Unlike an equilibrium gas, the distribution of active particles can be very sensitive to what happens at the boundaries of their container. Experiments and simulations have previously highlighted the possibility of exploiting this behavior for the geometric control of active particles, although a general theoretical framework is lacking. Here we propose a boundary method based on the flux transfer formalism typical of radiometry problems, where surface elements transmit and receive "rays" of active particles with infinite persistence length. As in the case of blackbody radiation, a Lambert scattering law results in a uniform distribution of active particles within the cavity, while other scattering laws result in specific patterns of particle accumulation in the bulk or over the boundary walls. We validate our method's predictions with numerical simulations and demonstrate its practical utility by spatially controlling swimming microalgae confined in light-defined arenas. The presented boundary method offers a simple and efficient way to predict particle distributions when both the geometry of the boundaries and the scattering law are known. In addition, it provides a general design principle for engineering container shapes optimized for transport, accumulation, and sorting of self-propelled colloids and microorganisms.

Novel imaging techniques utilizing nondegenerate, correlated photon pairs sparked intense interest during the last couple of years among scientists of the quantum optics community and beyond. It is a key property of such "ghost imaging" or "quantum interference" methods that they use those photons of the correlated pairs for imaging that never interacted with the sample, allowing detection in a spectral range different from that of the illumination of the object. Extensive applications of these techniques in spectroscopy and microscopy are envisioned, however, their limited spatial resolution to date has not yet supported real-life microscopic investigations of tiny biological objects. Here we report a modification of the method based on quantum interference by using a seeding laser and confocal scanning, that allows the improvement of the resolution of imaging with undetected photons by more than an order of magnitude, and we also present examples of application in the microscopy of biological samples.

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.

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.

Whispering gallery mode (WGM) resonators are promising optical structures for microfluidic label-free biosensors mainly due to their high sensitivity, but from a practical point of view they present numerous constraints that make their use in real laboratory diagnosis application difficult. Herein we report on a monolithic lab on a chip fabricated by a hybrid femtosecond laser micromachining approach, for label-free biosensing. It consists of a polymer WGM microresonator sensor integrated inside a glass microfluidic chip, presenting a refractive index change sensitivity of 61 nm per RIU. The biosensing capabilities of the device have been demonstrated by exploiting the biotin-streptavidin binding affinity, obtaining a measurable minimum surface density increase of 67 × 103 molecules per μm2.

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.

The extremely high peak intensity associated with ultrashort pulse width of femtosecond laser allows us to induce nonlinear interaction such as multiphoton absorption and tunneling ionization with materials that are transparent to the laser wavelength. More importantly, focusing the femtosecond laser beam inside the transparent materials confines the nonlinear interaction only within the focal volume, enabling three-dimensional (3D) micro- and nanofabrication. This 3D capability offers three different schemes, which involve undeformative, subtractive, and additive processing. The undeformative processing preforms internal refractive index modification to construct optical microcomponents including optical waveguides. Subtractive processing can realize the direct fabrication of 3D microfluidics, micromechanics, microelectronics, and photonic microcomponents in glass. Additive processing represented by two-photon polymerization enables the fabrication of 3D polymer micro- and nanostructures for photonic and microfluidic devices. These different schemes can be integrated to realize more functional microdevices including lab-on-a-chip devices, which are miniaturized laboratories that can perform reaction, detection, analysis, separation, and synthesis of biochemical materials with high efficiency, high speed, high sensitivity, low reagent consumption, and low waste production. This review paper describes the principles and applications of femtosecond laser 3D micro-and nanofabrication for lab-on-a-chip applications. A hybrid technique that promises to enhance functionality of lab-on-a-chip devices is also introduced.

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.

Mechanical manipulation of live cells is crucial to numerous biological experiments. Optical trapping and manipulation is a popular and widely used approach, however, direct optical trapping suffers from serious limitations: The intensive laser illumination damages the cells, in addition, since trapping occurs due to interaction of light with high index of refraction regions in the cells, the trapping is not defined sufficiently well for high precision manipulation. To overcome these problems microbeads have already been used for trapping cells indirectly thereby reducing the irradiation damage and increasing trapping efficiency with their high refractive index contrast. The drawbacks can be completely eliminated by the use of tailor made microtools designed specifically for this task, so that indirect trapping and manipulation is applied. The trapping light interacts only with the tools, and the shape of the tool can be optimized for accurate and efficient manipulation around all spatial coordinates as well as for optimal cell handling. The microtools of complex shape are fabricated by two-photon excitation photopolymerization. Appropriate chemical surface functionalization establishes controlled and strong attachment of the cells. Holographic optical traps are used to precisely control the position of the cells in 6D, the positional accuracy is in the order of 10 nm. The power of the approach is demonstrated by greatly improved imaging of live 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.

