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Nanodiamonds (NDs) with NV−defect centers are great probes for bionanotechnology applications, with potential to act as biomarkers for cell differentiation. To explore this concept, uptake of NDs (~120nm) by THP-1 monocytes and monocyte-derived M0-macrophages is studied. The time course analysis of ND uptake by monocytes confirms differing ND-cell interactions and a positive time-dependence. No effect on cell viability, proliferation and differentiation potential into macrophages is observed, while cells saturated with NDs, unload the NDs completely by 25 cell divisions and subsequently take up a second dose effectively. ND uptake variations by THP-1 cells at early exposure-times indicate differing phagocytic capability. The cell fraction that exhibits relatively enhanced ND uptake is associated to a macrophage phenotype which derives from spontaneous monocyte differentiation. In accordance, chemical-differentiation of the THP-1 cells into M0-macrophages triggers increased and homogeneous ND uptake, depleting the fraction of cells that were non-responsive to NDs. These observations verify that ND uptake allows for distinction between the two cell subtypes based on phagocytic capacity. Overall, NDs demonstrate effective cell labeling of monocytes and macrophages, and are promising candidates for tracking biological processes that involve cell differentiation.

Defects in solid-state materials provide an ideal, robust platform for quantum sensing. To deliver maximum sensitivity, a large ensemble of noninteracting defects hosting quantum states with long coherence is required. Control of such an ensemble is challenging due to the spatial variation in both the defect energy levels and in any control field across a macroscopic sample. In this work, we experimentally demonstrate that we can overcome these challenges using Floquet theory and optimal control optimization methods to efficiently and coherently control a large defect ensemble, suitable for sensing. We apply our methods experimentally to a spin ensemble of up to 4×109 nitrogen-vacancy centers in diamond. By explicitly including the hyperfine interaction to the intrinsic 14N nuclear spin in the optimization, we design shaped microwave control pulses that can outperform conventional (π) pulses when applied to sensing schemes, with an improvement in the strength of ensemble response of between 11% and 78%. Through simulation of the ensemble dynamics, we shed light on the bandwidth limitations of large-ensemble reinitialization and propose alternative routes for further improvement.

The ability to measure the passage of electrical current with high spatial and temporal resolution is vital for applications ranging from inspection of microscopic electronic circuits to biosensing. The ability to image such signals passively and remotely is of great importance, in order to measure without invasive disruption of the system under study or the signal itself. A recent approach to achieving this utilizes point defects in solid-state materials; in particular, nitrogen-vacancy centers in diamond. Acting as a high-density array of independent sensors, addressable opto-electronically and highly sensitive to factors including temperature and magnetic field, these are ideally suited to microscopic wide-field imaging. In this work, we demonstrate simultaneous spatially and temporally resolved recovery signals from a microscopic lithographically patterned circuit. Through application of a lock-in amplifier camera, we demonstrate micrometer-scale imaging resolution with a millimeter-scale field of view with simultaneous spatially resolved submillisecond (up to 3500 frames s-1) recovery of dc to kilohertz alternating and broadband pulsed-current electrical signals, without aliasing or undersampling. We demonstrate as examples of our method the recovery of synthetic signals replicating digital pulses in integrated circuits and signals that would be observed in a biological neuronal network in the brain.

Flow cytometry (FC) is a pivotal tool for studying the physical and chemical properties of particles. State-of-the-art FC systems are highly advanced, yet they are expensive, bulky, and require high sample volume, qualified operators, and periodic maintenance. The manipulation of particles suspended in viscoelastic fluids has received increasing attention, especially for miniaturized flow cytometry technologies. This study presents a miniaturized optical capillary FC device using the viscoelastic focusing technique. A straight, one inlet/outlet microcapillary device is precisely aligned to a fiber-coupled laser source and detectors. Forward scattered, side scattered, and fluorescently emitted light signals are collected and analyzed in a real-time environment. The developed platform fits onto an inverted microscope stage enabling real-time microscopy imaging of the particles of interest together with the flow cytometry analysis. We achieved stable viscoelastic focusing and performed FC measurements for rigid polystyrene beads (diameters: 2 - 15 μm), non-spherical human erythrocytes, and canonical shape metaphase human chromosomes. We performed cytometry measurements with a throughput of 100 events/s yielding a coefficient of variation of 2%. This newly developed FC device is a versatile tool and can be operated with any inverted microscope to get the mutual benefits of optical and imaging FC measurements. Furthermore, it is possible to extend these benefits by adding more back-end tools, such as optical trapping and Raman spectroscopy.

Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nanoparticle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration., Comment: 181 pages, 61 figures

We report the first magnetic measurements of electrical activity from in vitro mammalian muscle and brain tissue. By using Nitrogen-Vacancy centers in diamond, these measurements can be carried out non-invasively and in an unshielded laboratory.

A compact microfluidic flow cytometer is demonstrated, comprising viscoelastic flow focusing in fused silica capillaries and a fiber optical interface. Viscoelastic flow focusing enables simple device design and operation with a single-inlet/outlet fluidic configuration. Fused silica capillaries with different inner diameters are effortlessly interchanged to eliminate blockage ratio limitations and enable single-train particle focusing for a wide range of particle sizes and geometries. The compact system is mounted on an inverted microscope for easy integration with optical imaging and other optofluidic modalities, such as optical trapping and particle sorting. A real-time cytometric analysis of three channels, forward scattering, side scattering, and fluorescence detection, is performed on LABVIEW. A throughput of 3500 events s(-1) is performed on particles of sizes ranging from 2 to 20 mu m, using capillaries of different inner diameters ranging from 30 to 75 mu m. The outer diameter of all capillaries is identical to the cladding diameter of the applied optical fibers. This enables easy exchange and precise optical alignment of fibers and capillaries on a microfabricated jig. The performance of the microfluidic flow cytometer is benchmarked using polystyrene calibration beads, poly(lactic-co-glycolic acid) particles, erythrocytes, THP-1 leukemic monocytes, and human metaphase chromosomes.

The detection of physiological activity at the microscopic level is key for understanding the function of biosystems and relating this to their physical structure. Current sensing methods for in vitro study of living tissue often rely on invasive probes to stimulate and detect activity, bearing the risk of inducing damage in the target system. In recent years, a new type of quantum sensor based on color centers in diamond has begun to offer the possibility to instead passively sense and image living biological systems. Here, we use such a sensor to realize the recording of the biomagnetic field generated by tightly focused, high intensity pulsed laser optogenetic neuromuscular stimulation of extensor digitorum longus muscles, dissected from mice and kept alive in carbogenated solution. Recordings captured a compound action potential response and a slow signal component, which we seek to explain using a detailed model of the biological system. We show proof-of-principle experimental recording of localized neuromuscular activity from the laser stimulation site without photovoltaic or fluorescence artifacts associated with alternative techniques. Our work represents a further step toward passive sensing and imaging at the microscopic level with quantum sensing, enabling further research into mapping of neural activity and intracellular processes.

Stimulated Raman scattering (SRS) microscopes are a standard technique to image cells, tissues or track the uptake of drugs [1] . The sensitivity in these devices is directly linked to the laser power. While an increase of sensitivity is desired, increasing the laser power is challenging and squeezed light offers a solution to this challenge. Recently squeezed light has been used to enhance the sensitivity of continuous-wave and pulsed SRS [2] , [3] setups and stimulated emission spectroscopy [4] .

Nitrogen vacancy (NV) centers in diamond have attracted considerable recent interest for use in quantum sensing, promising increased sensitivity for applications ranging from geophysics to biomedicine. Conventional sensing schemes involve monitoring the change in red fluorescence from the NV center under green laser and microwave illumination. Due to the strong fluorescence background from emission in the NV triplet state and low relative contrast of any change in output, sensitivity is severely restricted by a high optical shot noise level. Here, we propose a means to avoid this issue, by using the change in green pump absorption through the diamond as part of a semiconductor external cavity laser run close to the lasing threshold. We show that theoretical sensitivity to the magnetic field on the pT/$\sqrt{\mathrm{Hz}}$ level is possible using a diamond with an optimal density of NV centers. We discuss the physical requirements and limitations of the method, particularly the role of amplified spontaneous emission near threshold and explore realistic implementations using current technology.

Stimulated Raman spectroscopy has become a powerful tool to study the spatio-dynamics of molecular bonds with high sensitivity, resolution, and speed. However, the sensitivity and speed of stimulated Raman spectroscopy are ultimately limited by the shot-noise of the light beam probing the Raman process. Here, we demonstrate an enhancement of the sensitivity of stimulated Raman spectroscopy by reducing the noise below the shot-noise limit by means of squeezed states of light. Our demonstration constitutes the first step towards a new generation of quantum-enhanced Raman microscopes.

