Wide-field Optical Microscopy Research Articles

Analysis of Amyloid Fibrillation of Two Family 1 Glycoside Hydrolases.

The formation and analysis of amyloid fibers by two β-glucosidases, BglA and BglB, belonging to the GH1 enzyme family, are reported. Both proteins have the (β/α)8 TIM-barrel fold, which is characteristic of this family and is also the most common protein structure. BglA is an octamer, whereas BglB is a monomer. Amyloid fibrillation using pH and temperature as perturbing agents was investigated using fluorescence spectroscopy as a preliminary approach and corroborated using wide-field optical microscopy, confocal microscopy, and field-emission scanning electron microscopy. These analyses showed that both enzymes fibrillate at a wide range of acidic and alkaline conditions and at several temperature conditions, particularly at acidic pH (3-4) and at temperatures between 45 and 65 °C. Circular dichroism spectroscopy corroborated the transition from an α-helix to a β-sheet secondary structure of both proteins in conditions where fibrillation was observed. Overall, our results suggest that fibrillation is a rather common phenomenon caused by protein misfolding, driven by a transition from an α-helix to a β-sheet secondary structure, that many proteins can undergo if subjected to conditions that disturb their native conformation.

Experimental study on subwavelength focusing optical field detection methods for micro-Fresnel zone plate

In this paper, the detection methods and detection systems for the subwavelength focusing optical field of the micro-Fresnel zone plate (FZP) are experimentally studied. First, a comparison is made between the micro/nanofabrication methods for micro-FZP, namely, focused ion beam (FIB) and electron beam lithography, and the results show that FIB is better suited for the amplitude-type micro-FZP fabrication. Subsequently, the experimental detection devices based on the wide-field microscopy amplification (WFMA) imaging method (indirect detection method) and scanning near-field optical microscopy (direct detection method) are, respectively, constructed for the detection of the subwavelength focusing optical field of micro-FZP. The experimental results are compared and analyzed with theoretical calculation results, indicating that the WFMA method is more suitable for the detection of micro-FZP subwavelength focusing optical field that is not sensitive to radial components. This study provides an experimental reference for the micro/nanofocusing optical field detection of micro/nano-optical components similar to micro-FZP and promotes the practical application of micro-FZP.

Optical detection of infectious SARS-CoV-2 virions by counting spikes.

Existing methods for the mass detection of viruses are limited to the registration of small amounts of a viral genome or specific protein markers. In spite of high sensitivity, the applied methods cannot distinguish between virulent viral particles and non-infectious viral particle debris. We report an approach to solve this long-standing challenge using the SARS-CoV-2 virus as an example. We show that wide-field optical microscopy with the state-of-the-art mesoscopic fluorescent labels, formed by a core-shell plasmonic nanoparticle with fluorescent dye molecules in the core-shell that are strongly coupled to the plasmonic nanoparticle, not only rapidly, i.e. in less than 20 minutes after sampling, detects SARS-CoV-2 virions directly in a patient sample without a pre-concentration step, but can also distinguish between infectious and non-infectious virus strains by counting the spikes on the lipid envelope of individual viral particles.

Optical projection tomography of fluorescent microscopic specimens using lateral translation of tube lens.

Optical projection tomography (OPT) is a three-dimensional (3D) fluorescence imaging technique, in which projection images are acquired for varying orientations of a sample using a large depth of field. OPT is typically applied to a millimeter-sized specimen, because the rotation of a microscopic specimen is challenging and not compatible with live cell imaging. In this Letter, we demonstrate fluorescence optical tomography of a microscopic specimen by laterally translating the tube lens of a wide-field optical microscope, which allows for high-resolution OPT without rotating the sample. The cost is the reduction of the field of view to about halfway along the direction of the tube lens translation. Using bovine pulmonary artery endothelial cells and 0.1 µm beads, we compare the 3D imaging performance of the proposed method with that of the conventional objective-focus scan method.

Single-spot spiral phase plate for tracking 3D particle motion.

