Viewing early life without labels: optical approaches for imaging the early embryo†.

Embryo quality is an important determinant of successful implantation and a resultant live birth. Current clinical approaches for evaluating embryo quality rely on subjective morphology assessments or an invasive biopsy for genetic testing. However, both approaches can be inherently inaccurate and crucially, fail to improve the live birth rate following the transfer of in vitro produced embryos. Optical imaging offers a potential non-invasive and accurate avenue for assessing embryo viability. Recent advances in various label-free optical imaging approaches have garnered increased interest in the field of reproductive biology due to their ability to rapidly capture images at high resolution, delivering both morphological and molecular information. This burgeoning field holds immense potential for further development, with profound implications for clinical translation. Here, our review aims to: (1) describe the principles of various imaging systems, distinguishing between approaches that capture morphological and molecular information, (2) highlight the recent application of these technologies in the field of reproductive biology, and (3) assess their respective merits and limitations concerning the capacity to evaluate embryo quality. Additionally, the review summarizes challenges in the translation of optical imaging systems into routine clinical practice, providing recommendations for their future development. Finally, we identify suitable imaging approaches for interrogating the mechanisms underpinning successful embryo development.

Snapshot hyperspectral imaging of intracellular lasers.

Intracellular lasers are emerging as powerful biosensors for multiplexed tracking and precision sensing of cells and their microenvironment. This sensing capacity is enabled by quantifying their narrow-linewidth emission spectra, which is presently challenging to do at high speeds. In this work, we demonstrate rapid snapshot hyperspectral imaging of intracellular lasers. Using integral field mapping with a microlens array and a diffraction grating, we obtain images of the spatial and spectral intensity distribution from a single camera acquisition. We demonstrate widefield hyperspectral imaging over a 3 × 3 mm2 field of view and volumetric imaging over 250 × 250 × 800 µm3 (XYZ) volumes with a lateral (XY) resolution of 5 µm, axial (Z) resolution of 10 µm, and a spectral resolution of less than 0.8 nm. We evaluate the performance and outline the challenges and strengths of snapshot methods in the context of characterizing the emission from intracellular lasers. This method offers new opportunities for a diverse range of applications, including high-throughput and long-term biosensing with intracellular lasers.

To focus-match or not to focus-match inverse spatially offset Raman spectroscopy: a question of light penetration.

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 center 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 compromising the suppression of the container signal. Using a flexible experimental setup which can realize 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.

Fabrication on the microscale: a two-photon polymerized device for oocyte microinjection.

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.

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device.

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.

The effect of discrete wavelengths of visible light on the developing murine embryo.

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.

BPM-Matlab: an open-source optical propagation simulation tool in MATLAB.

We present the use of the Douglas-Gunn Alternating Direction Implicit finite difference method for computationally efficient simulation of the electric field propagation through a wide variety of optical fiber geometries. The method can accommodate refractive index profiles of arbitrary shape and is implemented in a tool called BPM-Matlab. We validate BPM-Matlab by comparing it to published experimental, numerical, and theoretical data and to commercially available state-of-the-art software. It is user-friendly, fast, and is available open-source. BPM-Matlab has a broad scope of applications in modeling a variety of optical fibers for diverse fields such as imaging, communication, material processing, and remote sensing.

Femtometer-resolved simultaneous measurement of multiple laser wavelengths in a speckle wavemeter.

Many areas of optical science require an accurate measurement of optical spectra. Devices based on laser speckle promise compact wavelength measurement, with attometer-level sensitivity demonstrated for single wavelength laser fields. The measurement of multimode spectra using this approach would be attractive, yet this is currently limited to picometer resolution. Here, we present a method to improve the resolution and precision of speckle-based multi-wavelength measurements. We measure multiple wavelengths simultaneously, in a device comprising a single 1-m-long step-index multimode fiber and a fast camera. Independent wavelengths separated by as little as 1 fm are retrieved with 0.2 fm precision using principal component analysis. The method offers a viable way to measure sparse spectra containing multiple individual lines and may find application in the tracking of multiple lasers in fields such as quantum technologies and optical telecommunications.

