Fluorescence-Based Detection of Ferrous Iron in Senescent Cells.

A major limitation in aging research is the lack of reliable biomarkers to assess phenotypic changes with age or monitor response to antiaging interventions. This study investigates the role of intracellular ferrous iron (Fe2+) as a potential biomarker of senescence. Iron is known to accumulate in various tissues with age and recent studies have demonstrated that its level increases dramatically in senescent cells. The current techniques used to measure the accumulation of iron are cumbersome and only measure total iron not specific isotopes such as the redox reactive Fe2+. It is still to be determined whether the damaging form of iron (Fe2+) is specifically elevated in senescent cells. In this study, we assessed the potential use of a newly discovered Fe2+ reactive probe (SiRhoNox-1) for selective labeling of senescent cells in vitro. For this we have generated various senescent cell models and subjected them to SiRhoNox-1 labeling. Our results indicate that SiRhoNox-1 selectivity labels live senescent cells and was more specific and faster than current staining such as SA-ßGal or a derived fluorescent probe C12FDG. Together these findings suggest that SiRhoNox-1 may serve as a convenient tool to detect senescent cells based on their ferrous iron level.

Copine A Interacts with Actin Filaments and Plays a Role in Chemotaxis and Adhesion.

Copines make up a family of calcium-dependent, phospholipid-binding proteins found in numerous eukaryotic organisms. Copine proteins consist of two C2 domains at the N-terminus followed by an A domain similar to the von Willebrand A domain found in integrins. We are studying copine protein function in the model organism, Dictyostelium discoideum, which has six copine genes, cpnA-cpnF. Previous research showed that cells lacking the cpnA gene exhibited a cytokinesis defect, a contractile vacuole defect, and developmental defects. To provide insight into the role of CpnA in these cellular processes, we used column chromatography and immunoprecipitation to isolate proteins that bind to CpnA. These proteins were identified by mass spectrometry. One of the proteins identified was actin. Purified CpnA was shown to bind to actin filaments in a calcium-dependent manner in vitro. cpnA- cells exhibited defects in three actin-based processes: chemotaxis, cell polarity, and adhesion. These results suggest that CpnA plays a role in chemotaxis and adhesion and may do so by interacting with actin filaments.

Optical catapulting of microspheres in mucus models-toward overcoming the mucus biobarrier.

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.

Natural convection induced by an optically fabricated and actuated microtool with a thermoplasmonic disk.

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.

Light-driven micro-tool equipped with a syringe function.

Leveraging developments in microfabrication open new possibilities for optical manipulation. With the structural design freedom from three-dimensional printing capabilities of two-photon polymerization, we are starting to see the emergence of cleverly shaped 'light robots' or optically actuated micro-tools that closely resemble their macroscopic counterparts in function and sometimes even in form. In this work, we have fabricated a new type of light robot that is capable of loading and unloading cargo using photothermally induced convection currents within the body of the tool. We have demonstrated this using silica and polystyrene beads as cargo. The flow speeds of the cargo during loading and unloading are significantly larger than when using optical forces alone. This new type of light robot presents a mode of material transport that may have a significant impact on targeted drug delivery and nanofluidics injection.

GPC light shaping a supercontinuum source.

Generalized Phase Contrast (GPC) is a versatile tool for efficiently rerouting and managing photon energy into speckle-free contiguous spatial light distributions. We have previously shown theoretically and numerically that a GPC Light Shaper shows robustness to shift in wavelength and can maintain both projection length scale and high efficiency over a range [0.75λ(0); 1.5λ(0)] with λ(0) as the characteristic design wavelength. With this performance across multiple wavelengths and the recent availability of tabletop supercontinuum lasers, GPC light shaping opens the possibility for creatively incorporating various multi-wavelength approaches into spatially shaped excitations that can enable new broadband light applications. We verify this new approach using a supercontinuum light source, interfaced with a compact GPC light shaper. Our experiments give ~70% efficiency, ~3x intensity gain, and ~85% energy savings, limited, however, by the illumination equipment, but still in very good agreement with theoretical and numerical predictions.

GPC light shaper: static and dynamic experimental demonstrations.

