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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.

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 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.

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.

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.

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.

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.

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...

We demonstrate the use of a two-beam optical trap (an optical stretcher) in a low-cost microfluidic system with the purpose of measuring the mechanical properties of cells and vesicles. Delivery of micrometer-sized particles and cells to the optical stretcher is obtained by acoustophoretic prefocusing. This focusing mechanism aims for target particles to always flow in the correct height relative to the optical stretcher, and is induced by a piezo-electric ultrasound transducer attached underneath the chip and driven at a frequency leading to a vertical standing ultrasound wave in the microchannel. Trapping and manipulation is demonstrated for dielectric beads. In addition, we show trapping, manipulation and stretching of red blood cells and vesicles, whereby we extract the elastic properties of these objects. Our design points towards the construction of a low-cost, high-throughput lab-on-a-chip device for measurement of mechanical properties of cells and vesicles.

The application of optical traps to manipulate or monitor intracellular processes in in vivo systems calls for precise procedures both to evaluate the characteristics of the trap and the viscoelastic properties of the intracellular medium. Here, we verify experimentally the authenticity of a calibration procedure, specifically developed for use in calibration of optical tweezers in complex, viscoelastic media (Fischer and Berg-Sorensen, J. Opt. A: Pure Appl. Opt. 9, S239 (2007)).We demonstrate the application of the procedure in i) water and in ii) entangled F-actin without cross-linkers. As the theoretical background of the procedure is essentially convenient formulations of the fluctuation-dissipation theorem, we name the procedure as the “FDT-method”. The analysis of the measurements in water may be validated directly: We can compare the results for the spring constant to results obtained with well-established methods and those for the viscoelastic properties to theoretical predictions. In the actin solution, we may similarly compare our results with results of microrheology experiments in the literature.

In a typical optical tweezers detection system, the position of a trapped object is determined from laser light impinging on a quadrant photodiode. When the laser is infrared and the photodiode is of silicon, they can act together as an unintended low-pass filter. This parasitic effect is due to the high transparency of silicon to near-infrared light. A simple model that accounts for this phenomenon (Berg-Sorensen et al., J. Appl. Phys., 93, 3167-3176 (2003)) is here solved for frequencies up to 100 kHz, and for laser wavelengths between 750 and 1064 nm. The solution is applied to experimental data in the same range, and is demonstrated to give this detection system of optical tweezers a bandwidth, accuracy, and precision that is limited only by the data acquisition board's band-width and bandpass ripples, here 96.7 kHz, resp. 0.005 dB., Comment: 12 pages, 8 figures. Submitted to Review of Scientific Instruments

In a typical position detection system for optical tweezers, laser light impinges on a quadrant photodiode, and the signal from the four quadrants of the diode is used to determine the position of a trapped object. A widely used position detection system consists of a Si-PIN photodiode and an infrared laser. In previous work we have demonstrated with two distinct experimental methods how such a system may act as an unintended low-pass filter and we modeled its physical origin mathematically. Here we demonstrate that the general solution to this model can account precisely for the "parasitic" filter's effects up to as large frequencies as we can measure, approximately 100 kHz. Thus we increase the useful bandwidth of tweezers experiments by nearly two decades. This opens for investigations of phenomena in biophysics, soft matter, and polymer science at much higher frequencies than before.

We present a procedure for a precise power spectral analysis of optical tweezers data. This procedure uses the entire frequency range of the experimental power spectrum. We apply this procedure to experimental data from a biological system, data for the motion of a microsphere attached with a biotin-streptavidin linker to a single protein, the λ-receptor of E. coli. As in [Oddershede et al, Biophys. J. 83, 3152 (2002)] and [Oddershede et al, J. Phys.: Condens. Mat. 15, S1737 (2003)], we find that the λ-receptor moves diffusively in the outer membrane, but is confined to a particular region by a harmonic potential. Here, we show that since the power spectrum is known with precision up to its Nyquist frequency of 8 kHz, one can resolve the relative motion of the λ-receptor and the microsphere attached to it. We find that the biotin-streptavidin link between them can be described as a Hookean spring. Since we can see only the microsphere, these results are based on an interpretation of the power spectrum of its motion. This interpretation is based on a model. We use the simplest model that is necessary and sufficient to interpret the power spectrum in its full range. This model fits the spectrum well and the fit yields the model's parameters with some precision. These results indicate that optical tweezers have the potential to become a tool of precision. We discuss improvements of experiments and analysis that are necessary in order to realize this potential.

The viscoelastic properties of the cytoplasm of living yeast cells were investigated by studying the motion of lipid granules naturally occurring in the cytoplasm. A large frequency range of observation was obtained by a combination of video-based and laser-based tracking methods. At time scales from 10(-4) to 10(2) s, the granules typically perform subdiffusive motion with characteristics different from previous measurements in living cells. This subdiffusive behavior is thought to be due to the presence of polymer networks and membranous structures in the cytoplasm. Consistent with this hypothesis, we observe that the motion becomes less subdiffusive upon actin disruption.

We investigate the axial position detection of a trapped microsphere in an optical trap by using a quadrant photodiode. By replacing the photodiode with a CCD camera, we obtain detailed information on the light scattered by the microsphere. The correlation of the interference pattern with the axial position displays complex behavior with regions of positive and negative interference. By analyzing the scattered light intensity as a function of the axial position of the trapped sphere, we propose a simple method to increase the sensitivity and control the linear range of axial position detection.