Over the years, different approaches to obtaining antireflective surfaces have been explored, such as using index-matching, interference, or micro- and nanostructures. Structural super black colors are ubiquitous in nature, and biomimicry thus constitutes an interesting way to develop antireflective surfaces. Moth-eye nanostructures, for example, are well known and have been successfully replicated using micro- and nanofabrication. However, other animal species, such as birds of paradise and peacock spiders, have evolved to display larger structures with antireflective features. In peacock spiders, the antireflective properties of their super black patches arise from relatively simple microstructures with lens-like shapes organized in tightly packed hexagonal arrays, which makes them a good candidate for cheap mass replication techniques. In this paper, we present the fabrication and characterization of antireflective microarrays inspired by the peacock spider’s super black structures encountered in nature. Firstly, different microarrays 3D models are generated from a surface equation. Secondly, the arrays are fabricated in a polyacrylate resin by super-resolution 3D printing using two-photon polymerization. Thirdly, the resulting structures are inspected using a scanning electron microscope. Finally, the reflectance and transmittance of the printed structures are characterized at normal incidence with a dedicated optical setup. The bioinspired microlens arrays display excellent antireflective properties, with a measured reflectance as low as 0.042 ± 0.004% for normal incidence, a wavelength of 550 nm, and a collection angle of 14.5°. These values were obtained using a tightly-packed array of slightly pyramidal lenses with a radius of 5 µm and a height of 10 µm.

The ability to perform noninvasive and non-contact measurements of electric signals produced by action potentials is essential in biomedicine. A key method to do this is to remotely sense signals by the magnetic field they induce. Existing methods for magnetic field sensing of mammalian tissue, used in techniques such as magnetoencephalography of the brain, require cryogenically cooled superconducting detectors. These have many disadvantages in terms of high cost, flexibility and limited portability as well as poor spatial and temporal resolution. In this work we demonstrate an alternative technique for detecting magnetic fields generated by the current from action potentials in living tissue using nitrogen vacancy centres in diamond. With 50 pT/$$\sqrt{\text {Hz}}$$ Hz sensitivity, we show the first measurements of magnetic sensing from mammalian tissue with a diamond sensor using mouse muscle optogenetically activated with blue light. We show these proof of principle measurements can be performed in an ordinary, unshielded lab environment and that the signal can be easily recovered by digital signal processing techniques. Although as yet uncompetitive with probe electrophysiology in terms of sensitivity, we demonstrate the feasibility of sensing action potentials via magnetic field in mammals using a diamond quantum sensor, as a step towards microscopic imaging of electrical activity in a biological sample using nitrogen vacancy centres in diamond.

Sensing of signals from biological processes, such as action potential propagation in nerves, are essential for clinical diagnosis and basic understanding of physiology. Sensing can be performed electrically by placing sensor probes near or inside a living specimen or dissected tissue using well-established electrophysiology techniques. However, these electrical probe techniques have poor spatial resolution and cannot easily access tissue deep within a living subject, in particular within the brain. An alternative approach is to detect the magnetic field induced by the passage of the electrical signal, giving the equivalent readout without direct electrical contact. Such measurements are performed today using bulky and expensive superconducting sensors with poor spatial resolution. An alternative is to use nitrogen vacancy (NV) centers in diamond that promise biocompatibilty and high sensitivity without cryogenic cooling. In this work we present advances in biomagnetometry using NV centers, demonstrating magnetic field sensitivity of similar to 100 pT/root Hz in the DC/low frequency range using a setup designed for biological measurements. Biocompatibility of the setup with a living sample (mouse brain slice) is studied and optimized, and we show work toward sensitivity improvements using a pulsed magnetometry scheme. In addition to the bulk magnetometry study, systematic artifacts in NV-ensemble widefield fluorescence imaging are investigated.

The biomolecule is among the most important building blocks of biological systems, and a full understanding of its function forms the scaffold for describing the mechanisms of higher order structures as organelles and cells. Force is a fundamental regulatory mechanism of biomolecular interactions driving many cellular processes. The forces on a molecular scale are exactly in the range that can be manipulated and probed with single molecule force spectroscopy. The natural environment of a biomolecule is inside a living cell, hence, this is the most relevant environment for probing their function. In vivo studies are, however, challenged by the complexity of the cell. In this review, we start with presenting relevant theoretical tools for analyzing single molecule data obtained in intracellular environments followed by a description of state-of-the art visualization techniques. The most commonly used force spectroscopy techniques, namely optical tweezers, magnetic tweezers, and atomic force microscopy, are described in detail, and their strength and limitations related to in vivo experiments are discussed. Finally, recent exciting discoveries within the field of in vivo manipulation and dynamics of single molecule and organelles are reviewed.

