A low-cost glass-based microfluidic flow cell with a piezo actuator is built using off-the-shelf parts (total cost €9 per device) to apply acoustophoretic force on polystyrene micro-beads. The main challenge in the fabrication of these devices was to ensure their leak tightness, which we solved using double-sided tape and nail polish. Beads with 1.5 μm diameter flowing in a 100 μm deep channel were trapped at 7.5 MHz using a 23.7 peak-to-peak voltage (V_pp) sinusoidal input. The trap located at 50 ± 0.1 μm depth was measured to have a stiffness of approximately 0.6 pN/μm. With this simple device we can trap and control the axial position of micrometer scale objects, which allows for the manipulation of beads and cells. We intend to use the device for force spectroscopy on micro-bead tethered DNA. This can be combined with super-resolution imaging techniques to study mechanics and binding of protein structures along a DNA strand as a function of induced tension.

Three dimensional modulation-enhanced single-molecule localization techniques, such as ModLoc, offer advancements in axial localization precision across the entire field of view and axial capture range, by applying phase shifting to the illumination pattern. However, this improvement is limited by the pitch of the illumination pattern that can be used and requires registration between separate regions of the camera. To overcome these limitations, we present ZIMFLUX, a method that combines astigmatic point-spread-function (PSF) engineering with a structured illumination pattern in all three spatial dimensions. In order to achieve this we address challenges such as optical aberrations, refractive index mismatch, supercritical angle fluorescence (SAF), and imaging at varying depths within a sample, by implementing a vectorial PSF model. In scenarios involving refractive index mismatch between the sample and immersion medium, the astigmatic PSF loses its ellipticity at greater imaging depths, leading to a deterioration in axial localization precision. In contrast, our simulations demonstrate that ZIMFLUX maintains high axial localization precision even when imaging deeper into the sample. Experimental results show unbiased localization of 3D 80 nm DNA-origami nanostructures in SAF conditions with a 1.5-fold improvement in axial localization precision when comparing ZIMFLUX to conventional SMLM methods that rely solely on astigmatic PSF engineering.

To better understand the interactions between biological molecules, a high optical resolution in all three dimensions is crucial. The intrinsically lower axial resolution of microscopes however, is a limiting factor in fluorescence imaging, correspondingly in fluorescence based single molecule localization microscopy (SMLM). Here, we present a method to improve the axial localization precision in SMLM by combining point-spread-function engineering with total internal reflection fluorescence (TIRF) fields with decay lengths that vary within the on-time of a fluorophore. Such time-varying illumination field intensity allows one to extract additional axial location information from the emitted photons. With this time varying illumination approach, we show that axial localization is improved two-fold over TIRF-based SMLM using astigmatic PSFs. We calculate theoretical resolution gains for various imaging conditions via the Cramér Rao Lower Bound (CRLB), a commonly used metric to compute the best attainable localization precision in SMLM.

Kinetic inductance detectors (KIDs) are superconducting energy-resolving detectors, sensitive to single photons from the near-infrared to ultraviolet. We study a hybrid KID design consisting of a β-phase tantalum (β-Ta) inductor and a Nb-Ti-N interdigitated capacitor. The devices show an average intrinsic quality factor Qi of 4.3×105±1.3×105. To increase the power captured by the light-sensitive inductor, we 3D print an array of 150×150μm resin microlenses on the backside of the sapphire substrate. The shape deviation between design and printed lenses is smaller than 1μm, and the alignment accuracy of this process is δx=+5.8±0.5μm and δy=+8.3±3.3μm. We measure a resolving power for 1545-402 nm that is limited to 4.9 by saturation in the KID's phase response. We can model the saturation in the phase response with the evolution of the number of quasiparticles generated by a photon event. An alternative coordinate system that has a linear response raises the resolving power to 5.9 at 402 nm. We verify the measured resolving power with a two-line measurement using a laser source and a monochromator. We discuss several improvements that can be made to the devices on a route towards KID arrays with high resolving powers.

Individual cells and multicellular systems respond to cell-scale curvatures in their environments, guiding migration, orientation, and tissue formation. However, it remains largely unclear how cells collectively explore and pattern complex landscapes with curvature gradients across the Euclidean and non-Euclidean spectra. Here, we show that mathematically designed substrates with controlled curvature variations induce multicellular spatiotemporal organization of preosteoblasts. We quantify curvature-induced patterning and find that cells generally prefer regions with at least one negative principal curvature. However, we also show that the developing tissue can eventually cover unfavorably curved territories, can bridge large portions of the substrates, and is often characterized by collectively aligned stress fibers. We demonstrate that this is partly regulated by cellular contractility and extracellular matrix development, underscoring the mechanical nature of curvature guidance. Our findings offer a geometric perspective on cell-environment interactions that could be harnessed in tissue engineering and regenerative medicine applications.

