Microscope Objective Research Articles

Active Synthetic Microrotors: Design Strategies and Applications.

Microrotors are microscopic objects that convert energy stored in the environment into spontaneous rotation, in the form of spinning along an axis, rolling on a surface, or orbiting in circles. Because of its distinct dynamics and the vertical flows around it, a microrotor is potentially useful for applications, including drug delivery, minimally invasive surgery, fluid mixing, and sensing. It is also useful as a model system to probe the collective behaviors among rotating micro-objects. In this review article, we comprehensively review the recent experimental progress in designing, synthesizing, and using microrotors. For applications, particular emphasis is placed on microfluidic mixing, biomedicine, and collective behaviors. In the end, we comment on how microrotors can be made more biocompatible and more controllable and rotate in more ways and the challenges therein. A key feature of this review article is to introduce three ways in which to classify a microrotor: the nature of its rotational behavior (spinners, rollers, or orbiters), the cause of its rotation (whether chiral symmetry is broken by shapes, chemical compositions, or the way energy is applied), and its power source (whether powered by chemical reactions, electric or magnetic fields, light, or ultrasound). This review article will help materials scientists and chemists in designing micromachines and microrotors, help engineers in finding appropriate microrotors for a specific application, and help physicists in finding appropriate model systems.

Microscopy with heralded Fock states.

We consider a microscopy setting where quantum light is used for illumination. Spontaneous parametric down conversion (SPDC) is used as a source of a heralded single photon, which is quantum light prepared in a Fock state. We present analytical formulas for the spatial mode tracking along with the heralded and non-heralded mode widths. The obtained analytical results are supported by numerical calculations and the following discussion taking into account realistic setup parameters such as finite-size optics and finite-size single-photon detectors. This allows us to observe that the diffraction limit can be approached with simultaneous alleviation of the photon loss leading to increased signal-to-noise ratio - a factor limiting practical applications of quantum light. Additionally, it is shown that the spatial resolution can be manipulated by carefully preparing the amplitude and phase of the spatial mode profile of the single photon at the input to the microscope objective. Here, the spatial entanglement of the biphoton wavefunction or adaptive optics can be applied for spatial mode shaping. Analytical dependencies between the incident and focused spatial mode profiles parameters are provided.

In vivo spatial-spectral photoacoustic microscopy enabled by optical evanescent wave sensing

In addition to offering morphological visualizations via capture of the spatial distributions of optical absorption, photoacoustic imaging technology can reveal abundant physical information about biological particles, including their orientation, density, and viscoelasticity, through analysis of the pressure transients in the spectral domain. However, the low-amplitude wideband photoacoustic signals of intrinsic microscopic optically-absorbing objects under the action of confined photoacoustic excitation power continue to hinder simultaneous photoacoustic structural imaging and spectroscopic analysis of the nonfluorescent chromophores in living biological tissues because of the inadequate responses to photoacoustic impulses observed in most photoacoustic imaging setups that include piezoelectric transducers. Building upon a recently-developed optical evanescent wave sensor that can respond to ultrasound with high sensitivity over a broad frequency range, we propose in vivo spatial-spectral photoacoustic microscopy for recovery of structural imaging in three dimensions and characterization of anatomical features in the acoustic frequency domain. Label-free photoacoustic images of a living zebrafish are acquired in which spectroscopically-resolved differentiation of the microarchitecture is accessed, along with isometric micrometer-scale volumetric visualizations. The proposed imaging technology could potentially provide more comprehensive evaluations of the physiopathological status of living small animals.

Rethinking microscope objectives.

Rethinking microscope objectives.