Photopolymerization with high-order Bessel light beams.

We study photopolymerization with high-order Bessel light beams with phase singularities on-axis. Self-trapping and self-focusing of propagation-invariant light beams in a photopolymer allow the fabrication of extended helical microfibers with a length scale of a centimeter, which is more than an order of magnitude larger than the propagation distance of the Bessel light beams. We show the evolution of microfibers rotating at a rate proportional to the incident optical power, while the periodicity of the helical structures remains constant, irrespective of the laser power. This suggests that optical momentum transfer plays a predominant role in the growth and rotation of such fiber structures.

Label-free optical hemogram of granulocytes enhanced by artificial neural networks.

An outstanding challenge for immunology is the classification of immune cells in a label-free fashion with high speed. For this purpose, optical techniques such as Raman spectroscopy or digital holographic microscopy have been used successfully to identify immune cell subsets. To achieve high accuracy, these techniques require a post-processing step using linear methods of multivariate processing, such as principal component analysis. Here we demonstrate for the first time a comparison between artificial neural networks and principal component analysis (PCA) to classify the key granulocyte cell lineages of neutrophils and eosinophils using both digital holographic microscopy and Raman spectroscopy. Artificial neural networks can offer advantages in terms of classification accuracy and speed over a PCA approach. We conclude that digital holographic microscopy with convolutional neural networks based analysis provides a route to a robust, stand-alone and high-throughput hemogram with a classification accuracy of 91.3 % at a throughput rate of greater than 100 cells per second.

Overcoming the speckle correlation limit to achieve a fiber wavemeter with attometer resolution.

The measurement of the wavelength of light using speckle is a promising tool for the realization of compact and precise wavemeters and spectrometers. However, the resolution of these devices is limited by strong correlations between the speckle patterns produced by closely spaced wavelengths. Here, we show how principal component analysis of speckle images provides a route to overcome this limit. Using this, we demonstrate a compact wavemeter that measures attometer-scale wavelength changes of a stabilized diode laser, eight orders of magnitude below the speckle correlation limit.

Optimal compressive multiphoton imaging at depth using single-pixel detection.

Compressive sensing can overcome the Nyquist criterion and record images with a fraction of the usual number of measurements required. However, conventional measurement bases are susceptible to diffraction and scattering, prevalent in high-resolution microscopy. In this Letter, we explore the random Morlet basis as an optimal set for compressive multiphoton imaging, based on its ability to minimize the space-frequency uncertainty. We implement this approach for wide-field multiphoton microscopy with single-pixel detection, which allows imaging through turbid media without correction. The Morlet basis promises a route for rapid acquisition with lower photodamage.

Three-photon light-sheet fluorescence microscopy.

We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single- and two-photon modes has emerged as a powerful wide-field, low-photodamage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime, enhancing its penetration depth. Our present study uses a conventional femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 µm diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams.

Optical trapping with planar silicon metalenses.

Contactless manipulation of micron-scale objects in a microfluidic environment is a key ingredient for a range of applications in the biosciences, including sorting, guiding, and analysis of cells and bacteria. Optical forces are powerful for this purpose but, typically, require bulky focusing elements to achieve the appropriate optical field gradients. To this end, realizing the focusing optics in a planar format would be very attractive and conducive to the integration of such microscale devices, either individually or as arrays. Here we report on, to the best of our knowledge, the first experimental demonstration of optical trapping using planar silicon metalenses illuminated with a collimated laser beam. The structures consist of high-contrast gratings with a locally varying period and duty cycle. They are designed to mimic parabolic reflectors with a numerical aperture of 0.56 at a vacuum wavelength of 1064 nm. We achieve both two- and three-dimensional trapping in water, with the latter realized by omitting the central Fresnel zones. This Letter highlights the versatility of such lithographically defined metastructures for exerting optical forces without the need for traditional optical elements.