High-NA light sheet illumination can improve the resolution of single-molecule localization microscopy (SMLM) by reducing the background fluorescence. These approaches currently require custom-made sample holders or additional specialized objectives, which makes the sample mounting or the optical system complex and therefore reduces the usability of these approaches. Here, we developed a single-objective lens-inclined light sheet microscope (SOLEIL) that is capable of 2D and 3D SMLM in thick samples. SOLEIL combines oblique illumination with point spread function PSF engineering to enable dSTORM imaging in a wide variety of samples. SOLEIL is compatible with standard sample holders and off-the-shelve optics and standard high NA objectives. To accomplish optimal optical sectioning we show that there is an ideal oblique angle and sheet thickness. Furthermore, to show what optical sectioning delivers for SMLM we benchmark SOLEIL against widefield and HILO microscopy with several biological samples. SOLEIL delivers in 15 μm thick Caco2-BBE cells a 374% higher intensity to background ratio and a 54% improvement in the estimated CRLB compared to widefield illumination, and a 184% higher intensity to background ratio and a 20% improvement in the estimated CRLB compared to HILO illumination.

Single-photon avalanche diode (SPAD) arrays can be used for single-molecule localization microscopy (SMLM) because of their high frame rate and lack of readout noise. SPAD arrays have a binary frame output, which means photon arrivals should be described as a binomial process rather than a Poissonian process. Consequentially, the theoretical minimum uncertainty of the localizations is not accurately predicted by the Poissonian Cramér-Rao lower bound (CRLB). Here, we derive a binomial CRLB and benchmark it using simulated and experimental data. We show that if the expected photon count is larger than one for all pixels within one standard deviation of a Gaussian point spread function, the binomial CRLB gives a 46% higher theoretical uncertainty than the Poissonian CRLB. For typical SMLM photon fluxes, where no saturation occurs, the binomial CRLB predicts the same uncertainty as the Poissonian CRLB. Therefore, the binomial CRLB can be used to predict and benchmark localization uncertainty for SMLM with SPAD arrays for all practical emitter intensities.

Optical sectioning technologies achieve high precision localization by reducing the background photon count. We use tilted light-sheet microscopy to achieve optical sectioning in localization microscopy, enabling thick sample observation and low background photon count images. A deformable mirror was incorporated to generate a tetrapod point spread function (PSF), enabling high resolution 3D localization. DNA-PAINT was imaged with 15 nm transverse and 60 nm axial resolution.

Optical microrobotics is an emerging field that has the potential to improve upon current optical tweezer studies through avenues such as limiting the exposure of biological molecules of interest to laser radiation and overcoming the current limitations of low forces and unwanted interactions between nearby optical traps. However, optical microrobotics has been historically limited to rigid, single-body end-effectors rather than even simple machines, limiting the tasks that can be performed. Additionally, while multi-body machines such as microlevers exist in the literature, they have not yet been successfully demonstrated as tools for biological studies, such as molecule stretching. In this work we have taken a step towards moving the field forward by developing two types of microlever, produced using two-photon absorption polymerisation, to perform the first lever-assisted stretches of double-stranded DNA. The aim of the work is to provide a proof of concept for using optical micromachines for single molecule studies. Both styles of microlevers were successfully used to stretch single duplexes of DNA, and the results were analysed with the worm-like chain model to show that they were in good agreement.

Organ-on-chip (OoC) technology is increasingly used for biomedical research and to speed up the process of bringing a drug from lab to the market. The main fluidic components of an OoC device are microfluidic channels and porous membranes arranged in three dimensions. Current chips are often assembled from several parts. In the development phase a small change in design will cause a delay in the research because a new prototype has to be built and assembled again step-by-step. The research discussed in this paper addresses this point by targeting a monolithic 3D device that can be fabricated in a single lithography and development step, enabling rapid prototyping. Two-photon lithography (TPL) was used in combination with a positive photoresist AZ 4562. The exposure process was characterized, which included an experimental and theoretical study of the voxel size and shape. It was found that the voxel has an hourglass-shape for the laser power settings that were required for process stability. The smallest pores we could produce with these settings measured 250 nm in diameter. The TPL process was then used to fabricate a microfluidic device featuring two crossed channels each one on a separate height-level, connected by a membrane in the centre. Access to the channels was provided through 4 reservoirs from the top-side of the device. The device was successfully filled with water and dried to see whether it can withstand the corresponding capillary forces.

Advances in nanofabrication over the past twenty years have enabled the creation and use of ever-more interesting and useful micromachines. Optical micromachines are a particularly attractive subset of these for researchers in biological and soft-matter sciences, due to their potential to aid in optical tweezer studies of laser-sensitive samples. However, the development of multi-component micromachines is made difficult due to the dominance of surface forces at this scale, which is made all the more relevant in the high-salt concentrations used for biological studies. This study concerns the design of simple, first-class lever micromachines for use in environments with different salt concentrations, in an attempt to provide a guideline for design requirements of functional optical micromachines for use in physiological conditions.

Recently, we have experimentally demonstrated the generation of mW average power, in the 100-600GHz bandwidth, by photoconductive sources using a connected array of dipoles coherently fed via a free-standing micro-lens array. In this contribution, we extend these sources to a much wider bandwidth reaching 5THz, without compromising the radiated power, by resorting to a tight array periodicity of 15Ϝm between the array elements and the use of an integrated 3D-printed micro-lens array to focus the laser power.