We have previously proposed and demonstrated the targeted-light delivery capability of wave-guided optical waveguides (WOWs). The full strength of this structure-mediated paradigm can be harnessed by addressing multiple WOWs and manipulating them to work in tandem. We propose the use of diffractive techniques to create multiple focal spots that can be coupled into light manipulated WOWs. This is done by using a spatial light modulator to project the necessary phase to generate the multiple coupling light spots. We incorporate a diffractive setup in our Biophotonics Workstation (BWS) and demonstrate holographic shaping, tracking of light in 3D with the purpose of coupling light in the WOWs.

Brettanomyces bruxellensis yeast produces ethyl phenols, which cause wine spoilage and severe economic losses. Ethyl phenols are produced by a class of polyphenols that are present in grapes and musts, called hydrocinnamic acids, which are capable of inhibiting yeasts and bacteria. The viability and intracellular pH changes in B. bruxellensis DSM 7001, in response to extracellular pH, as well as to the presence of an energy source and hydroxycinnamic acids, have been investigated in this paper by means of Fluorescent Ratio Imaging Microscopy (FRIM). The results show that B. bruxellensis DSM 7001 is able to maintain viability and increase its pH gradient with decreasing external pH values, whereas it is not able to maintain a pH gradient at high external pH values (i.e. pH 8) and, as a consequence, dies. The growth inhibitory effects of ferulic and p-coumaric acid do not seem to be caused by a weak-acid inhibition mechanism, since both acids induce a similar, or even higher, intracellular acidification at a high external pH than at a low external pH. The results presented have to be confirmed by using other strains of B. bruxellensins in order to validate the outcomes obtained in this study. (C) 2014 Elsevier Ltd. All rights reserved.

Generalized Phase Contrast (GPC) is an efficient method for generating speckle-free contiguous optical distributions useful in diverse applications such as static beam shaping, optical manipulation and recently, for excitation in two-photon optogenetics. To fully utilize typical Gaussian lasers in such applications, we analytically derive conditions for photon efficient light shaping with GPC. When combined with the conditions for optimal contrast developed in previous works, our analysis further simplifies GPC's implementation. The results of our analysis are applied to practical illumination shapes, such as a circle and different rectangles commonly used in industrial or commercial applications. We also show simple and efficient beam shaping of arbitrary shapes geared towards biophotonics research and other contemporary applications. Optimized GPC configurations consistently give ~84% efficiency and ~3x intensity gain. Assessment of the energy savings when comparing to conventional amplitude masking show that ~93% of typical energy losses are saved with optimized GPC configurations.

Optically micromanipulated waveguides can be arbitrarily positioned and oriented for targeted light delivery. At the same time, controlled light deflection in designed waveguides can be exploited to exert optical forces for new optical micromanipulation modalities.

We demonstrate the efficient generation of line patterns using matched-filtering Generalized Phase Contrast (mGPC). So far, the main emphasis of mGPC light addressing has been on the creation of rapidly reconfigurable focused spots. This has recently been extended to encoding extended line patterns for structured light applications and advanced microscopy.

This work discusses the use of matched filtering Generalized Phase Contrast (mGPC) as an efficient and cost-effective beam shaper for applications such as in biophotonics, optical micromanipulation, microscopy and two-photon polymerization. The theoretical foundation of mGPC is described as a combination of Generalized Phase Contrast and phase-only correlation. Such an analysis makes it convenient to optimize an mGPC system for different setup conditions. Results showing binary-only phase generation of dynamic spot arrays and line patterns are presented.

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.