Light-sheet microscopy using an Airy beam.

Light-sheet microscopy facilitates rapid, high-contrast, volumetric imaging with minimal sample exposure. However, the rapid divergence of a traditional Gaussian light sheet restricts the field of view (FOV) that provides innate subcellular resolution. We show that the Airy beam innately yields high contrast and resolution up to a tenfold larger FOV. In contrast to the Bessel beam, which also provides an increased FOV, the Airy beam's characteristic asymmetric excitation pattern results in all fluorescence contributing positively to the contrast, enabling a step change for light-sheet microscopy.

Raman imaging through a single multimode fibre.

Vibrational spectroscopy is a widespread, powerful method of recording the molecular spectra of constituent molecules within a sample in a label-free manner. As an example, Raman spectroscopy has major applications in materials science, biomedical analysis and clinical studies. The need to access deep tissues and organs in vivo has triggered major advances in fibre Raman probes that are compatible with endoscopic settings. However, imaging in confined geometries still remains out of reach for the current state of art fibre Raman systems without compromising the compactness and flexibility. Here we demonstrate Raman spectroscopic imaging via complex correction in single multimode fibre without using any additional optics and filters in the probe design. Our approach retains the information content typical to traditional fibre bundle imaging, yet within an ultra-thin footprint of diameter 125 µm which is the thinnest Raman imaging probe realised to date. We are able to acquire Raman images, including for bacteria samples, with fields of view exceeding 200 µm in diameter.

Wide-field three-dimensional optical imaging using temporal focusing for holographically trapped microparticles.

A contemporary challenge across the natural sciences is the simultaneous optical imaging or stimulation of small numbers of cells or colloidal particles organized into arbitrary geometries. We demonstrate the use of temporal focusing with holographic optical tweezers in order to achieve depth-resolved two-photon imaging of trapped objects arranged in arbitrary three-dimensional (3D) geometries using a single objective. Trapping allows for the independent position control of multiple objects by holographic beam shaping. Temporal focusing of ultrashort pulses provides the wide-field two-photon depth-selective activation of fluorescent samples. We demonstrate the wide-field depth-resolved illumination of both trapped fluorescent beads and trapped HL60 cells in suspension with full 3D positioning control. These approaches are compatible with implementation through scattering media and can be beneficial for emergent studies in colloidal science and particularly optogenetics, offering targeted photoactivation over a wide area with micrometer-precision depth control.

Rotation of two trapped microparticles in vacuum: observation of optically mediated parametric resonances.

We demonstrate trapping and rotation of two mesoscopic particles in vacuum using a spatial-light-modulator-based approach to trap more than one particle, induce controlled rotation of individual particles, and mediate interparticle separation. By trapping and rotating two vaterite particles, we observe intensity modulation of the scattered light at the sum and difference frequencies with respect to the individual rotation rates. This first demonstration of optical interference between two microparticles in vacuum leads to a platform to potentially explore optical binding and quantum friction effects.

Modulated Raman Spectroscopy for Enhanced Cancer Diagnosis at the Cellular Level.

Raman spectroscopy is emerging as a promising and novel biophotonics tool for non-invasive, real-time diagnosis of tissue and cell abnormalities. However, the presence of a strong fluorescence background is a key issue that can detract from the use of Raman spectroscopy in routine clinical care. The review summarizes the state-of-the-art methods to remove the fluorescence background and explores recent achievements to address this issue obtained with modulated Raman spectroscopy. This innovative approach can be used to extract the Raman spectral component from the fluorescence background and improve the quality of the Raman signal. We describe the potential of modulated Raman spectroscopy as a rapid, inexpensive and accurate clinical tool to detect the presence of bladder cancer cells. Finally, in a broader context, we show how this approach can greatly enhance the sensitivity of integrated Raman spectroscopy and microfluidic systems, opening new prospects for portable higher throughput Raman cell sorting.