Shining Light in Mechanobiology: Optical Tweezers, Scissors, and Beyond.

Mechanobiology helps us to decipher cell and tissue functions by looking at changes in their mechanical properties that contribute to development, cell differentiation, physiology, and disease. Mechanobiology sits at the interface of biology, physics and engineering. One of the key technologies that enables characterization of properties of cells and tissue is microscopy. Combining microscopy with other quantitative measurement techniques such as optical tweezers and scissors, gives a very powerful tool for unraveling the intricacies of mechanobiology enabling measurement of forces, torques and displacements at play. We review the field of some light based studies of mechanobiology and optical detection of signal transduction ranging from optical micromanipulation-optical tweezers and scissors, advanced fluorescence techniques and optogenentics. In the current perspective paper, we concentrate our efforts on elucidating interesting measurements of forces, torques, positions, viscoelastic properties, and optogenetics inside and outside a cell attained when using structured light in combination with optical tweezers and scissors. We give perspective on the field concentrating on the use of structured light in imaging in combination with tweezers and scissors pointing out how novel developments in quantum imaging in combination with tweezers and scissors can bring to this fast growing field.

Tired and stressed: direct holographic quasi-static stretching of aging echinocytes and discocytes in plasma using optical tweezers [Invited].

Red blood cells (RBCs) undergo a progressive morphological transformation from smooth biconcave discocytes into rounder echinocytes with spicules on their surface during cold storage. The echinocytic morphology impacts RBCs' ability to flow through narrow sections of the circulation and therefore transfusion of RBC units with a high echinocytic content are thought to have a reduced efficiency. We use an optical tweezers-based technique where we directly trap and measure linear stiffness of RBCs under stress without the use of attached spherical probe particles or microfluidic flow to induce shear. We study RBC deformability with over 50 days of storage performing multiple stretches in blood plasma (serum with cold agglutinins removed to eliminate clotting). In particular, we find that discocytes and echinocytes do not show significant changes in linear stiffness in the small strain limit (∼20% change in length) up to day 30 of the storage period, but do find differences between repeated stretches. By day 50 the linear stiffness of discocytes had increased to approximately that measured for echinocytes throughout the entire period of measurements. These changes in stiffness corresponded to recorded morphological changes in the discocytes as they underwent storage lesion. We believe our holographic trapping and direct measurement technique has applications to directly control and quantify forces that stretch other types of cells without the use of attached probes.

Roadmap on Deep Learning for Microscopy.

Through digital imaging, microscopy has evolved from primarily being a means for visual observation of life at the micro- and nano-scale, to a quantitative tool with ever-increasing resolution and throughput. Artificial intelligence, deep neural networks, and machine learning are all niche terms describing computational methods that have gained a pivotal role in microscopy-based research over the past decade. This Roadmap is written collectively by prominent researchers and encompasses selected aspects of how machine learning is applied to microscopy image data, with the aim of gaining scientific knowledge by improved image quality, automated detection, segmentation, classification and tracking of objects, and efficient merging of information from multiple imaging modalities. We aim to give the reader an overview of the key developments and an understanding of possibilities and limitations of machine learning for microscopy. It will be of interest to a wide cross-disciplinary audience in the physical sciences and life sciences.

Enhanced Signal-to-Noise and Fast Calibration of Optical Tweezers Using Single Trapping Events.

The trap stiffness us the key property in using optical tweezers as a force transducer. Force reconstruction via maximum-likelihood-estimator analysis (FORMA) determines the optical trap stiffness based on estimation of the particle velocity from statistical trajectories. Using a modification of this technique, we determine the trap stiffness for a two micron particle within 2 ms to a precision of ∼10% using camera measurements at 10 kfps with the contribution of pixel noise to the signal being larger the level Brownian motion. This is done by observing a particle fall into an optical trap once at a high stiffness. This type of calibration is attractive, as it avoids the use of a nanopositioning stage, which makes it ideal for systems of large numbers of particles, e.g., micro-fluidics or active matter systems.

Optical Tweezers Exploring Neuroscience.

Over the past decade, optical tweezers (OT) have been increasingly used in neuroscience for studies of molecules and neuronal dynamics, as well as for the study of model organisms as a whole. Compared to other areas of biology, it has taken much longer for OT to become an established tool in neuroscience. This is, in part, due to the complexity of the brain and the inherent difficulties in trapping individual molecules or manipulating cells located deep within biological tissue. Recent advances in OT, as well as parallel developments in imaging and adaptive optics, have significantly extended the capabilities of OT. In this review, we describe how OT became an established tool in neuroscience and we elaborate on possible future directions for the field. Rather than covering all applications of OT to neurons or related proteins and molecules, we focus our discussions on studies that provide crucial information to neuroscience, such as neuron dynamics, growth, and communication, as these studies have revealed meaningful information and provide direction for the field into the future.

