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Embryo quality is a crucial factor affecting live birth outcomes. However, an accurate diagnostic for embryo quality remains elusive in thein vitrofertilization clinic. Determining physical parameters of the embryo may offer key information for this purpose. Here, we demonstrate that digital holographic microscopy (DHM) can rapidly and non-invasively assess the refractive index of mouse embryos. We showed that DHM can detect spatio-temporal changes in refractive index during embryo development that are reflective of its lipid content. As accumulation of intracellular lipid is known to compromise embryo health, DHM may prove beneficial in developing an accurate, non-invasive, multimodal diagnostic.

Optical imaging has the potential to reveal high-resolution information with minimal photodamage. The recent renaissance of super-resolution, widefield, ultrafast, and computational imaging methods has broadened its horizons even further. However, a remaining grand challenge is imaging at depth over a widefield and with a high spatiotemporal resolution. This achievement would enable the observation of fast collective biological processes, particularly those underpinning neuroscience and developmental biology. Multiphoton imaging at depth, combining temporal focusing and single-pixel detection, is an emerging avenue to address this challenge. The novel physics and computational methods driving this approach offer great potential for future advances. This chapter articulates the theories of temporal focusing and single-pixel detection and details the specific approach of TempoRAl Focusing microscopy with single-pIXel detection (TRAFIX), with a particular focus on its current practical implementation and future prospects.

Purpose Vitrification permits long-term banking of oocytes and embryos. It is a technically challenging procedure requiring direct handling and movement of cells between potentially cytotoxic cryoprotectant solutions. Variation in adherence to timing, and ability to trace cells during the procedure, affects survival post-warming. We hypothesized that minimizing direct handling will simplify the procedure and improve traceability. To address this, we present a novel photopolymerized device that houses the sample during vitrification. Methods The fabricated device consisted of two components: the Pod and Garage. Single mouse oocytes or embryos were housed in a Pod, with multiple Pods docked into a Garage. The suitability of the device for cryogenic application was assessed by repeated vitrification and warming cycles. Oocytes or early blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Post-warming, we assessed survival rate, oocyte developmental potential (fertilization and subsequent development) and metabolism (autofluorescence). Results Vitrification within the device occurred within ~ 3 nL of cryoprotectant: this volume being ~ 1000-fold lower than standard vitrification. Compared to standard practice, vitrification and warming within our device showed no differences in viability, developmental competency, or metabolism for oocytes and embryos. The device housed the sample during processing, which improved traceability and minimized handling. Interestingly, vitrification-warming itself, altered oocyte and embryo metabolism. Conclusion The Pod and Garage system minimized the volume of cryoprotectant at vitrification—by ~ 1000-fold—improved traceability and reduced direct handling of the sample. This is a major step in simplifying the procedure.

This work was supported by the National Research Foundation of Korea (NRF) grant by the Korea government (MSIT) (No. 2019R1A2C2011293) and University of Sydney – Yonsei University Partnership Collaboration Awards. M. P. acknowledges European Union's Horizon 2020 Marie Sklodowska-Curie Actions (MSCA) individual fellowship under Contract No. 846218. KD thanks the UK Engineering and Physical Sciences Research Council for funding (grant EP/P030017/1). From mechanical syringes to electric field-assisted injection devices, precise control of liquid droplet generation has been sought after, and the present state-of-the-art technologies have provided droplets ranging from nanoliter to subpicoliter volume sizes. In this study, we present a new laser-driven method to generate liquid droplets with a zeptoliter volume, breaking the fundamental limits of previous studies. We guided an infrared laser beam through a hollow optical fiber (HOF) with a ring core whose end facet was coated with single-walled carbon nanotubes. The laser light was absorbed by this nanotube film and efficiently generated a highly localized microring heat source. This evaporated the liquid inside the HOF, which rapidly recondensed into zeptoliter droplets in the surrounding air at room temperature. We spectroscopically confirmed the chemical structures of the liquid precursor maintained in the droplets by atomizing dye-dissolved glycerol. Moreover, we explain the fundamental physical principles as well as functionalities of the optical atomizer and perform a detailed characterization of the droplets. Our approach has strong prospects for nanoscale delivery of biochemical substances in minuscule zeptoliter volumes. Postprint Publisher PDF

