Microscope Objective Research Articles

Spinning disk interferometric scattering confocal microscopy captures millisecond timescale dynamics of living cells.

Interferometric scattering (iSCAT) microscopy is a highly sensitive imaging technique that uses common-path interferometry to detect the linear scattering fields associated with samples. However, when measuring a complex sample, such as a biological cell, the superposition of the scattering signals from various sources, particularly those along the optical axis of the microscope objective, considerably complicates the data interpretation. Herein, we demonstrate high-speed, wide-field iSCAT microscopy in conjunction with confocal optical sectioning. Utilizing the multibeam scanning strategy of spinning disk confocal microscopy, our iSCAT confocal microscope acquires images at a rate of 1,000 frames per second (fps). The configurations of the spinning disk and the background correction procedures are described. The iSCAT confocal microscope is highly sensitive-individual 10 nm gold nanoparticles are successfully detected. Using high-speed iSCAT confocal imaging, we captured the rapid movements of single nanoparticles on the model membrane and single native vesicles in the living cells. Label-free iSCAT confocal imaging enables the detailed visualization of nanoscopic cell dynamics in their most native forms. This holds promise to unveil cell activities that are previously undescribed by fluorescence-based microscopy.

Module for zooming in extended depth of focus in digital holographic microscopy

Microscopes with continuous magnification and a quick zoom reaction time are much needed for imaging of biological specimen. Here, we are proposing a module consisting of two electrically tunable lenses (ETL) to facilitate the digital holographic microscopy with a continuous variable zoom/magnification within the extended depth of focus of the microscope objective. The proposed module provides a large working distance for the movement of the sample as well as the elimination of break-off while switching objectives. The major benefit is that the image plane remains stationary throughout the zooming range within the extended focal depth. The range of both ETL currents for the continuous zoom operation is discussed. Post experimental calculations, over a 3.5 mm expanded focal depth, the zoom precision is determined to be 12 µm with a response time of ∼7 ms. The zoom ratio has been found to be in the range -28.75X to -122.5X experimentally. In the expanded depth of focus, the zoom ratios at different focus planes are presented with simulation and experimental results. Moreover, the quantitative information of micro samples at deep depths with high zoom ratios without sacrificing resolution over a field of view of 1.77° is provided. The results are provided for human red blood cells and a standard USAF chart present at different depths to demonstrate the feasibility of utilizing the proposed configuration for zooming into extended focal depths of objective for optically transparent samples.

Creating stable trapping force and switchable optical torque with tunable phase of light.

Light-induced rotation of microscopic objects is of general interest as the objects may serve as micromotors. Such rotation can be driven by the angular momentum of light or recoil forces arising from special light-matter interactions. However, in the absence of intensity gradient, simultaneously controlling the position and switching the rotation direction is challenging. Here, we report stable optical trapping and switchable optical rotation of nanoparticle (NP)-assembled micromotors with programmed phase of light. We imprint customized phase gradients into a circularly polarized flat-top (i.e., no intensity gradient) laser beam to trap and assemble metal NPs into reconfigurable clusters. Modulating the phase gradients allows direction-switchable and magnitude-tunable optical torque in the same cluster under fixed laser wavelength and handedness. This work provides a valuable method to achieve reversible optical torque in micro/nanomotors, and new insights for optical trapping and manipulation using the phase gradient of a spatially extended light field.

Miniaturized structured illumination microscopy using two 3-axis MEMS micromirrors.

We present the development and performance characterisation of a novel structured illumination microscope (SIM) in which the grating pattern is generated using two optical beams controlled via 2 micro-electro-mechanical system (MEMS) three-axis scanning micromirrors. The implementation of MEMS micromirrors to accurately and repeatably control angular, radial and phase positioning delivers flexible control of the fluorescence excitation illumination, with achromatic beam delivery through the same optical path, reduced spatial footprint and cost-efficient integration being further benefits. Our SIM architecture enables the direct implementation of multi-color imaging in a compact and adaptable package. The two-dimensional SIM system approach is enabled by a pair of 2 mm aperture electrostatically actuated three-axis micromirrors having static angular tilt motion along the x- and y-axes and static piston motion along the z-axis. This allows precise angular, radial and phase positioning of two optical beams, generating a fully controllable spatial interference pattern at the focal plane by adjusting the positions of the beam in the back-aperture of a microscope objective. This MEMS-SIM system was applied to fluorescent bead samples and cell specimens, and was able to obtain a variable lateral resolution improvement between 1.3 and 1.8 times the diffraction limited resolution.

Scalable-resolution structured illumination microscopy.