We have imaged and tracked submicron fluorescent particles in three dimensions by inserting a spiral phase plate (SPP) of our design into a conventional wide-field optical microscope. Our novel SPP consists of concentric annular zones that each impose a unique quantized orbital angular momentum (OAM) on the ring of fluorescent light it intercepts. Each ring of the SPP consists of a vortex surface that produces one additional unit of OAM than the smaller ring just inside it, with the seven successive rings each contributing n = 1 to 7 quantum units of OAM respectively. The light rings interfere to produce a single-spot rotating point spread function (SS-RPSF). This image ‘spot’ rotates with the axial depth of a particle, permitting 3D mapping of the particle position. The SPP is easily incorporated into existing microscopes. We used the DIC slider here for the SPP. 3D positions of fluorescent beads were retrieved using template matching. Bead positions could be inferred over a 6 µm depth range. Best results were obtained over a depth range of 2.3 μm, with a mean absolute error of 20 nm in this range. As proof-of-concept for live-cell imaging we tracked DNA loci and determined their diffusion coefficients in the 3D environment of the nucleus of U2OS cells. We are pursuing chromatin tracking experiments to better understand how DNA damage affects chromatin motions and, reciprocally, how chromatin dynamics impacts the DNA damage response. Beyond this specific application, our SPP-based imaging approach should enable researchers to track dynamic fluorescent objects in samples with high temporal resolution because x, y, and z are determined at exactly the same time.

Spectroscopic photoacoustic/ultrasound/optical-microscopic multimodal intrarectal endoscopy for detection of centimeter-scale deep lesions.

The inadequacy of existing colorectal imaging tools has significantly obstructed the efficient detection of colorectal cancer. To address this issue, this work presents the cross-scale endoscopic imaging of rectal tumors with a combined photoacoustic/ultrasound tomography system and wide-field optical microscopy. This multimodal system combines the merits of centimeter-scale deep penetration, multi-spectral imaging, cross-scale imaging ability, low system cost, and 360° view in a single modality. Results indicated that the proposed system could reliably depict the location of the cancer invasion depth spectroscopically with indocyanine green The tumor angiogenesis can be well identified in the wide-field optical imaging mode, which helps to localize the tumors and guide the following photoacoustic/ultrasound scan. This work may facilitate the accurate characterization of colorectal cancer and promote the clinical translation of photoacoustic-based colorectal endoscopy.

Quantitative phase imaging with single-path phase-shifting digital holography using a light-emitting diode

We propose a quantitative phase imaging technique with single-path phase-shifting digital holography using a light-emitting diode (LED). A reference wave is generated from an object wave in the Fourier plane using a single-path interferometer, based on self-reference digital holography. The object wave interferes with the reference wave, and the quantitative phase information of the object wave is recorded as a digital hologram. Quantitative phase images of objects are obtained by applying a phase-shifting interferometry technique. All the light diffracted from the objects can be utilized to generate a digital hologram. Its validity is experimentally demonstrated by constructing an optical system with a wide-field optical microscope.

Widefield quantum microscopy with nitrogen-vacancy centers in diamond: Strengths, limitations, and prospects

A dense layer of nitrogen-vacancy (NV) centers near the surface of a diamond can be interrogated in a widefield optical microscope to produce spatially resolved maps of local quantities such as magnetic field, electric field, and lattice strain, providing potentially valuable information about a sample or device placed in proximity. Since the first experimental realization of such a widefield NV microscope in 2010, the technology has seen rapid development and demonstration of applications in various areas across condensed matter physics, geoscience, and biology. This Perspective analyzes the strengths and shortcomings of widefield NV microscopy in order to identify the most promising applications and guide future development. We begin with a brief review of quantum sensing with ensembles of NV centers and the experimental implementation of widefield NV microscopy. We then compare this technology to alternative microscopy techniques commonly employed to probe magnetic materials and charge flow distributions. Current limitations in spatial resolution, measurement accuracy, magnetic sensitivity, operating conditions, and ease of use are discussed. Finally, we identify the technological advances that solve the aforementioned limitations and argue that their implementation would result in a practical, accessible, high-throughput widefield NV microscope.

BALM: Watching the Formation of Tethered Bilayer Lipid Membranes with Submicron Lateral Resolution.