Shark skin has for many years inspired engineers to produce biomimetic structures reducing surface drag or acting as an anti-fouling layer. Both effects are presumed to be consequences of the structure of shark skin that is composed of arrays of so-called dermal denticles. However, the understanding of the full functional role of the dermal denticles is still a topic of research. We report optical microscopy and scanning electron microscopy of dermal denticles from three slowly swimming shark species for which the functional role of the dermal denticles is suggested as one of defense (possibly understood as anti-fouling) and/or abrasion strength. The three species are Greenland shark (Somnosius microcephalus), small-spotted catshark (Scyliorhinus canicula) and spiny dogfish (Squalus acanthias). Samples were taken at over 30 different positions on the bodies of the sharks. In addition, we demonstrate that the flow pattern near natural shark skin can be measured by micro-PIV (particle image velocimetry). The microfluidic experiments are complemented by numerical flow simulations. Both visualize unsteady flow, small eddies, and recirculation bubbles behind the natural dermal denticles.

An optical two-beam trap composed from two counter propagating laser beams is an interesting setup due to the ability of the system to trap, hold, and stretch soft biological objects like vesicles or single cells. Because of this functionality, the system was also named the optical stretcher by Jochen Guck, Josep Kaas and co-workers almost 20 years ago. In a favorable setup, the two opposing laser beams meet with equal intensities in the middle of a fluidic channel in which cells may ow past, be trapped, stretched, and allowed to move on, giving the promise of a high throughput device. Yet, single beam optical traps, aka optical tweezers, by far outnumber the existing optical stretchers in research labs throughout the world. The ability to easily construct an optical stretcher setup in a low-cost material would possibly imply more frequent use of the optical stretching technique. Here, we discuss advantages and disadvantages of choice of material and methodology for chip assembly and chip production. For high throughput investigations of stretching deformation of single cells, optical stretching is, however, out-performed by hydrodynamic deformability assays. As we will discuss, injection molded polymer chips may with advantage be applied both for optical stretching and for hydrodynamic deformability experiments.

Stimulated Raman spectroscopy has become a powerful tool to study the spatiodynamics of molecular bonds with high sensitivity, resolution, and speed. However, the sensitivity and speed of state-of-the-art stimulated Raman scattering spectroscopy are currently limited by the shot-noise of the light beam probing the Raman process. Here, we demonstrate in a proof-of-principle experiment an enhancement of the sensitivity of continuous-wave stimulated Raman spectroscopy by reducing the quantum noise of the probing light below the shot-noise limit by means of amplitude squeezed states of light. Probing polymer samples with Raman shifts around 2950 c m − 1 with squeezed states, we demonstrate a quantum enhancement of the stimulated Raman signal-to-noise ratio (SNR) of 3.60 dB relative to the shot-noise limited SNR. Our proof-of-concept demonstration of quantum-enhanced continuous-wave Raman spectroscopy paves the way for more elaborate demonstrations using state-of-the-art stimulated Raman scattering microscopes, and thus constitutes the very first step towards a new generation of Raman microscopes, where weak Raman transitions can be imaged without the use of markers or an increase in the total optical power.

Acoustophoresis is a widely reported and used technique for microparticle manipulation and separation. In the study described here, acustophoresis is employed to prefocus the flow (i.e., focusing occurring upstream of the analysis region) in a microfluidic chip intended for optical trapping and stretching. The whole microchip is made of silica with optical waveguides integrated by femtosecond laser writing. The acoustic force is produced by driving an external piezoelectric ceramic attached underneath the microchip at the chip resonance frequency. Thanks to an efficient excitation of acoustic waves in both water and glass, acoustophoretic focusing is observed along the channel length (>40 mm) and it is successfully demonstrated both with polystyrene beads, swollen red blood cell, and cells from mouse fibroblast cellular lines (L929). Moreover, by comparing results of cell stretching measurements, we demonstrate that acoustic waves do not alter the optical deformability of the cells and that the acoustic prefocusing results in a considerable enhancement of throughput in optical stretching experiments.