Structured illumination microscopy suffers from the need of sophisticated instrumentation and precise calibration. This makes structured illumination microscopes costly and skill-dependent. We present a novel approach to realize super-resolution structured illumination microscopy using an alignment non-critical illumination system and a reconstruction algorithm that does not need illumination information. The optical system is designed to encode higher order frequency components of the specimen by projecting PSF-modulated binary patterns for illuminating the sample plane, which do not have clean Fourier peaks conventionally used in structured illumination microscopy. These patterns fold high frequency content of sample into the measurements in an obfuscated manner, which are de-obfuscated using multiple signal classification algorithm. This algorithm eliminates the need of clean peaks in illumination and the knowledge of illumination patterns, which makes instrumentation simple and flexible for use with a variety of microscope objective lenses. We present a variety of experimental results on beads and cell samples to demonstrate resolution enhancement by a factor of 2.6 to 3.4 times, which is better than the enhancement supported by the conventional linear structure illumination microscopy where the same objective lens is used for structured illumination as well as collection of light. We show that the same system can be used in SIM configuration with different collection objective lenses without any careful re-calibration or realignment, thereby supporting a range of resolutions with the same system.

Tunable critical Casimir forces counteract Casimir–Lifshitz attraction

In developing micro- and nanodevices, stiction between their parts, that is, static friction preventing surfaces in contact from moving, is a well-known problem. It is caused by the finite-temperature analogue of the quantum electrodynamical Casimir–Lifshitz forces, which are normally attractive. Repulsive Casimir–Lifshitz forces have been realized experimentally, but their reliance on specialized materials severely limits their applicability and prevents their dynamic control. Here we demonstrate that repulsive critical Casimir forces, which emerge in a critical binary liquid mixture upon approaching the critical temperature, can be used to counteract stiction due to Casimir–Lifshitz forces and actively control microscopic and nanoscopic objects with nanometre precision. Our experiment is conducted on a microscopic gold flake suspended above a flat gold-coated substrate immersed in a critical binary liquid mixture. This may stimulate the development of micro- and nanodevices by preventing stiction as well as by providing active control and precise tunability of the forces acting between their constituent parts.

An Agent-Based Traffic Recommendation System: Revisiting and Revising Urban Traffic Management Strategies

Strategic traffic management is crucial for combating traffic congestion at the macroscopic level. However, such a field is still relatively unexplored, particularly for microscopic control objects, such as intersections and coordinated intersection groups. This article proposes a human-in-the-loop recommendation system for strategic urban traffic management, which follows an agent-based structure. A regional agent dispatcher is defined to assign agents for operation whenever “operation on-demand” is required. Such a requirement is identified by a daily-dependent operational mode on strategic traffic operations at a control object level. The strategic management scheme for each control object is guided by a strategic agent (customized), which is essentially a deep recommender model with a specific architecture. By featuring the multiagent design, a customized operational scheme can be generated at the intersection level, which instructs the corresponding controller to take specific operations. The utility of the recommendation system is demonstrated via a case study using real-world traffic data. In both offline and online evaluations, the system performs consistently at traffic operational recommendations in different scenarios and has the potential to provide more reasonable traffic operational strategies than a human-operated system.

Manipulation of Microscopic Objects with Two-Lobe Light Fields

Manipulation of Microscopic Objects with Two-Lobe Light Fields

Methods and instruments for the measurement of numerical aperture for microscope objective lens: A mini review.

Numerical aperture (NA) of objective lens is an important parameter for the design of microscope systems and evaluation of imaging characteristics. The present mini review presents and summarizes the methods and instruments used in the NA measurement of objective lens. Five different categories of methods are introduced, which are original versions of apertometer measurement for angular aperture, method based on the working of Abbe apertometer and its modified versions, geometry-based methods, focal-plane (FP)-imaging-based methods, and back-FP-imaging-based methods, respectively. The methodology, devices, applied scenarios, and characteristics of methods (instruments) are summarized. Finally, some issues and potential areas of application are indicated as well.

3D-printed machines that manipulate microscopic objects using capillary forces.