On the characterization of bias errors in defocusing-based 3D particle tracking velocimetry for microfluidics

In this work, we provide a systematic theoretical and experimental characterization of bias errors in defocusing particle tracking (DPT) methods based on a single calibration function, with respect to microfluidic applications, in which it is not possible to use calibration targets inside the measurement volume. This approach is widely used in microfluidic experiments, but bias errors are often neglected and to date only few works reported empirical procedures to compensate for that. A systematic characterization of the impact of such error in DPT measurements is still lacking. We show that the field curvature aberration and the refractive index mismatch are the main sources of bias error in these applications. We present a correction methodology for the bias error based on the determination of a reference surface, and in addition we propose a procedure based on a reference measurement of a Poiseuille flow to determine the reference surface on microfluidic channels with constant cross section. We discuss the impact of the refractive index mismatch and how to correctly compensate for it. We validated our methodology and quantified the bias errors on 10 different experimental setups, using different working fluids, materials, geometries, and microscope objective lenses ranging from 5times to 40times magnification. Our results indicate that the impact of this type of bias errors is in general not predictable and must be evaluated case by case. The proposed methodology allows to estimate and minimize the bias error in most microfluidic setups and is suitable for any single-camera DPT approach.

From a Dinuclear System to Close Binary Cosmic Objects

Applying the ideas from microscopic objects to macroscopic stellar and galactic systems, the evolution of compact di-stars and di-galaxies is studied in the mass asymmetry coordinate. The formation of stable binary systems is analyzed. The role of symmetrization of an initially asymmetric binary system is revealed in the transformation of gravitational energy into internal energy of stars or galaxies accompanied by the release of a huge amount of energy. For the contact binary stars, the change of the orbital period is explained by evolution to symmetry in mass asymmetry coordinates. The matter transfer in binary black holes is studied. The conditions for the merger of black holes in a binary system are analyzed regarding the radiation of gravitational waves. Using the model based on the Regge-like laws, the Darwin instability effect in binary systems is discussed. New analytical formulas are derived for the period of orbital rotation and the relative distance between the components of a binary system. The impossibility of the appearance of a binary cosmic object from a single cosmic object is revealed.

Whole-brain imaging of freely-moving zebrafish.

One of the holy grails of neuroscience is to record the activity of every neuron in the brain while an animal moves freely and performs complex behavioral tasks. While important steps forward have been taken recently in large-scale neural recording in rodent models, single neuron resolution across the entire mammalian brain remains elusive. In contrast the larval zebrafish offers great promise in this regard. Zebrafish are a vertebrate model with substantial homology to the mammalian brain, but their transparency allows whole-brain recordings of genetically-encoded fluorescent indicators at single-neuron resolution using optical microscopy techniques. Furthermore zebrafish begin to show a complex repertoire of natural behavior from an early age, including hunting small, fast-moving prey using visual cues. Until recently work to address the neural bases of these behaviors mostly relied on assays where the fish was immobilized under the microscope objective, and stimuli such as prey were presented virtually. However significant progress has recently been made in developing brain imaging techniques for zebrafish which are not immobilized. Here we discuss recent advances, focusing particularly on techniques based on light-field microscopy. We also draw attention to several important outstanding issues which remain to be addressed to increase the ecological validity of the results obtained.

High numerical aperture imaging allows chirality measurement in individual collagen fibrils using polarization second harmonic generation microscopy.

Second harmonic generation (SHG) microscopy is a commonly used technique to study the organization of collagen within tissues. However, individual collagen fibrils, which have diameters much smaller than the resolution of most optical systems, have not been extensively investigated. Here we probe the structure of individual collagen fibrils using polarization-resolved SHG (PSHG) microscopy and atomic force microscopy. We find that longitudinally polarized light occurring at the edge of a focal volume of a high numerical aperture microscope objective illuminated with linearly polarized light creates a measurable variation in PSHG signal along the axis orthogonal to an individual collagen fibril. By comparing numerical simulations to experimental data, we are able to estimate parameters related to the structure and chirality of the collagen fibril without tilting the sample out of the image plane, or cutting tissue at different angles, enabling chirality measurements on individual nanostructures to be performed in standard PSHG microscopes. The results presented here are expected to lead to a better understanding of PSHG results from both collagen fibrils and collagenous tissues. Further, the technique presented can be applied to other chiral nanoscale structures such as microtubules, nanowires, and nanoribbons.