The science fiction inspired shrinking of macro-scale robotic manipulation and handling down to the micro- and nano-scale regime open new doors for exploiting the forces and torques of light for micro- and nanobiologic probing, actuation and control [1]. Advancing light-driven micro-robotics requires the optimization of optical forces and torques that, in turn, requires optimization of the underlying light-matter interaction. The requirement of having tightly focused beams in optical tweezing systems exemplifies the need for optimal light-shaping in optical trapping, manipulation and sorting [2]. On the other hand, the recent report on stable optical lift shows that optical manipulation can be achieved, even when using unshaped light, by using an appropriately shaped structure instead [3]. Therefore, a generic approach for optimizing light-matter interaction would involve the combination of optimal light-sculpting techniques [4] with the use of optimized shapes in micro-robotics structures [5]. Micro-fabrication processes such as two-photon photo-polymerization offer three-dimensional resolutions for creating custom-designed monolithic microstructures that can be equipped with optical trapping handles for convenient mechanical control using only optical forces [6]. These microstructures can be effectively handled with simultaneous top- and side-view on our BioPhotonics Workstation to carry out proof-of-principle experiments illustrating the six-degree-of-freedom optical actuation of two-photon polymerised microstructures equipped with features easily entering the submicron-regime. Furthermore, we exploited the light shaping capabilities available on the BioPhotonics Workstation to demonstrate a new strategy for controlling microstructures that goes beyond the typical refractive light deflections that are utilized in conventional optical trapping and manipulation. We took this approach to extend the opto-mechanical

The synergy between photonics, nanotechnology and biotechnology is spawning the emerging fields of nano-biotechnology and nano-biophotonics. Photonic innovations already hurdle the diffraction barrier for imaging with nanoscopic resolutions. However, scientific hypothesis testing demands tools, not only for observing nanoscopic phenomena, but also for reaching into and manipulating nanoscale constituents in this domain. This report is two-fold desribing the new use of proprietary strongholds we currently are establishing at DTU Fotonik on new means of sculpting of both light and matter for bio-probing at the smallest scales.

We are presenting so-called Wave-guided Optical Waveguides (WOWs) fabricated by two-photon polymerization and capable of being optically manipulated into any arbitrary orientation. By integrating optical waveguides into the structures we have created freestanding waveguides which can be positioned anywhere in a sample at any orientation using real-time 3D optical micromanipulation with six degrees of freedom. One of the key aspects of our demonstrated WOWs is the change in direction of in-coupled light and the marked increase in numerical aperture of the out-coupled light. Hence, each light propelled WOW can tap from a relatively broad incident beam and generate a much more tightly confined light at its tip. The presentation contains both numerical simulations related to the propagation of light through a WOW and preliminary experimental demonstrations on our BioPhotonics Workstation. In a broader context, this research shows that optically trapped micro-fabricated structures can potentially help bridge the diffraction barrier. This structure-mediated paradigm may be carried forward to open new possibilities for exploiting beams from far-field optics down to the sub-wavelength domain.

We propose the use of consumer pico projectors as cost effective spatial light modulators in cell sorting applications. The matched filtering Generalized Phase Contrast (mGPC) beam shaping method is used to produce high intensity optical spots for trapping and catapulting cells. A pico projector’s liquid crystal on silicon (LCoS) chip was used as a binary phase spatial light modulator (SLM) wherein correlation target patterns are addressed. Experiments using the binary LCoS phase SLM with a fabricated Pyrex matched filter demonstrate the generation of intense optical spots that can potentially be used for cell sorting. Numerical studies also show mGPC’s robustness to phase aberrations in the LCoS device, and its ability to outperform a top hat beam with the same power.

Robotics can use optics feedback in vision-based control of intelligent robotic guidance systems. With light's miniscule momentum, shrinking robots down to the microscale regime creates opportunities for exploiting optical forces and torques in microrobotic actuation and control. Indeed, the literature on optical trapping and micromanipulation attests to the possibilities for optical microrobotics. This work presents an optical microrobotics perspective on the optical microfabrication and micromanipulation work that we performed. We designed different three-dimensional microstructures and fabricated them by two-photon polymerization. These microstructures were then handled using our biophotonics workstation (BWS) for proof-of-principle demonstrations of optical actuation, akin to 6DOF actuation of robotic micromanipulators. Furthermore, we also show an example of dynamic behavior of the trapped microstructure that can be achieved when using static traps in the BWS. This can be generalized, in the future, towards a structural shaping optimization strategy for optimally controlling microstructures to complement approaches based on light-shaping. We also show that light channeled to microfabricated, free-standing waveguides can be used not only to redirect light for targeted delivery of optical energy but can also for targeted delivery of optical force, which can serve to further extend the manipulation arms in optical robotics. Moreover, light deflection with waveguide also creates a recoil force on the waveguide, which can be exploited for controlling the optical force.

