were calculated real-time on an NVIDIA CUDA GPU, and displayed on an electronically addressed SLM. A 3D demonstrational structure was designed that is built up from a nested set of dodecahedron frames of decreasing size. Each individual microstructure was fabricated with the parallel and coordinated motion of 5 holographic foci. The reproducibility and the high uniformity of features of the microstructures were verified by scanning electron microscopy.

High-speed integrated optical logic based on the protein bacteriorhodopsin.

The principle of all-optical logical operations utilizing the unique nonlinear optical properties of a protein was demonstrated by a logic gate constructed from an integrated optical Mach-Zehnder interferometer as a passive structure, covered by a bacteriorhodopsin (bR) adlayer as the active element. Logical operations were based on a reversible change of the refractive index of the bR adlayer over one or both arms of the interferometer. Depending on the operating point of the interferometer, we demonstrated binary and ternary logical modes of operation. Using an ultrafast transition of the bR photocycle (BR-K), we achieved high-speed (nanosecond) logical switching. This is the fastest operation of a protein-based integrated optical logic gate that has been demonstrated so far. The results are expected to have important implications for finding novel, alternative solutions in all-optical data processing research.

Optical forces through guided light deflections.

Optical trapping and manipulation typically relies on shaping focused light to control the optical force, usually on spherical objects. However, one can also shape the object to control the light deflection arising from the light-matter interaction and, hence, achieve desired optomechanical effects. In this work we look into the object shaping aspect and its potential for controlled optical manipulation. Using a simple bent waveguide as example, our numerical simulations show that the guided deflection of light efficiently converts incident light momentum into optical force with one order-of-magnitude improvement in the efficiency factor relative to a microbead, which is comparable to the improvement expected from orthogonal deflection with a perfect mirror. This improvement is illustrated in proof-of-principle experiments demonstrating the optical manipulation of two-photon polymerized waveguides. Results show that the force on the waveguide exceeds the combined forces on spherical trapping handles. Furthermore, it shows that static illumination can exert a constant force on a moving structure, unlike the position-dependent forces from harmonic potentials in conventional trapping.

Anisotropic organization and microscopic manipulation of self-assembling synthetic porphyrin microrods that mimic chlorosomes: bacterial light-harvesting systems.

Being able to control in time and space the positioning, orientation, movement, and sense of rotation of nano- to microscale objects is currently an active research area in nanoscience, having diverse nanotechnological applications. In this paper, we demonstrate unprecedented control and maneuvering of rod-shaped or tubular nanostructures with high aspect ratios which are formed by self-assembling synthetic porphyrins. The self-assembly algorithm, encoded by appended chemical-recognition groups on the periphery of these porphyrins, is the same as the one operating for chlorosomal bacteriochlorophylls (BChl's). Chlorosomes, rod-shaped organelles with relatively long-range molecular order, are the most efficient naturally occurring light-harvesting systems. They are used by green photosynthetic bacteria to trap visible and infrared light of minute intensities even at great depths, e.g., 100 m below water surface or in volcanic vents in the absence of solar radiation. In contrast to most other natural light-harvesting systems, the chlorosomal antennae are devoid of a protein scaffold to orient the BChl's; thus, they are an attractive goal for mimicry by synthetic chemists, who are able to engineer more robust chromophores to self-assemble. Functional devices with environmentally friendly chromophores-which should be able to act as photosensitizers within hybrid solar cells, leading to high photon-to-current conversion efficiencies even under low illumination conditions-have yet to be fabricated. The orderly manner in which the BChl's and their synthetic counterparts self-assemble imparts strong diamagnetic and optical anisotropies and flow/shear characteristics to their nanostructured assemblies, allowing them to be manipulated by electrical, magnetic, or tribomechanical forces.

Protein-based ultrafast photonic switching.

Several inorganic and organic materials have been suggested for utilization as nonlinear optical material performing light-controlled active functions in integrated optical circuits, however, none of them is considered to be the optimal solution. Here we present the first demonstration of a subpicosecond photonic switch by an alternative approach, where the active role is performed by a material of biological origin: the chromoprotein bacteriorhodopsin, via its ultrafast BR->K and BR->I transitions. The results may serve as a basis for the future realization of protein-based integrated optical devices that can eventually lead to a conceptual revolution in the development of telecommunications technologies.

Optical tweezers with tips grown at the end of fibers by photopolymerization.

We present a method to build an optical tip at the end of a single-mode optical fiber. The tip is grown by a self-writing process: photopolymerization by the light coming from the optical fiber. We developed a technique to produce a flat end surface on the tip. The good optical quality of the tip and the output laser beam was demonstrated by the fact that a counterpropagating optical trap could be constructed by using the tips with parameters comparable to regular fiber traps. Because of the small size of the tips, the tweezers require a much smaller space than regular fiber traps.