Enhancement of Raman signal of monolayer graphene films using a single optical microsphere-assisted Raman microscopic technique

For the first time, we report the enhancement of the Raman scattering signal of monolayer graphene films (MGFs) on Cu foils using a single optical microsphere-assisted Raman microscopic (SOMRM) technique. Initially, the Raman scattering spectra of MGF on Cu foil are recorded using the conventional Raman microscopic (CRM) technique, where the excitation laser is directly focused on the MGFs with the help of a different microscopic objective lens. The obtained spectra are observed to consist of only the low-intensity G and 2D bands but not the D band, known as the disorder or defect band. However, the intensity of all three bands is enhanced significantly using the SOMRM technique. Finally, the numerical investigation is performed on the SOMRM technique to understand the origin of the enhancement of the Raman scattering signal of MGF on the Cu substrates. The role of the substrate for MGF and the radius of the microsphere on the enhancement of the Raman scattering signal of MGFs is also investigated numerically in detail.

Multiplexed superresolution phase microscopy with transport of intensity equation

Resolving power in imaging systems, optical microscopy in particular, is limited by several factors being diffraction probably the most important one. Digital holographic microscopy (DHM) allows diffraction-limited quantitative phase imaging (QPI) with high accuracy by retrieving complex fields using interferometric recording, and a bunch of superresolution (SR) techniques have been widely reported through the past. However, there are additional ways to retrieve the complex amplitude distribution (such as the transport of intensity equation – TIE), having additional advantages over interferometric recording. We present how to increase the resolution limit in low/medium numerical aperture (NA) microscope objectives by using time and angular multiplexing while providing quantitative phase imaging (QPI) retrieved by TIE algorithm. This is, to the best of our knowledge, the first time that the resolution limit is experimentally improved in combination with TIE for phase retrieval in QPI. The proposed approach is validated in a real microscope embodiment (Olympus IX81) using resolution targets for quantitative resolution analysis and verifying a resolution gain factor of 2 from an experimental point of view.

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope

Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion ‘Schmidt objective’ consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy.

Broadband Antireflective Hybrid Micro/Nanostructure on Zinc Sulfide Fabricated by Optimal Bessel Femtosecond Laser.

Enhancing the infrared window transmittance of zinc sulfide (ZnS) is important to improve the performance of infrared detector systems. In this work, a new hybrid micro/nanostructure was fabricated by an optimal Bessel femtosecond laser on ZnS substrate. The surface morphologies and profiles of ASS ablated by a 20× microscope objective Bessel beam are described, indicating that the nanoripples on the micropore were formed by the SPP interference and the SPP scattering in a particular direction. Further, the maximum average transmittance of ASS increased by 9.7% and 12.3% in the wavelength ranges of 5~12 μm and 8~12 μm, respectively. Finally, the antireflective mechanism of the hybrid micro/nanostructure is explored using the novel electromagnetic field model based on the FDTD method, and we attribute the stable antireflective performance of ASS in broadband to the interface effective dielectric effect and LLFE.

On-Chip Optical Trapping with High NA Metasurfaces.

Optical trapping of small particles typically requires the use of high NA microscope objectives. Photonic metasurfaces are an attractive alternative to create strongly focused beams for optical trapping applications in an integrated platform. Here, we report on the design, fabrication, and characterization of optical metasurfaces with a numerical aperture up to 1.2 and trapping stiffness greater than 400 pN/μm/W. We demonstrate that these metasurfaces perform as well as microscope objectives with the same numerical aperture. We systematically analyze the impact of the metasurface dimension on the trapping performance and show efficient trapping with metasurfaces with an area as small as 0.001 mm2. Finally, we demonstrate the versatility of the platform by designing metasurfaces able to create multisite optical tweezers for the trapping of extended objects.