Tethered bilayer lipid membranes (tBLMs) are artificial membranes largely used for the in situ study of biological membranes and membrane-associated proteins. To date, the formation of these membranes was essentially monitored by surface averaging techniques like surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D), which cannot provide both local and real-time information in a single approach. Here, we report an original application of backside absorbing layer microscopy (BALM), a novel white-light wide-field optical microscopy, to study tBLMs. Thanks to the combination of sensitivity and resolution, BALM not only allowed the real-time quantitative monitoring of tBLM formation but also enabled the high-resolution visualization of local fluxes and matter exchanges taking place at each step of the process. Quantitative BALM measurements of the final layer thickness, reproduced in parallel with SPR, were consistent with the achievement of a continuous lipid bilayer. This finding was confirmed by BALM imaging, which additionally revealed the heterogeneity of the bilayer during its formation. While established real-time techniques, like SPR or QCM-D, view the surface as homogeneous, BALM showed the presence of surface patterns appearing in the first step of the tBLM formation process and governing subsequent matter adsorption or desorption steps. Finally, matter fluxes persisting even after rinsing at the end of the tBLM formation demonstrated the lasting presence of dispersed vesicular pockets with laterally fluctuating positions over the final single and continuous lipid bilayer. These new mechanistic insights into the tBLM formation process demonstrate the great potential of BALM in the study of complex biological systems.

Far-field optical imaging of surface plasmons with a subdiffraction limited separation.

When an ultrathin silver nanowire with a diameter less than 100 nm is placed on a photonic band gap structure, surface plasmons can be excited and propagate along two side-walls of the silver nanowire. Although the diameter of the silver nanowire is far below the diffraction limit, two bright lines can be clearly observed at the image plane by a standard wide-field optical microscope. Simulations suggest that the two bright lines in the far-field are caused by the unique phase distribution of plasmons on the two side-walls of the silver nanowire. Combining with the sensing ability of surface plasmons to its environment, the configuration reported in this work is capable of functioning as a sensing platform to monitor environmental changes in the near-field region of this ultrathin nanowire.

Sequential Colocalization of ERa, PR, and AR Hormone Receptors Using Confocal Microscopy Enables New Insights into Normal Breast and Prostate Tissue and Cancers.

Simple SummaryAt present, platforms for multiplex immunohistochemistry (e.g., Opal) identify markers in distinct cell populations within a tissue section using multispectral fluorescence and optic microscopy. However, the optic resolution is not enough to colocalize markers at the subcellular level in the main epithelial or cancer population. We use confocal microscopy in multiplex detection of nuclear hormone receptors since they are an important part of the diagnosis and treatment of breast and prostate cancer. Moreover, we increased the quantitative dynamic range and resolution through increasing the signal/noise ration through reducing autofluorescence and increased longer antibody incubations. ColNu mIHCF identified distinct patterns of nuclear receptor colocalization in breast cancers. Furthermore, in prostate cancer all cancer epithelium was positive for ERa at the plasma membrane; and in normal prostate a small ERa+/p63+/AR− basal population suggest stem cell commitment to differentiation. ColNu mIHCF could be used for improving diagnosis and treatment in cancer.Multiplex immunohistochemistry (mIHC) use markers staining different cell populations applying widefield optical microscopy. Resolution is low not resolving subcellular co-localization. We sought to colocalize markers at subcellular level with antibodies validated for clinical diagnosis, including the single secondary antibody (combination of anti-rabbit/mouse-antibodies) used for diagnostic IHC with any primary antibody, and confocal microscopy. We explore colocalization in the nucleus (ColNu) of nuclear hormone receptors (ERa, PR, and AR) along with the baseline marker p63 in paired samples of breast and prostate tissues. We established ColNu mIHCF as a reliable technique easily implemented in a hospital setting. In ERa+ breast cancer, we identified different colocalization patterns (nuclear or cytoplasmatic) with PR and AR on the luminal epithelium. A triple-negative breast-cancer case expressed membrane-only ERa. A PR-only case was double positive PR/p63. In normal prostate, we identified an ERa+/p63+/AR-negative distinct population. All prostate cancer cases characteristically expressed ERa on the apical membrane of the AR+ epithelium. We confirmed this using ERa IHC and needle-core biopsies. ColNu mIHCF is feasible and already revealed a new marker for prostate cancer and identified sub-patterns in breast cancer. It could be useful for pathology as well as for functional studies in normal prostate and breast tissues.

Optical imaging of antiferromagnetic domains in ultrathin CoO(001) films

Antiferromagnetic (AFM) domains in ultrathin CoO(001) films are imaged by a wide-field optical microscopy using magneto-optical birefringence effect. The magnetic origin of observed optical contrast is confirmed by the spin orientation manipulation through exchange coupling in Fe/CoO(001) bilayer. The finite size effect of ordering temperature for ultrathin single crystal CoO film is revealed by the thickness and temperature dependent measurement of birefringence contrast. The magneto-optical birefringence effect is found to strongly depend on the photon energy of incident light, and a surprising large polarization rotation angle up to 168.5 mdeg is obtained from a 4.6 nm CoO film with a blue light source, making it possible to further investigate the evolution of AFM domains in AFM ultrathin film under external field.