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. GPC allows efficient utilization of typical Gaussian lasers in such applications using binary-only phase modulation. In this work, we experimentally verify previously derived conditions for photon-efficient light shaping with GPC [Opt. Express22(5), 5299 (2014)]. We demonstrate a compact implementation of GPC for creating practical illumination shapes that can find use in light-efficient industrial or commercial applications. Using a dynamic spatial light modulator, we also show simple and efficient beam shaping of reconfigurable shapes geared towards materials processing, biophotonics research and other contemporary applications. Our experiments give ~80% efficiency, ~3x intensity gain, and ~90% energy savings which are in good agreement with previous theoretical estimations.

Dynamic diffraction-limited light-coupling of 3D-maneuvered wave-guided optical waveguides.

We have previously proposed and demonstrated the targeted-light delivery capability of wave-guided optical waveguides (WOWs). As the WOWs are maneuvered in 3D space, it is important to maintain efficient light coupling through the waveguides within their operating volume. We propose the use of dynamic diffractive techniques to create diffraction-limited spots that will track and couple to the WOWs during operation. This is done by using a spatial light modulator to encode the necessary diffractive phase patterns to generate the multiple and dynamic coupling spots. The method is initially tested for a single WOW and we have experimentally demonstrated dynamic tracking and coupling for both lateral and axial displacements.

GPC light shaper for speckle-free one- and two-photon contiguous pattern excitation.

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.

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.

Matched filtering Generalized Phase Contrast using binary phase for dynamic spot- and line patterns in biophotonics and structured lighting.

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.

Matched-filtering generalized phase contrast using LCoS pico-projectors for beam-forming.

We report on a new beam-forming system for generating high intensity programmable optical spikes using so-called matched-filtering Generalized Phase Contrast (mGPC) applying two consumer handheld pico-projectors. Such a system presents a low-cost alternative for optical trapping and manipulation, optical lattices and other beam-shaping applications usually implemented with high-end spatial light modulators. Portable pico-projectors based on liquid crystal on silicon (LCoS) devices are used as binary phase-only spatial light modulators by carefully setting the appropriate polarization of the laser illumination. The devices are subsequently placed into the object and Fourier plane of a standard 4f-setup according to the mGPC spatial filtering configuration. Having a reconfigurable spatial phase filter, instead of a fixed and fabricated one, allows the beam shaper to adapt to different input phase patterns suited for different requirements. Despite imperfections in these consumer pico-projectors, the mGPC approach tolerates phase aberrations that would have otherwise been hard to overcome by standard phase projection.

BioPhotonics workstation: a versatile setup for simultaneous optical manipulation, heat stress, and intracellular pH measurements of a live yeast cell.

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 (pH(i)) response and membrane integrity of an optically trapped Saccharomyces cerevisiae cell at 5 mW subject to increasing temperatures. The pH(i) 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 pH(i) 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.

Experimental demonstration of Generalized Phase Contrast based Gaussian beam-shaper.

We report the first experimental demonstration of Gaussian beam-shaping based on the Generalized Phase Contrast (GPC) approach. We show that, when using a dynamic spatial light modulator (SLM), this approach can rapidly generate arbitrarily shaped beams. Moreover, we demonstrate that low-cost binary-phase optics fabricated using photolithography and chemical etching techniques can replace the SLM in static and high power beam shaping applications. The design parameters for the binary-phase elements of the module are chosen according to the results of our previously conducted analysis and numerical demonstrations [Opt. Express 15, 11971 (2007)]. Beams with a variety of cross-sections such as circular, rectangular and square, with near flat-top intensity distributions are demonstrated. GPC-based beam shaping is inherently speckle-free and the shaped beams maintain a flat output phase. The non-absorbing components used in this beam-shaping approach have a high-damage-threshold and are thus ideally suited for high power applications.

Dynamic axial stabilization of counter-propagating beam-traps with feedback control.

Optical trapping in a counter-propagating (CP) beam-geometry provides unique advantages in terms of working distance, aberration requirements and intensity hotspots. However, its axial performance is governed by the wave propagation of the opposing beams, which can limit the practical geometries. Here we propose a dynamic method for controlling axial forces to overcome this constraint. The technique uses computer-vision object tracking of the axial position, in conjunction with software-based feedback, for dynamically stabilizing the axial forces. We present proof-of-concept experiments showing real-time rapid repositioning coupled with a strongly enhanced axial trapping for a plurality of particles of varying sizes. We also demonstrate the technique's adaptability for real-time reconfigurable feedback-trapping of a dynamically growing structure that mimics a continuously dividing cell colony. Advanced implementation of this feedback-driven approach can help make CP-trapping resistant to a host of perturbations such as laser fluctuations, mechanical vibrations and other distortions emphasizing its experimental versatility.