The barrier properties of cellular membranes are increasingly attracting attention as a source of inspiration for designing biomimetic membranes. The broad range of potential technological applications makes the use of lipid and lately also polymeric materials a popular choice for constructing biomimetic membranes, where the barrier properties can be controlled by the composition of the membrane constituent elements. Here we investigate the membrane properties reported by the light-induced proton pumping activity of bacteriorhodopsin (bR) reconstituted in three vesicle systems of different membrane composition. Specifically we quantify how the resulting proton influx and efflux rates are influenced by the membrane composition using a variety of membrane modulators. We demonstrate that by adding hydrocarbons to vesicles with reconstituted bR formed from asolectin lipids the resulting transmembrane proton fluxes changes proportional to the carbon chain length when compared against control. We observe a similar proportionality in single-component 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) model membranes when using cholesterol. Lastly we investigate the effects of adding the amphiphilic di-block co-polymer polybutadiene-polyethyleneoxide (PB12-PEO10) to phospholipid membranes formed from DOPC, 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and 1,2-Dioleoyl-sn-glycero-3-phosphatidylserine (DOPS). The proton pumping activity of bR (measured as a change in extra-vesicular pH) in mixed lipid/PB12-PEO10 lipid systems is up to six-fold higher compared to that observed for bR containing vesicles made from PB12-PEO10 alone. Interestingly, bR inserts with apparent opposite orientation in pure PB12-PEO10 vesicles as compared to pure lipid vesicles. Addition of equimolar amounts of lipids to PB12-PEO10 results in bR orientation similar to that observed for pure lipids. In conclusion our results show how the barrier properties of the membranes can be controlled by the composition of the membrane. In particular the use of mixed lipid-polymer systems may pave the way for constructing biomimetic membranes tailored for optimal properties in various applications including drug delivery systems, biosensors and energy conservation technology.

The rapid development of microfluidic circuits and lab-on-chip devices for cell studies opened new perspectives for cellular biology, in particular for the biophysics and biomechanics of single cells. At the state of the art, a lot of attention is being paid to the possible integration of optical and acoustic actuators within microfluidic system because they both provide a contactless way for cell manipulation and analysis; additionally, they both offer other advantages such as rapid actuation, programmable capability and simple operation. In this work we exploit a microfluidic setup, combining optical and acoustic actuators, to perform cell mechanics characterization in terms of both cellular optical deformability (OD) and acoustic compressibility (AC). The OD and AC measurements reliability is obtained thanks to a precise determination of many parameters, as culture medium refractive index, viscosity and density, laser beam waist values and cell size, which are required for a proper estimation of cellular OD and AC. Cellular OD values are obtained by cell optical stretching procedure [1] while AC values are derived by fitting the cell acoustophoretic trajectory [2]. We apply this technique to measure the OD and AC of two human breast cancer cell populations, MCF7 and MDA, which are characterized by different metastatic potential. These two measurements are firstly performed separately and the results are reported in Fig.1. It can be seen that the two cell lines have similar cell size distribution, however, MDA shows both higher optical deformation and higher acoustic compressibility than MCF7, which is in good agreement with literature and can be associated with its elevated metastatic nature and stronger migratory ability. Afterwards, combined measurements of these two parameters were directly carried out on the same cell and it is found that there is no clear relationship between them in each cell sample, i.e., higher compressibility does not imply higher optical deformability [3]. Furthermore, we demonstrated that these two measurements do not affect each other. In conclusion, the chip presented here, together with the proposed measurement protocols, constitute a step forward in the characterization of cellular mechanical properties and thus in the possible identification of cells with specific biological properties. Additionally, we will present the experimental results of the currently on-going experiments showing the impact of drugs treatments on tumour-cells mechanical properties.

Mechanical properties, like deformability or elasticity, of cells can in some cases be indicative of the health of the organism they originate from. In this work, we explore the potential of deformability and other mechanical parameters of individual red blood cells (RBCs) from humans as a marker for the state of health of the human source, patient or donor. In particular, we have investigated the use of different experimental strategies implemented in injection molded plastic microfluidic devices. One strategy is to optically stretch the red blood cells in an optical two-beam trap, also known as an optical stretcher, in a microfluidic chip in which optical fibers have been placed during a post-processing step. Another strategy is to exert hydrodynamic shear forces on the cells by forcing the cells through a narrow constriction. The latter method has the advantage of a considerably higher throughput but does so far not allow for subsequent investigations of single “interesting” cells. The paper is a progress report with preliminary results based on the different strategies, we have pursued.

A monolithic polymer optofluidic chip for manipulation of microbeads in flow is demonstrated. On this chip, polymer waveguides induced by Deep UV lithography are integrated with microfluidic channels. The optical propagation losses of the waveguides are measured to be 0.66+/-0.13dB/mm at a wavelength of @l=808nm. An optimized bead tracking algorithm is implemented, allowing for determination of the optical forces acting on the particles. The algorithm features a spatio-temporal mapping of coordinates for uniting partial trajectories, without increased processing time. With an external laser power of 250mW, a maximum scattering force of 0.84 pN is achieved for 5@mm diameter polystyrene beads in water.