Strong Transient Flows Generated by Thermoplasmonic Bubble Nucleation.

The challenge of inducing and controlling localized fluid flows for generic force actuation and for achieving efficient mass transport in microfluidics is key to the development of next-generation miniaturized systems for chemistry and life sciences. Here we demonstrate a methodology for the robust generation and precise quantification of extremely strong flow transients driven by vapor bubble nucleation on spatially isolated plasmonic nanoantennas excited by light. The system is capable of producing peak flow speeds of the order mm/s at modulation rates up to ∼100 Hz in water, thus allowing for a variety of high-throughput applications. Analysis of flow dynamics and fluid viscosity dependence indicates that the transient originates in the rapid bubble expansion that follows nucleation rather than being strictly thermocapillary in nature.

Sound generation in zebrafish with Bio-Opto-Acoustics.

Hearing is a crucial sense in underwater environments for communication, hunting, attracting mates, and detecting predators. However, the tools currently used to study hearing are limited, as they cannot controllably stimulate specific parts of the auditory system. To date, the contributions of hearing organs have been identified through lesion experiments that inactivate an organ, making it difficult to gauge the specific stimuli to which each organ is sensitive, or the ways in which inputs from multiple organs are combined during perception. Here, we introduce Bio-Opto-Acoustic (BOA) stimulation, using optical forces to generate localized vibrations in vivo, and demonstrate stimulation of the auditory system of zebrafish larvae with precise control. We use a rapidly oscillated optical trap to generate vibrations in individual otolith organs that are perceived as sound, while adjacent otoliths are either left unstimulated or similarly stimulated with a second optical laser trap. The resulting brain-wide neural activity is characterized using fluorescent calcium indicators, thus linking each otolith organ to its individual neuronal network in a way that would be impossible using traditional sound delivery methods. The results reveal integration and cooperation of the utricular and saccular otoliths, which were previously described as having separate biological functions, during hearing.

Optical-trapping of particles in air using parabolic reflectors and a hollow laser beam.

We present an advanced optical-trapping method that is capable of trapping arbitrary shapes of transparent and absorbing particles in air. Two parabolic reflectors were used to reflect the inner and outer parts of a single hollow laser beam, respectively, to form two counter-propagating conical beams and bring them into a focal point for trapping. This novel design demonstrated high trapping efficiency and strong trapping robustness with a simple optical configuration. Instead of using expensive microscope objectives, the parabolic reflectors can not only achieved large numerical aperture (N.A.) focusing, but were also able to focus the beam far away from optical surfaces to minimize optics contamination. This design also offered a large free space for flexible integration with other measuring techniques, such as optical-trapping Raman spectroscopy, for on-line single particle characterization.

Giant vortex clusters in a two-dimensional quantum fluid.

Adding energy to a system through transient stirring usually leads to more disorder. In contrast, point-like vortices in a bounded two-dimensional fluid are predicted to reorder above a certain energy, forming persistent vortex clusters. In this study, we experimentally realize these vortex clusters in a planar superfluid: a 87Rb Bose-Einstein condensate confined to an elliptical geometry. We demonstrate that the clusters persist for long time periods, maintaining the superfluid system in a high-energy state far from global equilibrium. Our experiments explore a regime of vortex matter at negative absolute temperatures and have relevance for the dynamics of topological defects, two-dimensional turbulence, and systems such as helium films, nonlinear optical materials, fermion superfluids, and quark-gluon plasmas.

High-speed transverse and axial optical force measurements using amplitude filter masks.

Direct optical force measurement is a versatile method used in optical tweezers experiments, providing accurate measurements of forces for a wide range of particles and trapping beams. It is based on the detection of the change of the momentum of light scattered by a trapped object. A digital micromirror device can be used to selectively reflect light in different directions using an appropriately defined mask. We have developed position-sensitive masked detection (PSMD) for measuring transverse (radial) and axial forces. The method is comparable in performance to the fastest split detectors, while maintaining the linearity and customizability similar to duo-lateral position-sensitive detectors (PSD) and cameras. We show an order of magnitude increase in the bandwidth compared to a conventional PSD for radial forces. We measure axial force and verify the measurement using the Stokes drag for the particle. Combining both detectors (PSMD and PSD), we can perform full 3-D optical force measurements in real time.