Optical micromanipulation in its original form involves the trapping of micrometer sized spheres in a high numerical aperture focused laser light. This arrangement permits the control of the location of the trapped sphere – with appropriate modifications the procedure proved to be a very useful micromanipulation tool. The capabilities of optical trapping can be vastly enhanced if instead of a sphere a complex structure, and instead of the simple focused light a shaped light pattern is used: this way most complicated manipulation schemes can be realized. The trapped objects can be rotated, they can be used like optical rotators, microprobes can be precisely directed to points of interest without mechanical contact, light driven micromechanical devices can be constructed to perform different tailored tasks. With appropriate surface modifications the optically manipulated microtools can be applied on microscopic test objects for mechanical actuation, spectroscopic investigations, etc. The possibilities are endless. In this article we will introduce the basic concepts of the technology: microstructure building by laser induced photopolymerization, the selective functionalization of the microtool surfaces, and the controlled optical micromanipulation with shaped optical fields. We will describe typical applications in diverse fields, like light driven micromechanical machines, light actuated microtools for local spectroscopy, technique for the torsional manipulation of biological macromolecules. The examples will illustrate the immense potential of the approach in the investigation and manipulation of microscopic objects.

Optical micro manipulation of live cells has been extensively used to study a wide range of cellular phenomena with relevance in basic research or in diagnostics. The approaches span from manipulation of many cells for high throughput measurement or sorting, to more elaborated studies of intracellular events on trapped single cells when coupled with modern imaging techniques. In case of direct cell trapping the damaging effects of light-cell interaction must be minimized, for instance with the choice of proper laser wavelength. Microbeads have already been used for trapping cells indirectly thereby reducing the irradiation damage and increasing trapping efficiency with their high refractive index contrast. We show here that such intermediate objects can be tailor-made for indirect cell trapping to further increase cell-to-focal spot distance while maintaining their free and fast maneuverability. Carefully designed structures were produced with two-photon polymerization with shapes optimized for effective manipulation and cell attachment. Functionalization of the microstructures is also presented that enables cell attachment to them within a few seconds with strength much higher that the optical forces. Fast cell actuation in 6 degrees of freedom is demonstrated with the outlook to possible applications in cell imaging.

Models of biological barriers are important to study physiological functions, transport mechanisms, drug delivery and pathologies. However, there are only a few integrated biochips which are able to monitor several of the crucial parameters of cell-culture-based barrier models. The aim of this study was to design and manufacture a simple but versatile device, which allows a complex investigation of barrier functions. The following functions and measurements are enabled simultaneously: co-culture of 2 or 3 types of cells; flow of culture medium; visualization of the entire cell layer by microscopy; real-time transcellular electrical resistance monitoring; permeability measurements. To this end, a poly(dimethylsiloxane)-based biochip with integrated transparent gold electrodes and with a possibility to connect to a peristaltic pump was built. Unlike previous systems, the structure of the device allowed a constant visual observation of cell growth over the whole membrane surface. Morphological characterization of the layers was also accomplished by immunohistochemical staining. The chip was applied to monitor and characterize models of the intestinal and lung epithelial barriers, and the blood-brain barrier. The models were established using human Caco-2 intestinal and A549 lung epithelial cell lines, hCMEC/D3 human brain endothelial cell line and primary rat brain endothelial cells co-cultured with primary astrocytes and brain pericytes. This triple primary co-culture blood-brain barrier model was assembled on a lab-on-a-chip device and investigated under fluid flow for the first time. Such a versatile tool is expected to facilitate the kinetic investigation of various biological barriers. (C) 2015 Elsevier B.V. All rights reserved.

We have studied the conditions of rotation induced by collimated light carrying no angular momentum. Objects of different shapes and optical properties were examined in the nontrivial case where the rotation axis is perpendicular to the direction of light propagation. This geometry offers important advantages for application as it fundamentally broadens the possible practical arrangements to be realised. We found that collimated light cannot drive permanent rotation of 2D or prism-like 3D objects (i.e., fixed cross-sectional profile along the rotation axis) in the case of fully reflective or fully transparent materials. Based on both geometrical optics simulations and theoretical analysis, we derived a general condition for rotation induced by collimated light carrying no angular momentum valid for any arrangement: Permanent rotation is not possible if the scattering interaction is two-dimensional and lossless. In contrast, light induced rotation can be sustained if partial absorption is present or the object has specific true 3D geometry. We designed, simulated, fabricated, and experimentally tested a microscopic rotor capable of rotation around an axis perpendicular to the illuminating light.

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.

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.