K.D. thanks the UK Engineering and Physical Sciences Research Council for funding (grant number EP/P030017/1). B.W.D. gratefully acknowledges funding from the Wolfson Foundation and the Royal Society. M.R.-M. gratefully acknowledges support from the Austrian Science Fund FWF (SFB-project F6806-N36). The application of acoustic and optical waves to exert non-contact forces on microscopic and mesoscopic objects has grown considerably in importance in the past few decades. Different physical principles govern the acoustic and optical forces, leading to diverse biomedical applications. Biocompatibility is crucial, and useful optical and acoustic forces can be applied in devices that maintain local heating to acceptable levels. Current acoustic and optical devices work on complementary length scales, with both modalities having useful capabilities at the scale of the cell. Optical devices also cover subcellular scales and acoustic devices also cover supercellular scales. This complementarity has led to the emergence of multimode manipulation, often with integrated imaging. In this Technical Review, we provide an overview of optical and acoustic forces, before comparing and contrasting the use of these modalities, or combinations thereof, in terms of sample manipulation and suitability for biomedical studies. We conclude with our perspective on the applications in which we expect to see notable developments in the near future. Postprint

Purpose Intracytoplasmic sperm injection (ICSI) addresses male sub-fertility by injecting a spermatozoon into the oocyte. This challenging procedure requires the use of dual micromanipulators, with success influenced by inter-operator expertise. We hypothesized that minimizing oocyte handling during ICSI will simplify the procedure. To address this, we designed and fabricated a micrometer scale device that houses the oocyte and requires only one micromanipulator for microinjection. Methods The device consisted of 2 components, each of sub-cubic millimeter volume: a Pod and a Garage. These were fabricated using 2-photon polymerization. Toxicity was evaluated by culturing single-mouse presumptive zygotes (PZs) to the blastocyst stage within a Pod, with several Pods (and embryos) docked in a Garage. The development was compared to standard culture. The level of DNA damage/repair in resultant blastocysts was quantified (γH2A.X immunohistochemistry). To demonstrate the capability to carry out ICSI within the device, PZs were microinjected with 4-μm fluorescent microspheres and cultured to the blastocyst stage. Finally, the device was assessed for oocyte traceability and high-throughput microinjection capabilities and compared to standard microinjection practice using key parameters (pipette setup, holding then injecting oocytes). Results Compared to standard culture, embryo culture within Pods and a Garage showed no differences in development to the blastocyst stage or levels of DNA damage in resultant blastocysts. Furthermore, microinjection within our device removes the need for a holding pipette, improves traceability, and facilitates high-throughput microinjection. Conclusion This novel device could improve embryo production following ICSI by simplifying the procedure and thus decreasing inter-operator variability.

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

The ability to identify the contents of a sealed container, without the need to extract a sample, is desirable in applications ranging from forensics to product quality control. One technique suited to this is inverse spatially offset Raman spectroscopy (ISORS) which illuminates a sample of interest with an annular beam of light and collects Raman scattering from the centre of the ring, thereby retrieving the chemical signature of the contents while suppressing signal from the container. Here we explore in detail the relative benefits of a recently developed variant of ISORS, called focus-matched ISORS. In this variant, the Fourier relationship between the annular beam and a tightly focused Bessel beam is exploited to focus the excitation light inside the sample and to match the focal point of excitation and collection optics to increase the signal from the contents without out compromising the suppression of the container signal. Using a flexible experimental setup which can realise both traditional and focus-matched ISORS, and Monte-Carlo simulations, we elucidate the relative advantages of the two techniques for a range of optical properties of sample and container., 11 pages, 4 figures, plus 9 supplementary figures