Machine learning wall effects of eccentric spheres for convenient computation.

In confined systems, such as the inside of a biological cell, the outer boundary or wall can affect the dynamics of internal particles. In many cases of interest both the internal particle and outer wall are approximately spherical. Therefore, quantifying the wall effects from an outer spherical boundary on the motion of an internal eccentric sphere is very useful. However, when the two spheres are not concentric, the problem becomes nontrivial. In this paper we improve existing analytical methods to evaluate these wall effects and then train a feed-forward artificial neural network within a broader model. The final model generally performed with ∼0.001% error within the training domain and ∼0.05% when the outer spherical wall was extrapolated to an infinite plane. Through this model, the wall effects of an outer spherical boundary on the arbitrary motion of an internal sphere for all experimentally achievable configurations can now be conveniently and efficiently determined.

Measuring local properties inside a cell-mimicking structure using rotating optical tweezers.

Exploring the rheological properties of intracellular materials is essential for understanding cellular and subcellular processes. Optical traps have been widely used for physical manipulation of micro and nano objects within fluids enabling studies of biological systems. However, experiments remain challenging as it is unclear how the probe particle's mobility is influenced by the nearby membranes and organelles. We use liposomes (unilamellar lipid vesicles) as a simple biomimetic model of living cells, together with a trapped particle rotated by optical tweezers to study mechanical and rheological properties inside a liposome both theoretically and experimentally. Here, we demonstrate that this system has the capacity to predict the hydrodynamic interaction between three-dimensional spatial membranes and internal probe particles within submicron distances, and it has the potential to aid in the design of high resolution optical micro/nanorheology techniques to be used inside living cells.

Quantitative Acoustic Models for Superfluid Circuits.

We experimentally realize a highly tunable superfluid oscillator circuit in a quantum gas of ultracold atoms and develop and verify a simple lumped-element description of this circuit. At low oscillator currents, we demonstrate that the circuit is accurately described as a Helmholtz resonator, a fundamental element of acoustic circuits. At larger currents, the breakdown of the Helmholtz regime is heralded by a turbulent shedding of vortices and density waves. Although a simple phase-slip model offers qualitative insights into the circuit's resistive behavior, our results indicate deviations from the phase-slip model. A full understanding of the dissipation in superfluid circuits will thus require the development of empirical models of the turbulent dynamics in this system, as have been developed for classical acoustic systems.

Cellular-Resolution Imaging of Vestibular Processing across the Larval Zebrafish Brain.

The vestibular system, which reports on motion and gravity, is essential to postural control, balance, and egocentric representations of movement and space. The motion needed to stimulate the vestibular system complicates studying its circuitry, so we previously developed a method for fictive vestibular stimulation in zebrafish, using optical trapping to apply physical forces to the otoliths. Here, we combine this approach with whole-brain calcium imaging at cellular resolution, delivering a comprehensive map of the brain regions and cellular responses involved in basic vestibular processing. We find responses broadly distributed across the brain, with unique profiles of cellular responses and topography in each region. The most widespread and abundant responses involve excitation that is graded to the stimulus strength. Other responses, localized to the telencephalon and habenulae, show excitation that is only weakly correlated to stimulus strength and that is sensitive to weak stimuli. Finally, numerous brain regions contain neurons that are inhibited by vestibular stimuli, and these neurons are often tightly localized spatially within their regions. By exerting separate control over the left and right otoliths, we explore the laterality of brain-wide vestibular processing, distinguishing between neurons with unilateral and bilateral vestibular sensitivity and revealing patterns whereby conflicting signals from the ears mutually cancel. Our results confirm previously identified vestibular responses in specific regions of the larval zebrafish brain while revealing a broader and more extensive network of vestibular responsive neurons than has previously been described. This provides a departure point for more targeted studies of the underlying functional circuits.

Calibration of force detection for arbitrarily shaped particles in optical tweezers.

Force measurement with an optical trap requires calibration of it. With a suitable detector, such as a position-sensitive detector (PSD), it is possible to calibrate the detector so that the force can be measured for arbitrary particles and arbitrary beams without further calibration; such a calibration can be called an "absolute calibration". Here, we present a simple method for the absolute calibration of a PSD. Very often, paired position and force measurements are required, and even if synchronous measurements are possible with the position and force detectors used, knowledge of the force-position curve for the particle in the trap can be highly beneficial. Therefore, we experimentally demonstrate methods for determining the force-position curve with and without synchronous force and position measurements, beyond the Hookean (linear) region of the trap. Unlike the absolute calibration of the force and position detectors, the force-position curve depends on the particle and the trapping beam, and needs to be determined in each individual case. We demonstrate the robustness of our absolute calibration by measuring optical forces on microspheres as commonly trapped in optical tweezers, and other particles such a birefringent vaterite microspheres, red blood cells, and a deformable "blob".