As described in the previous chapters, optical tweezers have become a tool of precision for in vitro single-molecule investigations, where the single molecule of interest most often is studied in purified form in an experimental assay with a well-controlled fluidic environment. A well-controlled fluidic environment implies that the physical properties of the liquid, most notably the viscosity, are known and the fluidic environment can, for calibrational purposes, be treated as a simple liquid.In vivo, however, optical tweezers have primarily been used as a tool of manipulation and not so often for precise quantitative force measurements, due to the unknown value of the spring constant of the optical trap formed within the cell's viscoelastic cytoplasm. Here, we describe a method for utilizing optical tweezers for quantitative in vivo force measurements. The experimental protocol and the protocol for data analysis rely on two types of experiments, passive observation of the thermal motion of a trapped object inside a living cell, followed by observations of the response of the trapped object when subject to controlled oscillations of the optical trap. One advantage of this calibration method is that the size and refractive properties of the trapped object and the viscoelastic properties of its environment need not be known. We explain the protocol and demonstrate its use with experiments of trapped granules inside live S. pombe cells.

As described in the previous chapters, optical tweezers have become a tool of precision for in vitro single-molecule investigations, where the single molecule of interest most often is studied in purified form in an experimental assay with a well-controlled fluidic environment. A well-controlled fluidic environment implies that the physical properties of the liquid, most notably the viscosity, are known and the fluidic environment can, for calibrational purposes, be treated as a simple liquid.In vivo, however, optical tweezers have primarily been used as a tool of manipulation and not so often for precise quantitative force measurements, due to the unknown value of the spring constant of the optical trap formed within the cell's viscoelastic cytoplasm. Here, we describe a method for utilizing optical tweezers for quantitative in vivo force measurements. The experimental protocol and the protocol for data analysis rely on two types of experiments, passive observation of the thermal motion of a trapped object inside a living cell, followed by observations of the response of the trapped object when subject to controlled oscillations of the optical trap. One advantage of this calibration method is that the size and refractive properties of the trapped object and the viscoelastic properties of its environment need not be known. We explain the protocol and demonstrate its use with experiments of trapped granules inside live S. pombe cells.

An optical two-beam trap, composed from two counter propagating laser beams, is an interesting setup due to the ability of the system to trap, hold, and stretch soft biological objects like vesicles or single cells. Because of this functionality, the system was also named "the optical stretcher" by Jochen Guck, Josep Kas and co-workers some 15 years ago. In a favorable setup, the two opposing laser beams meet with equal intensities in the middle of a fluidic channel in which cells may flow past, be trapped, stretched, and allowed to move on, giving the promise of a high throughput device. Yet, single beam optical traps, aka optical tweezers, by far outnumber the existing optical stretchers in research labs throughout the world. The ability to easily construct an optical stretcher setup in a low-cost material would possibly imply more frequent use of the optical stretching technique. Here, we will outline the design, the production procedures, and results obtained in a fiber-based experimental setup built within an injection molded microfluidic polymer chip. The microfluidic chip is constructed with a three layer technology in which we ensure both horizontal and vertical focusing of the cells we wish to trap, thereby preventing too many cells to flow below the line of focus of the two counter propagating laser beams that are positioned perpendicular to the direction of flow of the cells. Results will be compared to that from other designs from previous work in the group.

We realized an integrated microfluidic chip that allows measuring both optical deformability and acoustic compressibility on single cells, by optical stretching and acoustophoresis experiments respectively. Additionally, we propose a measurement protocol that allows evaluating the experimental apparatus parameters before performing the cell-characterization experiments, including a non-destructive method to characterize the optical force distribution inside the microchannel. The chip was used to study important cell-mechanics parameters in two human breast cancer cell lines, MCF7 and MDA-MB231. Results indicate that MDA-MB231 has both higher acoustic compressibility and higher optical deformability than MCF7, but statistical analysis shows that optical deformability and acoustic compressibility are not correlated parameters. This result suggests the possibility to use them to analyze the response of different cellular structures. We also demonstrate that it is possible to perform both measurements on a single cell, and that the order of the two experiments does not affect the retrieved values.