Funding: Engineering and Physical Sciences Research Council. Grant Number: EP/L015331/1 European Research Council. Grant Number: ERC-2016-AdG-742222 Leverhulme Trust. Grant Number: 11715R. Raman spectroscopy is a powerful technique that enables fingerprinting of materials, molecules, and chemical environments by probing vibrational resonances. In many applications, the desired Raman signals are masked by fluorescence, either from the molecular system being studied, or from adjacent metallic nanostructures. Here, it is shown that wavelength-modulated Raman spectroscopy provides a powerful way to significantly reduce the strength of the fluorescence background, thereby allowing the desired Raman signals to be clearly recorded. This approach is made use of to explore Raman scattering in the context of vibrational strong coupling, an area that has thus far been problematic to visualise. Specifically, strong coupling between the vibrational modes in a polymer and two types of confined light field, the fundamental mode of a metal-clad microcavity, and the surface-plasmon modes of an adjacent thin metal film are looked at. While clear advantages in using the wavelength-modulated Raman approach are found, these results on strong coupling are inconclusive, and highlight the need for more work in this exciting topic area. Publisher PDF

In general,the optical binding force between identical particles is thought to be symmetric.However,we demonstrate analytically a counter-intuitively asymmetric longitudinal optical binding force between two identical dual dipolar dielectric particles.This homodimer is confined in two counter-propagating incoherent plane waves along the dimer's axis.The force consists of the electric dipolar,magnetic dipolar,and electric-magnetic dipolar coupling interactions.The combined effect of these interactions is markedly different than the expected behavior in the Rayleigh approximation.The asymmetric force is a result of the asymmetric forward and backward scattering of the particles due to the dipolar hybridization and coupling interactions.Consequently,it leads to a harmonic driving force on the pair,which decays with the interparticle distance to the first power.We show the rich nonequilibrium dynamics of the dimer and of the two particles impelled by the driving and binding forces and discuss the ranges of particle refractive index and size in which the asymmetric binding force arises.Our results open perspectives for nonequilibrium light-driven multiparticle transport and self-assembly., 9 pages, 10 figures

Functionally distinct synapses exhibit diverse and complex organisation at molecular and nanoscale levels. Synaptic diversity may be dependent on developmental stage, anatomical locus and the neural circuit within which synapses reside. Furthermore, astrocytes, which align with pre and post-synaptic structures to form 'tripartite synapses', can modulate neural circuits and impact on synaptic organisation. In this study, we aimed to determine which factors impact the diversity of excitatory synapses throughout the lumbar spinal cord. We used PSD95-eGFP mice, to visualise excitatory postsynaptic densities (PSDs) using high-resolution and super-resolution microscopy. We reveal a detailed and quantitative map of the features of excitatory synapses in the lumbar spinal cord, detailing synaptic diversity that is dependent on developmental stage, anatomical region and whether associated with VGLUT1 or VGLUT2 terminals. We report that PSDs are nanostructurally distinct between spinal laminae and across age groups. PSDs receiving VGLUT1 inputs also show enhanced nanostructural complexity compared with those receiving VGLUT2 inputs, suggesting pathway-specific diversity. Finally, we show that PSDs exhibit greater nanostructural complexity when part of tripartite synapses, and we provide evidence that astrocytic activation enhances PSD95 expression. Taken together, these results provide novel insights into the regulation and diversification of synapses across functionally distinct spinal regions and advance our general understanding of the 'rules' governing synaptic nanostructural organisation.

Funding: We acknowledge the support from the Engineering and Physical Sciences Research Council (Grant No. EP/P030017/1), the European Regional Development Fund (Grant No. CZ.02.1.01/0.0/0.0/15_003/0000476), the Czech Science Foundation (Grant No. GA19-17765S), and the Czech Academy of Sciences (Praemium Academiae, Grant No. RVO:68081731). The forces acting on an isotropic microsphere in optical tweezers are effectively conservative. However, reductions in the symmetry of the particle or trapping field can break this condition. Here we theoretically analyse the motion of a particle in a linearly non-conservative optical vacuum trap, concentrating on the case where symmetry is broken by optical birefringence, causing non-conservative coupling between rotational and translational degrees of freedom. Neglecting thermal fluctuations, we first show that the underlying deterministic motion can exhibit a Hopf bifurcation in which the trapping point destabilizes and limit cycles emerge whose amplitude grows with decreasing viscosity. When fluctuations are included, the bifurcation of the underlying deterministic system is expressed as a transition in the statistical description of the motion. For high viscosities, the probability distribution is normal, with a kurtosis of three, and persistent probability currents swirl around the stable trapping point. As the bifurcation is approached the distribution and currents spread out in phase space. Following the bifurcation the probability distribution function hollows out, reflecting the underlying limit cycle, and the kurtosis halves abruptly. The system is seen to be a noisy self sustained oscillator featuring a highly uneven limit cycle. A variety of applications, from autonomous stochastic resonance to synchronization, are discussed. Publisher PDF