Modelling of Cavity Optomechanical Magnetometers.

Cavity optomechanical magnetic field sensors, constructed by coupling a magnetostrictive material to a micro-toroidal optical cavity, act as ultra-sensitive room temperature magnetometers with tens of micrometre size and broad bandwidth, combined with a simple operating scheme. Here, we develop a general recipe for predicting the field sensitivity of these devices. Several geometries are analysed, with a highest predicted sensitivity of 180 p T / Hz at 28 µ m resolution limited by thermal noise in good agreement with previous experimental observations. Furthermore, by adjusting the composition of the magnetostrictive material and its annealing process, a sensitivity as good as 20 p T / Hz may be possible at the same resolution. This method paves a way for future design of magnetostrictive material based optomechanical magnetometers, possibly allowing both scalar and vectorial magnetometers.

Impact of complex surfaces on biomicrorheological measurements using optical tweezers.

The characterisation of physical properties in biologically relevant processes and the development of novel microfluidic devices for this purpose are experiencing a great resurgence at present. In many of measurements of this type where a probe in a fluid is used, the strong influence of the boundaries of the volume used is a serious problem. In these geometries the proximity of a probe to a wall can severely influence the measurement. However, although much knowledge has been gained about flat walls, to date, the effect of non-planar surfaces at microscopic scale on rotational motion of micro-objects has not been studied. Here we present for the first time both experimental measurements and numerical computations which aim to study the drag torque on optically trapped rotating particles moving near 3D-printed conical and cylindrical walls on-chip. These results are essential for quantifying how curved walls can effect the torque on particles, and thus enable accurate hydrodynamic simulations at the micron-scale. This opens the potential for new sensing approaches under more complex conditions, allowing both dynamic and microrheological studies of biological systems and lab-on-chip devices.

Rotational control of computer generated holograms.

We develop a basis for three-dimensional rotation of arbitrary light fields created by computer generated holograms. By adding an extra phase function into the kinoform, any light field or holographic image can be tilted in the focal plane with minimized distortion. We present two different approaches to rotate an arbitrary hologram: the Scheimpflug method and a novel coordinate transformation method. Experimental results are presented to demonstrate the validity of both proposed methods.

Optical trapping of otoliths drives vestibular behaviours in larval zebrafish.

The vestibular system, which detects gravity and motion, is crucial to survival, but the neural circuits processing vestibular information remain incompletely characterised. In part, this is because the movement needed to stimulate the vestibular system hampers traditional neuroscientific methods. Optical trapping uses focussed light to apply forces to targeted objects, typically ranging from nanometres to a few microns across. In principle, optical trapping of the otoliths (ear stones) could produce fictive vestibular stimuli in a stationary animal. Here we use optical trapping in vivo to manipulate 55-micron otoliths in larval zebrafish. Medial and lateral forces on the otoliths result in complementary corrective tail movements, and lateral forces on either otolith are sufficient to cause a rolling correction in both eyes. This confirms that optical trapping is sufficiently powerful and precise to move large objects in vivo, and sets the stage for the functional mapping of the resulting vestibular processing.The neural circuits of the vestibular system, which detects gravity and motion, remain incompletely characterised. Here the authors use an optical trap to manipulate otoliths (ear stones) in zebrafish larvae, and elicit corrective tail movements and eye rolling, thus establishing a method for mapping vestibular processing.

Active rotational and translational microrheology beyond the linear spring regime.

Active particle tracking microrheometers have the potential to perform accurate broadband measurements of viscoelasticity within microscopic systems. Generally, their largest possible precision is limited by Brownian motion and low frequency changes to the system. The signal to noise ratio is usually improved by increasing the size of the driven motion compared to the Brownian as well as averaging over repeated measurements. New theory is presented here whereby error in measurements of the complex shear modulus can be significantly reduced by analyzing the motion of a spherical particle driven by nonlinear forces. In some scenarios error can be further reduced by applying a variable transformation which linearizes the equation of motion. This enables normalization that eliminates error introduced by low frequency drift in the particle's equilibrium position. Our measurements indicate that this can further resolve an additional decade of viscoelasticity at high frequencies. Using this method will easily increase the signal strength enough to significantly reduce the measurement time for the same error. Thus the method is more conducive to measuring viscoelasticity in slowly changing microscopic systems, such as a living cell.