We describe an indirect optical gripper for noninvasive micromanipulation of sensitive objects, such as cells. Our optical gripper, driven by dynamic holographic optical tweezers, consists of an arrangement of six silica beads, each held in place by an optical trap. The beads are moved so as to grip an object, which itself is not significantly exposed to laser light. Six beads are sufficient to grip an object reliably, consistent with robotics design rules. We find that improved gripping is achieved when beads are arranged into groups of triplets, since each triplet of trapped beads forms a weakly attractive pocket into which other objects are drawn.

Internal pH regulation is vital for many cell functions, including transport mechanisms and metabolic enzyme activity. More specifically, transport mechanisms are to a wide degree governed by internal pH distributions. We introduce the term standard deviation of the intracellular pH (s.d.(pH int )) to describe the internal pH distributions. The cellular pH distributional response to external stress such as heat has not previously been determined. In this study, the intracellular pH (pH i ) and the s.d.(pH int ) of Saccharomyces cerevisiae cells exposed to supralethal temperatures were measured using fluorescence ratio imaging microscopy (FRIM). An exponential decline in pH i was observed after an initial small decline. For the first time, we report the use of FRIM for determining in vivo plasma membrane proton permeability coefficients in yeast. Furthermore, the exponential decay of pH i and the rupture of the cell plasma membrane, as measured by propidium iodide staining, at 70°C were not simultaneous but were separated by a significant temporal difference. Finally, a nonlinear relationship between the pH i and s.d.(pH int ) was found; i.e. the s.d.(pH int ) was significantly more sensitive to supralethal temperatures than pH i . s.d.(pH int ) is therefore proposed as an early health/vitality indicator in S. cerevisiae cells exposed to heat stress.

In this study we have modified the BioPhotonics workstation (BWS), which allows for using long working distance objective for optical trapping, to include traditional epi-fluorescence microscopy, using the trapping objectives. We have also added temperature regulation of sample stage, allowing for fast temperature variations while trapping. Using this modified BWS setup, we investigated the internal pH (pHi) response and membrane integrity of an optically trapped Saccharomyces cerevisiae cell at 5 mW subject to increasing temperatures. The pHi of the cell is obtained from the emission of 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, at 435 and 485 nm wavelengths, while the permeability is indicated by the fluorescence of propidium iodide. We present images mapping the pHi and permeability of the cell at different temperatures and with enough spatial resolution to localize these attributes within the cell. The combined capability of optical trapping, fluorescence microscopy and temperature regulation offers a versatile tool for biological research.

The effect of a 1070-nm continuous and pulsed wave ytterbium fiber laser on the growth of Saccharomyces cerevisiae single cells is investigated over a time span of 4 to 5 h. The cells are subjected to optical traps consisting of two counterpropagating plane wave beams with a uniform flux along the x, y axis. Even at the lowest continuous power investigated-i.e., 0.7 mW-the growth of S. cerevisiae cell clusters is markedly inhibited. The minimum power required to successfully trap single S. cerevisiae cells in three dimensions is estimated to be 3.5 mW. No threshold power for the photodamage, but instead a continuous response to the increased accumulated dose is found in the regime investigated from 0.7 to 2.6 mW. Furthermore, by keeping the delivered dose constant and varying the exposure time and power-i.e. pulsing-we find that the growth of S. cerevisiae cells is increasingly inhibited with increasing power. These results indicate that growth of S. cerevisiae is dependent on both the power as well as the accumulated dose at 1070 nm.