Highly resolved determination of refractive index is vital in fields ranging from biosensing through to laser range finding. Laser speckle is known to be a sensitive probe of the properties of the light and the environment, but to date speckle-based refractive index measurements have been restricted to 10

Open Access funding enabled and organized by CAUL and its Member Institutions KRD is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58–2019). KD is supported by funding from the UK Engineering and Physical Sciences Research Council (EP/P030017/1) and the Australian Research Council (FL210100099). CC acknowledges the support of a PhD scholarship jointly from the University of Adelaide and University of Nottingham. This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). PR acknowledges funding through the RMIT Vice-Chancellor’s Research Fellowship and ARC DECRA Fellowship scheme (DE200100279). Purpose A current focus of the IVF field is non-invasive imaging of the embryo to quantify developmental potential. Such approaches use varying wavelengths to gain maximum biological information. The impact of irradiating the developing embryo with discrete wavelengths of light is not fully understood. Here, we assess the impact of a range of wavelengths on the developing embryo. Methods Murine preimplantation embryos were exposed daily to wavelengths within the blue, green, yellow, and red spectral bands and compared to an unexposed control group. Development to blastocyst, DNA damage, and cell number/allocation to blastocyst cell lineages were assessed. For the longer wavelengths (yellow and red), pregnancy/fetal outcomes and the abundance of intracellular lipid were investigated. Results Significantly fewer embryos developed to the blastocyst stage when exposed to the yellow wavelength. Elevated DNA damage was observed within embryos exposed to blue, green, or red wavelengths. There was no effect on blastocyst cell number/lineage allocation for all wavelengths except red, where there was a significant decrease in total cell number. Pregnancy rate was significantly reduced when embryos were irradiated with the red wavelength. Weight at weaning was significantly higher when embryos were exposed to yellow or red wavelengths. Lipid abundance was significantly elevated following exposure to the yellow wavelength. Conclusion Our results demonstrate that the impact of light is wavelength-specific, with longer wavelengths also impacting the embryo. We also show that effects are energy-dependent. This data shows that damage is multifaceted and developmental rate alone may not fully reflect the impact of light exposure. Publisher PDF

We demonstrate parametric feedback cooling of a levitated birefringent microsphere held in a circularly polarised Gaussian trap. We show efficient cooling of the birefringent microsphere to the temperature less than a millikelvin, which is orders of magnitude lower than that of a silica counterpart. We reveal that the transverse spin momentum present in circularly polarised light plays a role in the trapped particle dynamics, which exhibits a range of nonequilibrium phenomena, namely, (i) biased stochastic motion, (ii) coupling of mechanical modes, (iii) nonconservative rotation-translation coupling. We further discuss the temperature of the trapped particle in a nonconservative vacuum optical trap.

The authors G.B., F.T., and C.C acknowledge financial support by the POR of Apulia region, IT (FESR FSR 2014−2020, Action 10.4, “Research for Innovation” (REFIN) Initiative). The authors D.C., G.P., K.D., and T.F.K. acknowledge financial support by the EPSRC of the UK (Grant EP/P030017/1). Optical trapping has revolutionized our understanding of biology by manipulating cells and single molecules using optical forces. Moving to the near-field creates intense field gradients to trap very smaller particles, such as DNA fragments, viruses, and vesicles. The next frontier for such optical nanotweezers in biomedical applications is to trap multiple particles and to study their heterogeneity. To this end, we have studied dielectric metasurfaces that allow the parallel trapping of multiple particles. We have explored the requirements for such metasurfaces and introduce a structure that allows the trapping of a large number of nanoscale particles (>1000) with a very low total power P < 26 mW. We experimentally demonstrate the near-field enhancement provided by the metasurface and simulate its trapping performance. We have optimized the metasurface for the trapping of 100 nm diameter particles, which will open up opportunities for new biological studies on viruses and extracellular vesicles, such as studying heterogeneity, or to massively parallelize analyses for drug discovery. Publisher PDF