Objects that deform a liquid interface are subject to capillary forces, which can be harnessed to assemble the objects1-4. Once assembled, such structures are generally static. Here we dynamically modulate these forces to move objects in programmable two-dimensional patterns. We 3D-print devices containing channels that trap floating objects using repulsive capillary forces5,6, then move these devices vertically in a water bath. Because the channel cross-sections vary with height, the trapped objects can be steered in two dimensions. The device and interface therefore constitute a simple machine that converts vertical to lateral motion. We design machines that translate, rotate and separate multiple floating objects and that do work on submerged objects through cyclic vertical motion. We combine these elementary machines to make centimetre-scale compound machines that braid micrometre-scale filaments into prescribed topologies, including non-repeating braids. Capillary machines are distinct from mechanical, optical or fluidic micromanipulators in that a meniscus links the object to the machine. Therefore, the channel shapes need only be controlled on the scale of the capillary length (a few millimetres), even when the objects are microscopic. Consequently, such machines can be built quickly and inexpensively. This approach could be used to manipulate micrometre-scale particles or to braid microwires for high-frequency electronics.

Microfluidic synthesis of Janus-structured QD-encoded magnetic microbeads for multiplex immunoassay.

Uniform and monodisperse quantum dot (QD)-encoded magnetic microbeads with Janus structure were produced in a microfluidic device via photopolymerization. UV light through a microscope objective was used to solidify the microbeads which showed sharp interfaces and excellent magnetic responses. QDs with different emission peaks (450nm for blue and 640nm for red) were mixed at different ratios to provide three spectral codes. The QD-encoded microbeads can be distinguished by analyzing their fluorescent images in HSV color space. After hydrolysis of the anhydride group in alkaline solution, protein was immobilized on microbeads via activation of carboxyl groups using (1-ethyl-3(3-dimethylaminoprophyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). A microhole array in polydimethylsiloxane (PDMS) substrates with a specific size was fabricated to trap individual microbeads in a single microhole. The combination of Janus-structured QD-encoded magnetic microbeads and microhole arrays facilitates both flexibility, binding kinetics, sensitivity for suspension assay, and fluorescence mapping analysis for conventional biochips, thus providing a novel platform for multiplex bioanalysis. The capability of this integration for multiplex immunoassays was verified using three kinds of IgG and their corresponding anti-IgG. Adetection limit of 0.07ng/mL was achieved for human IgG, indicating practical applications in various fields.

Improved two-photon photopolymerisation and optical trapping with aberration-corrected structured light

We demonstrate the effectiveness of phase only aberration corrections of structured light and their application to versatile optical trapping setups. We calculate phase corrections before (ex-situ) and after (in-situ) a high numerical aperture microscope objective using a spatial light modulator (SLM), and investigate how these corrections can be used to improve the efficiency and resolution of micro-structures fabricated through two-photon-photopolymerisation (2PP). We apply a phase retrieval algorithm to correct for distortions in a femtosecond laser that enables the fabrication of 3D structures using as many as 50 simultaneous foci. The inclusion of aberration correction in the fabrication process shows improved confinement of optically trapped particles and more efficient polymerisation while minimising intensity variations at individual foci, which potentially damage the structure during fabrication. We find that phase corrections allow for consistent voxel sizes, increased sharpness, and an expanded effective printing range when using an SLM, while also allowing for closer proximity of individual trap foci, minimising interference effects that hinder fabrication resolution.

Adaptive Autonomous Navigation of Multiple Optoelectronic Microrobots in Dynamic Environments

The optoelectronic microrobot is an advanced light-controlled micromanipulation technology which has particular promise for collecting and transporting sensitive microscopic objects such as biological cells. However, wider application of the technology is currently limited by a reliance on manual control and a lack of methods for simultaneous manipulation of multiple microrobotic actuators. In this letter, we present a computational framework for autonomous navigation of multiple optoelectronic microrobots in dynamic environments. Combining closed-loop visual-servoing, SLAM, real-time visual detection of microrobots and obstacles, dynamic path-finding and adaptive motion behaviors, this approach allows microrobots to avoid static and moving obstacles, and perform a range of tasks in real-world dynamic environments. The capabilities of the system are demonstrated through micromanipulation experiments in simulation and in real conditions using a custom built optoelectronic tweezer system.

Spatial Nonreciprocal Transmission and Optical Bistability Based on Millimeter‐Scale Suspended Metasurface

AbstractWhen light interacts with microscopic objects at the micro‐ and nano‐scale, the momentum exchange between photons and objects can be very significant. While the optical radiation pressure at the same level is usually too weak to change the mechanical state of macro‐scale objects at millimeter scale or beyond. Suspended metasurfaces with excellent mechanical and optical properties are ideal candidates for light‐matter interactions. Here, without sound insulation conditions, the spatial optical nonreciprocity based on an optomechanical system with a 2 mm × 2 mm × 110 nm Si3N4 suspended metasurface at the telecom wavelength is demonstrated, with peak wavelength shift up to 1.1 pm, which is about 4.0 times of the full width at half maximum, together with optical bistability at only about 10 W cm−2 input intensities, which is more than 4 orders of magnitude lower than traditional nonlinear optical mechanism. This work may pave new avenues for spatially nonreciprocal devices and bistable devices, and benefit the applications such as precision sensing, cavity quantum optomechanics, and all‐optical switching.