Diagnostics and Characterization of Photonic Circuits by Wide-Field Spatiotemporal Imaging

The growing diffusion of integrated photonic technologies requires fast and non-invasive quality control techniques for mass-production. We present a general diagnostic technique for sub-ps imaging of photonic circuits combining wide-field optical microscopy and optical gating. The simultaneous access to multiple parameters of a photonic structure enables an unprecedented characterization of its functional design as opposed to typical single-domain techniques such as frequency or time domain reflectometry and near-field microscopy. The non-contact and non-perturbative nature of the technique makes it relevant for both planar and three-dimensional circuits, as well as for silicon, polymeric or hybrid platforms. We apply our technique to different photonic chip components fabricated by Direct Laser Writing, revealing the spatial and temporal hallmarks of fabrication imperfections causing losses or deviations from the intended device behavior. At the same time, the technique allows in situ probing of key properties of photonic devices as the local propagation constants of guided modes or the quality factor of resonant elements. Our method is relevant for both the scientific and the industrial communities as it lends itself to be scaled up to in-line quality control thanks to its non-scanning nature.

Improving the space-bandwidth product of structured illumination microscopy using a transillumination configuration

Applying structured instead of plane illumination in widefield optical fluorescence microscopy can improve the spatial resolution beyond what is known as the Abbe limit. In general it is not only the resolution of an imaging system that is of interest but also its field of view (FOV). These two parameters are expressed in the space-bandwidth product (SBP). Here we introduce a modified structured illumination microscopy (SIM) approach that offers a larger SBP than any other available implementation. This is achieved through a transillumination geometry instead of the typical epifluorescent configuration. Compared to conventional SIM, the illumination path is decoupled from the objective lens by using a multi-mirror setup to generate the sinusoidal interference pattern for structured illumination in transmission mode. The spatial frequency of the illumination pattern can be controlled by changing the angle of the mirrors, achieving comparably fine patterns over a large FOV. In this work simulation results demonstrate the potential resolution improvement to be expected by the suggested implementation. Preliminary experimental results demonstrate phase-shifting ability and the stability of fringe frequencies over a large FOV of (16 mm2) at different numerical apertures, fulfilling the prerequisites for SIM acquisition.

Imaging antiferromagnetic domains in nickel oxide thin films by optical birefringence effect

Recent demonstrations of electrical detection and manipulation of antiferromagnets (AFMs) have opened new opportunities towards robust and ultrafast spintronics devices. However, it is difficult to establish the connection between the spin-transport behavior and the microscopic AFM domain states due to the lack of the real-time AFM domain imaging technique under the electric field. Here we report a significant Voigt rotation up to 60 mdeg in thin NiO(001) films at room temperature. Such large Voigt rotation allows us to directly observe AFM domains in thin-film NiO by utilizing a wide-field optical microscope. Further complementary XMLD-PEEM measurement confirms that the Voigt contrast originates from the NiO AFM order. We examine the domain pattern evolution at a wide range of temperature and with the application of external magnetic field. Comparing to large-scale-facility techniques such as the X-ray photoemission electron microscopy, the use with a wide-field, tabletop optical imaging method enables straightforward access to domain configurations of single-layer AFMs.

3D measurements of micro-objects based on monocular wide-field optical microscopy with extended depth of field.

Monocular rotational microscopes suffer from shallow depth of field and reduced field of view. Consequently, we propose monocular wide-field optical microscopy with extended depth of field to enable accurate 3D measurements of micro-objects. We use a liquid lens and a 4f system to extend the system's depth of field and two additional rotational reflectors to capture multi-view image sequences. In addition, we increase the number of expansion directions in the patch-based multi-view stereo algorithm and optimize the expansion radius. Our approach outperforms that based on the patch-based multi-view stereo method and achieves a high relative measurement accuracy of 1.9%. RESEARCH HIGHLIGHTS: This study proposes 3D measurements of micro-objects via monocular rotational microscopy. The depth of field is extended by the use of a liquid lens and a 4f system. Our approach outperforms that based on the patch-based multi-view stereo method and achieves a high relative measurement accuracy of 1.9%.