Microlenses Fabricated by Two‐Photon Laser Polymerization for Cell Imaging with Non‐Linear Excitation Microscopy

AbstractNon‐linear excitation microscopy offers several advantages for in‐vivo imaging compared to conventional confocal techniques. However, tissue penetration can still be an issue due to scattering and spherical aberrations induced on focused beams by the tissue. The use of low numerical aperture objectives to pass through the outer layers of the skin, together with high dioptric power microlenses implanted in‐vivo close to the observation volume, can be beneficial to the reduction of optical aberrations. Here, Fibroblast cell culture plano‐convex microlenses to be used for non‐linear imaging of biological tissue are developed and tested. The microlenses can be used as single lenses or multiplexed in an array. A thorough test of the lenses wavefront is reported together with the modulation transfer function and wavefront profile. Magnified fluorescence images can be retrieved through the microlenses coupled to commercial confocal and two‐photon excitation scanning microscopes. The signal‐to‐noise ratio of the images is not substantially affected by the use of the microlenses and the magnification can be adjusted by changing the relative position of the microlens array to the microscope objective and the immersion medium. These results are opening the way to the application of implanted micro‐optics for optical in‐vivo inspection of biological processes.

MesoTIRF: A prism-based Total Internal Reflection Fluorescence illuminator for high resolution, high contrast imaging of large cell populations

Total Internal Reflection Fluorescence (TIRF) illumination bypasses the axial diffraction limit of light by using an evanescent field to excite fluorophores close to a sample substrate. However, standard TIRF imaging through the objective requires a high numerical aperture (NA) to generate the evanescent wave. Available lenses have a high magnification with a correspondingly small field of view—ranging from ∼50 μm to 1 mm in diameter. Switching to the older prism-TIRF configuration introduced by Axelrod in the 1980s might seem to remove the requirement for high objective NA and allow the use of existing large-field objectives. Unfortunately, these lenses are unsuitable because their throughput of light is too low for TIRF imaging. As such, high sensitivity TIRF imaging over a much larger mesoscopic field has yet to be demonstrated. We have developed a prism-based TIRF illuminator for the Mesolens—a highly corrected objective lens with an unparalleled ratio of NA to magnification. The imaging field of the Mesolens is 204 times larger than that of the TIRF objectives previously described, increasing the optical throughput of the optical system by a factor of 25 compared to an off-the-shelf microscope objective of the same magnification. We demonstrate MesoTIRF imaging of cell specimens and show the multi-wavelength capability of the modality across more than 700 cells in a single image.

Dynamics of focused ultrasound-enhanced glymphatic transportation in the mouse brain

The glymphatic system, a perivascular network in the brain, regulates the exchange of cerebrospinal fluid and interstitial fluid in the perivascular space. Focused ultrasound combined with microbubbles (FUSMB) has been recently shown feasible to manipulate the glymphatic transportation by enhancing the intranasal delivery of agents in the perivascular space. The objective of this study was to reveal the dynamics of FUSMB-enhanced agent transport in the glymphatic system. A ring-shaped FUS transducer was confocally aligned with an objective of a two-photon microscope (2PM). Fluorescently labeled albumin was administered to the mouse nose, where it transported along olfactory nerve and trigeminal nerve from nose to brain and then spread in perivascular space in the brain. FUS sonication was performed after intravenous injection of microbubbles. In vivo 2PM recorded the dynamics of agent transportation before, during, and after FUS sonication. Time-lapse recording showed that FUSMB enhanced the agent accumulation at the perivascular space. FUS significantly increased the accumulation of the agent in the perivascular space by 1.2-folds immediately after the sonication (p = 0.0325), and 1.6-folds at 5 min after the sonication (p < 0.0001). In conclusion, this study showed, for the first time, direct evidence of FUSMB-enhanced glymphatic transportation in the mouse brain.

Differentiable optimization of the Debye-Wolf integral for light shaping and adaptive optics in two-photon microscopy.