Optical microassembly platform for constructing reconfigurable microenvironments for biomedical studies.

Cellular development is highly influenced by the surrounding microenvironment. We propose user-reconfigurable microenvironments and bio-compatible scaffolds as an approach for understanding cellular development processes. We demonstrate a model platform for constructing versatile microenvironments by fabricating morphologically complex microstructures by two-photon polymerization (2PP) and then assembling these archetypal building blocks into various configurations using multiple, real-time configurable counterpropagating-beam (CB) traps. The demonstrated capacity for handling feature-rich microcomponents may be further developed into a generalized microassembly platform.

Dynamic fluctuation of proteins watched in real time.

The dynamic nature of protein function is a fundamental concept in the physics of proteins. Although the basic general ideas are well accepted most experimental evidence has an indirect nature. The detailed characterization of the dynamics is necessary for the understanding in detail. The dynamic fluctuations thought crucial for the function span an extremely broad time, starting from the picosecond regime. Recently, a few new experimental techniques emerged that permit the observation of dynamical phenomena directly. Notably, pulsed infrared (IR) spectroscopy has been applied with great success to observe structural changes with picosecond time resolution. Using two-dimensional-IR vibrational echo chemical exchange spectroscopy Ishikawa and co-workers [Ishikawa et al. (2008), Proc. Natl. Acad. Sci. U.S.A. 101, 14402-14407] managed to observe the transition between well defined conformational substrates of carbonmonoxy myoglobin directly. This is an important step in improving our insight into the details of protein function.

Integrated optical switching based on the protein bacteriorhodopsin.

According to our earlier pioneering study, a dry film containing native bacteriorhodopsin (bR) shows unique nonlinear optical properties (refractive index change, controllable by light of different colors, greater than 2 x 10(-3)) that are in many respects superior to those of the materials presently applied in integrated optics. Here, we report on the first integrated optical application based on a miniature Mach-Zehnder interferometer (see Figs. 1 and 2) demonstrating a real switching effect by bR (efficiency higher than 90%) due to the M-state. Our results also imply that the refractive index change of the K-state (9 x 10(-4)) is high enough for fast switching.

2D optical manipulation and assembly of shape-complementary planar microstructures.

Optical trapping and manipulation offer great flexibility as a non-contact microassembly tool. Its application to the assembly of microscale building blocks may open new doors for micromachine technology. In this work, we demonstrate all-optical assembly of microscopic puzzle pieces in a fluidic environment using programmable arrays of trapping beams. Identical shape-complimentary pieces are optically fabricated with submicron resolution using two-photon polymerization (2PP) technique. These are efficiently assembled into space-filling tessellations by a multiple-beam optical micromanipulation system. The flexibility of the system allows us to demonstrate both user-interactive and computer-automated modes of serial and parallel assembly of microscale objects with high spatial and angular positioning precision.

Parallel photopolymerisation with complex light patterns generated by diffractive optical elements.

Photopolymerisation by scanning a focused laser beam is a powerful method to build structures of arbitrary complexity with submicrometer resolution. We introduce parallel photopolymerisation to enhance the efficiency. Instead of multidimensional scanning of a single focus, the structure is generated simultaneously with diffractive patterns. We used fixed diffractive optical elements (DOEs), kinoforms, and Spatial Light Modulators (SLMs). The possibilities of photopolymerisation using SLM were investigated: the added flexibility using the programmable device is demonstrated. By using these DOEs, straight and helical cross shaped columns were produced with a single scan at a rate about an order of magnitude faster than by simple scanning. The produced helical structures could be rotated by optical tweezers.

Direct measurement of torque in an optical trap and its application to double-strand DNA.

We present a method that offers the possibility to directly apply and measure torque on particles in an optical trap. It can be used to rotationally manipulate biopolymers attached to appropriate particles. A flat object is trapped and oriented in the focus of a linearly polarized laser light. The direction and power of the orientational trap are controlled by the polarization state of the light. As a demonstration of the capabilities of the method, we examined the torsional stiffness of dsDNA (lambda-DNA) in its linear torsional regime by directly measuring the torque generated by the molecule.

Integrated optical motor.

A light-driven micrometer-sized mechanical motor is created by laser-light-induced two-photon photopolymerization. All necessary components of the engine are built upon a glass surface by an identical procedure and include the following: a rigid mechanical framework, a rotor freely rotating on an axis, and an integrated optical waveguide carrying the actuating light to the rotor. The resulting product is a most practical stand-alone system. The light introduced into the integrated optical waveguide input of the motor provides the driving force: neither optical tweezers or even a microscope are needed for the function. The power and efficiency of the motor are evaluated. The independent unit is expected to become an important component of more complex integrated lab-on-a-chip devices.