Subnanometer localization accuracy in widefield optical microscopy

The common assumption that precision is the limit of accuracy in localization microscopy and the typical absence of comprehensive calibration of optical microscopes lead to a widespread issue—overconfidence in measurement results with nanoscale statistical uncertainties that can be invalid due to microscale systematic errors. In this article, we report a comprehensive solution to this underappreciated problem. We develop arrays of subresolution apertures into the first reference materials that enable localization errors approaching the atomic scale across a submillimeter field. We present novel methods for calibrating our microscope system using aperture arrays and develop aberration corrections that reach the precision limit of our reference materials. We correct and register localization data from multiple colors and test different sources of light emission with equal accuracy, indicating the general applicability of our reference materials and calibration methods. In a first application of our new measurement capability, we introduce the concept of critical-dimension localization microscopy, facilitating tests of nanofabrication processes and quality control of aperture arrays. In a second application, we apply these stable reference materials to answer open questions about the apparent instability of fluorescent nanoparticles that commonly serve as fiducial markers. Our study establishes a foundation for subnanometer localization accuracy in widefield optical microscopy.

Graphene-based near-field optical microscopy: high-resolution imaging using reconfigurable gratings.

High-resolution and fast-paced optical microscopy is a requirement for current trends in biotechnology and materials industry. The most reliable and adaptable technique so far to obtain higher resolution than conventional microscopy is near-field scanning optical microscopy (NSOM), which suffers from a slow-paced nature. Stemming from the principles of diffraction imaging, we present fast-paced graphene-based scanning-free wide-field optical microscopy that provides image resolution that competes with NSOM. Instead of spatial scanning of a sharp tip, we utilize the active reconfigurable nature of graphene's surface conductivity to vary the diffraction properties of a planar digitized atomically thin graphene sheet placed in the near field of an object. Scattered light through various realizations of gratings is collected at the far-field distance and postprocessed using a transmission function of surface gratings developed on the principles of rigorous coupled wave analysis. We demonstrate image resolutions of the order of λ0/16 using computational measurements through binary graphene gratings and numerical postprocessing. We also present an optimization scheme based on the genetic algorithm to predesign the unit cell structure of the gratings to minimize the complexity of postprocessing methods. We present and compare the imaging performance and noise tolerance of both grating types. While the results presented in this article are at terahertz frequencies (λ0=10 μm), where graphene is highly plasmonic, the proposed microscopy principle can be readily extended to any frequency regime subject to the availability of tunable materials.

Meta-nanocavity model for dynamic super-resolution fluorescent imaging based on the plasmonic structure illumination microscopy method.

Biological research requires dynamic and wide-field optical microscopy with resolution down to nanometer to study the biological process in a sub-cell or single molecular level. To address this issue, we propose a dynamic wide-field optical nanoimaging method based on a meta-nanocavity platform (MNCP) model which can be incorporated in micro/nano-fluidic systems so that the samples to be observed can be confined in a nano-scale space for the ease of imaging. It is found that this platform can support standing wave surface plasmons (SW-SPs) interference pattern with a period of 105 nm for a 532 nm incident wavelength. Furthermore, the potential application of the NCP for wide-field super-resolution imaging was discussed and the simulation results show that an imaging resolution of sub-80 nm can be achieved.

Relocation is the key to successful correlative fluorescence and scanning electron microscopy.

In this chapter the authors report on an automated hardware and software solution enabling swift correlative sample array mapping of fluorescently stained molecules within cells and tissues across length scales. Samples are first observed utilizing wide-field optical and fluorescence microscopy, followed by scanning electron microscopy, using calibration points on a dedicated sample-relocation holder. We investigated HeLa cells in vitro, fluorescently labeled for monosialoganglioside one (GM-1), across both imaging platforms within tens of minutes of initial sample preparation. This resulted in a high-throughput and high spatially resolved correlative fluorescence and electron microscopy analysis and allowed us to collect complementary nanoscopic information on the molecular and structural composition of two differently distinct HeLa cell populations expressing different levels of GM-1. Furthermore, using the small zebrafish animal model Danio rerio, we showed the versatility and relocation accuracy of the sample-relocation holder to locate fluo-tagged macromolecular complexes within large volumes using long ribbons of serial tissue sections. The subsequent electron microscopy imaging of the tissue arrays of interest enabled the generation of correlated information on the fine distribution of albumin within hepatic and kidney tissue. Our approach underpins the merits that an automated sample-relocation holder solution brings in support of results-driven research, where relevant biological questions can be answered, and high-throughput data can be generated in a rigorous statistical manner.