Green plants are Earth’s primary solar energy collectors. They harvest the energy of the Sun by converting light energy into chemical energy stored in the bonds of sugar molecules. A multitude of carefully orchestrated transport processes are needed to move water and minerals from the soil to sites of photosynthesis and to distribute energy-rich sugars throughout the plant body to support metabolism and growth. The long-distance transport happens in the plants’ vascular system, where water and solutes are moved along the entire length of the plant. In this review, the current understanding of the mechanism and the quantitative description of these flows are discussed, connecting theory and experiments as far as possible. The article begins with an overview of low-Reynolds-number transport processes, followed by an introduction to the anatomy and physiology of vascular transport in the phloem and xylem. Next, sugar transport in the phloem is explored with attention given to experimental results as well as the fluid mechanics of osmotically driven flows. Then water transport in the xylem is discussed with a focus on embolism dynamics, conduit optimization, and couplings between water and sugar transport. Finally, remarks are given on some of the open questions of this research field.Green plants harvest the energy of the Sun in the leaves by converting light energy into chemical energy in the bonds of sugar molecules, using water from the soil and carbon dioxide from the air. This review provides an overview of the vascular anatomy of plants and the physical models that describe the long-distance transport of water and minerals from root to leaf, and, in particular, of sugars from the leaves to the entire body of the plant sustaining growth and communication throughout even the tallest tree.

An attractive solution for optical trapping and stretching by means of two counterpropagating laser beams is to embed waveguides or optical fibers in a microfluidic system. The microfluidic system can be constructed in different materials, ranging from soft polymers that may easily be cast in a rapid prototyping manner, to hard polymers that could even be produced by injection moulding, or to silica in which waveguides may either be written directly, or with grooves for optical fibers. Here, we review different solutions to the system and also show results obtained in a polymer chip with DUV written waveguides and in an injection molded polymer chip with grooves for optical fibers.

The transport of sugars in the phloem vascular system of plants is believed to be driven by osmotic pressure differences according to the Munch hypothesis. Thus, the translocation process is viewed as a passive reaction to the active sugar loading in the leaves and sugar unloading in roots and other places of growth or storage. The modelling of the loading and unloading mechanism is thus a key ingredient in the mathematical description of such flows, but the influence of particular choices of loading functions on the translocation characteristics is not well understood. Most of the work has relied on numerical solutions, which makes it difficult to draw general conclusions. Here, we present analytic solutions to the Munch–Horwitz flow equations when the loading and unloading rates are assumed to be linear functions of the concentration, thus allowing them to depend on the local osmotic pressure. We are able to solve the equations analytically for very small and very large Munch numbers (e.g., very small and very large viscosity) for the flow velocity and sugar concentration as a function of the geometric and material parameters of the system. We further show, somewhat surprisingly, that the constant loading case can be solved along the same lines and we speculate on possible universal properties of different loading and unloading functions applied in the literature.

We demonstrate the energy dependence of the motion of a porin, the lambda-receptor, in the outer membrane of living Escherichia coli by single molecule investigations. By poisoning the bacteria with arsenate and azide, the bacterial energy metabolism was stopped. The motility of individual lambda-receptors significantly and rapidly decreased upon energy depletion. We suggest two different causes for the ceased motility upon comprised energy metabolism: One possible cause is that the cell uses energy to actively wiggle its proteins, this energy being one order-of-magnitude larger than thermal energy. Another possible cause is an induced change in the connection between the lambda-receptor and the membrane structure, for instance by a stiffening of part of the membrane structure. Treatment of the cells with ampicillin, which directly targets the bacterial cell wall by inhibiting cross-linking of the peptidoglycan layer, had an effect similar to energy depletion and the motility of the lambda-receptor significantly decreased. Since the lambda-receptor is closely linked to the peptidoglycan layer, we propose that lambda-receptor motility is directly coupled to the constant and dynamic energy-consuming reconstruction of the peptidoglycan layer. The result of this motion could be to facilitate transport of maltose-dextrins through the porin.

At present, a major obstacle to the quantitative application of optical tweezers as a force spectrometer in living cells is the lack of a method to calibrate the tweezers. Calibration with approved methods such as the power spectrum method (Berg-Sorensen and Flyvbjerg 2004 Rev. Sci. Instrum. 75 594; Berg-Sorensen et al 2006 Rev. Sci. Instrum. 77 063106) is not possible as the viscoelastic properties of the bio-active medium are a priori unknown. Here, we present an approach that neither requires explicit assumptions about the size of the trapped particle nor about the viscoelastic properties of the medium. Instead, the interaction between the medium and the trapped particle is described in a general manner, through velocity and acceleration memory. Our method is applicable to general, at least locally homogeneous, viscoelastic media. The procedure combines active and passive approaches by the application of Onsager's regression hypothesis. It allows extraction of the trapping stiffness κ of the optical tweezers and of the response function χ(ω), which is the frequency-dependent effective inverse spring constant of the system. Finally, information about the viscoelastic properties of the medium may also be found. To test the method, we have performed simulations in which the system is driven sinusoidally. These simulations serve as an example of how to deal with real experimental data. For realistic parameters, we calibrate the trap stiffness κ with ~1% stochastic error.