High finesse bow-tie cavity for strong atom-photon coupling in Rydberg arrays.

We report a high-finesse bow-tie cavity designed for atomic physics experiments with Rydberg atom arrays. The cavity has a finesse of 51,000 and a waist of 7.1 μm at the cesium D2 line (852 nm). With these parameters, the cavity is expected to induce strong coupling between a single atom and a single photon, corresponding to a cooperativity per traveling mode of 35 at the cavity waist. To trap and image atoms, the cavity setup utilizes two in-vacuum aspheric lenses with a numerical aperture (NA) of 0.35 and is capable of housing NA = 0.5 microscope objectives. In addition, the large atom-mirror distance (≳1.5 cm) provides good optical access and minimizes stray electric fields at the position of the atoms. This cavity setup can operate in tandem with a Rydberg array platform, creating a fully connected system for quantum simulation and computation.

Quantum gas microscope assisted with T-shape vacuum viewports.

A quantum gas microscope plays an important role in cold-atom experiments, which provides a high-resolution imaging of the spatial distributions of cold atoms. Here we design, build and calibrate an integrated microscope for quantum gases with all the optical components fixed outside the vacuum chamber. It provides large numerical aperture (NA) of 0.75, as well as good optical access from side for atom loading in cold-atom experiments due to long working distance (7 mm fused silica+6 mm vacuum) of the microscope objective. We make a special design of the vacuum viewport with a T-shape window, to suppress the window flatness distortion introduced by the metal-glass binding process, and protect the high-resolution imaging from distortions due to unflattened window. The achieved Strehl ratio is 0.9204 using scanning-near-field microscopy (SNOM) fiber coupling incoherent light as point light source.

Analysis of assembly tolerance compensation in microscope objectives with a free-form element at the aperture stop.

We analyze the feasibility of using refractive free-form phase plates at the aperture stop of microscope objectives as an alternative to active alignment to compensate for assembly tolerances. The method involves the determination of misalignment-induced aberrations at the exit pupil, and transferring them to the aperture stop while taking pupil aberrations into consideration. We demonstrate that despite being able to correct only for field-independent aberrations, this method can restore near-diffraction-limited imaging performance of passively aligned systems with practical tolerances, given that the as-designed system is highly corrected. We confirm the results via numerical simulations for two different commercial objective designs.

Development of high-aperture microscope objectives with an enlarged field of view and image recording on CCD detectors

Development of high-aperture microscope objectives with an enlarged field of view and image recording on CCD detectors

X-ray Dark-Field Imaging for Improved Contrast in Historical Handwritten Literature.

If ancient documents are too fragile to be opened, X-ray imaging can be used to recover the content non-destructively. As an extension to conventional attenuation imaging, dark-field imaging provides access to microscopic structural object information, which can be especially advantageous for materials with weak attenuation contrast, such as certain metal-free inks in paper. With cotton paper and different self-made inks based on authentic recipes, we produced test samples for attenuation and dark-field imaging at a metal-jet X-ray source. The resulting images show letters written in metal-free ink that were recovered via grating-based dark-field imaging. Without the need for synchrotron-like beam quality, these results set the ground for a mobile dark-field imaging setup that could be brought to a library for document scanning, avoiding long transport routes for valuable historic documents.

Total Variation-Based Reconstruction and Phase Retrieval for Diffraction Tomography

In optical diffraction tomography (ODT), the three-dimensional scattering potential of a microscopic object rotating around its center is recovered by a series of illuminations with coherent light. Reconstruction algorithms such as the filtered backpropagation require knowledge of the complex-valued wave at the measurement plane, whereas often only intensities, i.e., phaseless measurements, are available in practice. We propose a new reconstruction approach for ODT with unknown phase information based on three key ingredients. First, the light propagation is modeled using Born's approximation enabling us to use the Fourier diffraction theorem. Second, we stabilize the inversion of the nonuniform discrete Fourier transform via total variation regularization utilizing a primal-dual iteration, which also yields a novel numerical inversion formula for ODT with known phase. The third ingredient is a hybrid input-output scheme. We achieved convincing numerical results, which indicate that ODT with phaseless data is possible. The so-obtained 2-dimensional and 3-dimensional reconstructions are even comparable to the ones with known phase.