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The affinity of T-cell receptors (TCRs) for major histocompatibility complex molecules (MHCs) presenting cognate antigens likely determines whether T cells initiate immune responses, or not. There exist few measurements of two-dimensional (2D) TCR-MHC interactions, and the effect of auxiliary proteins on binding is unexplored. Here, Jurkat T-cells expressing the MHC molecule HLA-DQ8-glia-α1 and the ligand of an adhesion protein (rat CD2) were allowed to bind supported lipid bilayers (SLBs) presenting fluorescently-labelled L3-12 TCR and rat CD2, allowing measurements of binding unconfounded by cell signaling effects or co-receptor binding. The 2D Kd for L3-12 TCR binding to HLA-DQ8-glia-α1, 14±5 molecules/μm2, was only marginally influenced by including CD2 up to ∼200 bound molecules/μm2 but higher CD2 densities reduced the affinity up to 1.9-fold. Cell-SLB contact size increased steadily with ligand density without affecting binding for contacts up to ∼20% of total cell area but beyond this lamellipodia appeared, giving an apparent increase in bound receptors of up to 50%. Our findings show how parameters other than the specific protein-protein interaction can influence binding behavior at cell-cell contacts.

The generalized phase contrast method is employed as an efficient "phase-only" laser beam-shaping technique in an optical setup built for catapulting microspheres through simple mucus models. The influence of the laser power and mucin concentration on the motion of the microspheres is investigated in terms of instant and average velocities on a 250-μm trajectory, corresponding to the mucus thickness in the human gastrointestinal tract. Increasing the laser power leads to higher velocities in all the tested samples, while increasing the mucin concentration leads to significant velocity decrease for similar laser input power. However, velocities of up to 95 μm · s - 1 are demonstrated in a 5% mucin simple mucus model using our catapulting system. This study contributes to understanding and overcoming the challenges of optical manipulation in mucus models. This paves the way for efficient optical manipulation of three-dimensional-printed light-controlled microtools with the ability to penetrate the mucus biobarrier for in vitro drug-delivery studies.

Two-photon polymerization was employed for fabricating microtools amenable to optical trapping and manipulation. A disk feature was included as part of the microtools and further functionalized by electron-beam deposition. The nanostructured gold layer on the disk facilitates off-resonant plasmonic heating upon illumination with a laser beam. As a consequence, natural convection characterized by the typical toroidal shape resembling that of Rayleigh–Bénard flow can be observed. A velocity of several μm·s−1 is measured for 2 μm microspheres dispersed in the surroundings of the microtool. To the best of our knowledge, this is the first time that thermoplasmonic-induced natural convection is experimentally demonstrated using a mobile heat source.

After years of working on light-driven trapping and manipulation, we can see that a confluence of developments is now ripe for the emergence of a new area that can contribute to nanobiophotonics - Light Robotics - which combines advances in microfabrication and optical micromanipulation together with intelligent control ideas from robotics, wavefront engineering and information optics. In the Summer 2017 we are publishing a 482 pages edited Elsevier book volume covering the fundamental aspects needed for Light Robotics including optical trapping systems, microfabrication and microassembly as well as underlying theoretical principles and experimental illustrations for optimizing optical forces and torques for Light Robotics

Microfluidic systems have gained much interest in the past decade as they tremendously reduce sample volume requirements for investigating different phenomena and for various medical, pharmaceutical and defense applications. Rapid heat transfer and efficient diffusive material transport are among the benefits of miniaturization. These have been achieved so far by tediously designing and fabricating application-specific microfluidic chambers or by employing microdevices that can be difficult to integrate in microfluidic systems. In this work, we present the fabrication and functionalization via two-photon polymerization and physical vapor deposition of microstructures that serve as heat sources in microfluidic devices upon laser illumination. In contrast to other existing methods that rely on photo-thermal effects, our microtools are amenable to optical manipulation and can be actuated in specific locations where heat generation is desired. Heating effects manifest in the presence of a temperature gradient, induced fluid flow and the formation of microbubbles.

Light Robotics is one of the newest progenies of the robotics family, bringing together advances in microfabrication and optical manipulation with intelligent control ideas from robotics and Fourier optics. The development of lightcontrollable microrobots capable of performing specific tasks at the microscale requires the ability to sculpt the two protagonists of the story: the light and the microrobots. Complex light sculpting for optical trapping has been in focus for over three decades, and its importance for controlling microscopic objects is well understood. Designing intricate microrobots for the task is a more recent development facilitated by state-of-the-art microfabrication techniques, and particularly by two-photon polymerization. The full 3D design freedom offered by two-photon polymerization opens the door for imagination, while at the same time bringing the responsibility of rationally designing microrobots tailored to specific tasks. In addition to shape and topology features, the surface chemistry of the microrobots can also help steer them towards specific applications. This paper will discuss strategies for the design and fabrication of light-controllable microrobots as a toolbox for biomedical applications.

Two-photon polymerization (2PP) enables the microfabrication of structures at a resolution of about 100 nm by using femtosecond laser pulses. Microrobotic structures that allow optical manipulation can be fabricated with the aid of 2PP in a suitable photoresist. This is particularly interesting for the emerging field of Light Robotics1, which combines the most recent technologies in microfabrication and nanobiophotonics. Optical trapping has been employed in the past 30 years for e.g. single molecule studies, microrheology measurements or even subcellular delivery and sampling. Complex sculpting of the light for different types of optical trapping and manipulation experiments has been thoroughly explored. However, most optical trapping experiments make use of simple microbeads, which have limited functionality. An important step forward in the field of Light Robotics is the use of 3D printed microrobotic structures instead of microbeads. Including bead-shaped “handles” in the microrobot structures allows optical manipulation in a similar manner to that of simple microbeads. By using Generalized Phase Contrast (GPC) or Holographic GPC (Holo-GPC) as beam shaping techniques, multiple optical traps can be simultaneously generated and thus a microrobot with multiple “handles” can be controlled with full spatio-temporal freedom. Sculpting the object2, in addition to sculpting the light, allows incorporating specific features into the design. Our group has previously reported microrobots equipped with a syringe function3. Here, we show the optical manipulation of a disk-tip based optical microrobot (Figure 1). In the future, the disktip feature will be employed for inducing local perturbations of biological samples, such as mucus biobarriers, lipid bilayers or even cell membranes. This could facilitate e.g. DNA transfection in plant cells or to burst open bacteria cells. By incorporating different features into the microrobot design we are working towards developing a seri