Control of light through a microscope objective with a high numerical aperture is a common requirement in applications such as optogenetics, adaptive optics, or laser processing. Light propagation, including polarization effects, can be described under these conditions using the Debye-Wolf diffraction integral. Here, we take advantage of differentiable optimization and machine learning for efficiently optimizing the Debye-Wolf integral for such applications. For light shaping we show that this optimization approach is suitable for engineering arbitrary three-dimensional point spread functions in a two-photon microscope. For differentiable model-based adaptive optics (DAO), the developed method can find aberration corrections with intrinsic image features, for example neurons labeled with genetically encoded calcium indicators, without requiring guide stars. Using computational modeling we further discuss the range of spatial frequencies and magnitudes of aberrations which can be corrected with this approach.

Strain-Relaxed Nanocrystalline Diamond Thin Films with Silicon Vacancy Centers Using Femtosecond Laser Irradiation for Photonic Applications

Nanocrystalline diamond thin (thickness of 100–500 nm) films with ensembles of negatively charged light-emitting silicon vacancy (SiV) centers might find an application in the field of photonics or biosensing due to their scalable and low-price fabrication process in comparison with monocrystalline diamond. The potential application of diamond thin films is, however, limited due to the presence of radiative and nonradiative defects. The former type is a source of an unwanted background in the photoluminescence spectra, the latter lowers the quantum efficiency of light emission, and both types of defects cause the reabsorption of SiV emission during its propagation within the layer. Here we show that femtosecond (fs) laser pulses focused via a microscope objective on a nanocrystalline diamond thin film can be employed to reduce background photoluminescence intensity by a factor of 5 with respect to the luminescence of SiV centers. Raman spectra show that this decrease is mainly caused by laser ablation of the sp2-related carbon phase present in-between the grains. The reduction of the sp2 carbon phase also leads to a local decrease in the optical absorption followed by an almost twofold increase in the SiV emission peak intensity. Moreover, the fs-irradiation also entails the release of strain inside such a layer as manifested by the observed shift of the Raman diamond peak toward the value measured in the monocrystalline diamond and by a blue-shift of the zero-phonon-line peak position of the SiV centers. Such a finding might enable the improvement of the optical quality of the nanocrystalline diamond-based photonic nanostructures or even might be employed over a larger area by defocusing the irradiation laser in order to create large-scale strain-relaxed nanocrystalline diamond thin films.

Generation of extremely high-angle Bessel beams.

We present a setup to generate tightly focused Bessel beams that is composed of a half-ball lens coupled with a relay lens. The system is simple and compact compared to conventional imaging of axicons based on microscope objectives. We experimentally demonstrate the generation of a Bessel beam with a 42° cone angle at 980nm in air with a typical beam length of 500µm and a central core radius of about 550nm. We numerically studied the effects of the misalignment of the different optical elements and the range of tilt and shift that are acceptable to obtain a regular Bessel beam.

A nanophotonic interferometer

The transmission of light through sub-wavelength apertures (zero-mode waveguides, ZMW) in metal films is well-explored. It introduces both an amplitude modulation as well as a phase shift to the oscillating electromagnetic field. We propose a nanophotonic interferometer by bringing two ZMW (∼100 nm diameter) in proximity and monitoring the distribution of transmitted light in the back-focal plane of collecting microscope objective (1.3 N.A.). We demonstrate that both an asymmetry induced by the binding of a quantum dot in one of the two ZMW, as well as an asymmetry in ZMW diameter yield qualitatively similar transmission patterns. We find that the complex pattern can be quantified through a scalar measure of asymmetry along the symmetry axis of the aperture pair. In a combined experimental and computational exploration of detectors with differing ZMW diameters, we find that the scalar asymmetry is a monotonous function of the diameter difference of the two apertures, and that the scalar asymmetry measure is higher if the sample is slightly displaced from the focal plane of the collecting microscope objective. An optimization of the detector geometry determined that the maximum response is achieved at an aperture separation that is comparable to the wavelength on the exit side of the sensor. For small separations of apertures, on the order of a quarter of the wavelength and less, the signal is strongly polarization dependent, while for larger separations, on the order of the wavelength or larger, the signal becomes essentially polarization-independent.