An optical trapping system with two opposing laser beams, also known as the optical stretcher, are naturally constructed inside a microfluidic lab-on-chip system. We present and compare two approaches to combine a simple microfluidic system with either waveguides directly written in the microfluidic chip or with optical fibers mounted in the chip.

Optical tweezers are a versatile tool in biophysics and have matured from a tool of manipulation to a tool of precise measurements. We argue here that the data analysis with advantage can be developed to a level of sophistication that matches that of the instrument. We review methods of analysis of optical tweezers data, primarily based on the power spectra of time series of positions for trapped spherical objects. The majority of precise studies in the literature are performed on in vitro systems, whereas in the present work, an example of an in vivo system is presented for which precise power spectral analysis is both useful and necessary. The biological system is the cytoplasm of fission yeast, Schizosaccharomyces pombe in which we observe subdiffusion of lipid granules. In a search for the cause of subdiffusion, we chemically disrupt the actin network in the cytoplasm and further consider in vitro networks of filamenteous actin undergoing similar chemical disruption.

The study of live biological systems requires the use of advanced techniques that provide high structural and chemical information and at the same time, avoid damage in the system and modification of the structural/chemical features. Techniques based on interaction with light have shown their capability to work in biosensor devices. For example, Raman spectroscopy can be non-invasive and can provide 1 μm of spatial resolution in 1 second of collection time, well suited for sensing. Moreover, it may give information at the single cell and even approaching the single molecule scale. Here we present the capability of different light based techniques for biosensing.As the first example, surface enhanced Raman spectroscopy (SERS) is performed in onion using silver plasmonic nanostructures. Our studies detect different molecular compounds present in the plant based on their SERS signals. SERS imaging allows us to monitor the location of nanoparticles and to image chemical compounds within the target. Moreover, a pH-sensitive reporter molecule, pMBA attached to the silver nanoparticles, is used to infer pH values in the extracellular space of an onion layer.As a second example, we explore how a membrane protein may be used as an efficient sensor in an organic environment via a biomimetic membrane model. The combination of both biomimetic membranes and protein membranes as a signal transduction medium has interesting applications in biology and medicine. It is crucial that the matrix where a protein is embedded is optimal in order to maintain the concentration gradient. Moreover, curvature and mechanical forces in the membrane may also affect the protein function. In this work, by inducing chemical and mechanical changes of the matrix we optimize the system via measuring variations of the gradient through the membrane.

The motion of a single protein, a λ-receptor in the outer membrane of E. coli, is studied with optical tweezers. We present improved measurements and an analysis that accounts for stochastic errors, filters in the detection system, aliasing and hydrodynamic effects. We test a simple model for the protein's motion and find it in agreement with the low-frequency part of the data. We thus determine physical parameter values for the system with improved precision. The diffusion coefficient describing the motion of the λ-receptor is now obtained with 2% relative error for individual receptors, and varies between bacteria with roughly 70% root-mean-square deviation about the mean. This improved analysis also reveals that the simple model must be revised in order to agree with the high-frequency part of the power spectrum.

We characterize the frequency-dependent response of a photo detection system based on a Si-PIN photodiode and a laser with wavelength 1064 nm, a system commonly used with optical tweezers. We chopped the laser beam with chopper frequencies from 200 Hz to 14 kHz, and found an exponentially delayed response of the detection system with a characteristic delay time of ∼20 μs. The physical mechanism causing this time delay is silicon’s transparency to 1064 nm light: Photons are absorbed and create charge carriers not only in the diode’s depletion layer, where they are detected within nano-seconds, but predominantly in the n-layer, where they remain undetected till transported out by thermal diffusion. The diode’s response is dominated by this delay which can be characterized as a first-order low-pass filter with a 3dB-frequency of 8–9 kHz, depending on laser intensity. Measurements exploiting frequencies near or above this 3dB-frequency must be corrected for this unintended filter effect. We describe how to do...

Optical tweezers are the only nano-tools capable of manipulating and performing force-measurements on individual molecules and organelles within the living cell without performing destructive penetration through the cell wall and without the need for inserting a non-endogenous probe. Here, we describe how optical tweezers are used to manipulate individual molecules and perform accurate force and distance measurements within the complex cytoplasm of the living cell. Optical tweezers can grab individual molecules or organelles, if their optical contrast to the medium is large enough, as is the case, e.g., for lipid granules or chromosomes. However, often the molecule of interest is specifically attached to a handle manipulated by the optical trap. The most commonly used handles, their insertion into the cytoplasm, and the relevant micro-rheology of the cell are discussed here and we also review recent and exciting results achieved through optical force manipulation of individual molecules in vivo.