Application Of Optical Microscopy Research Articles

Direct Observation of Different One- and Two-Photon Fluorescent States in a Pyrrolo[3,2-b]pyrrole Fluorophore.

Two-photon fluorophores are frequently employed to obtain superior spatial resolution in optical microscopy applications. To guide the rational design of these molecules, a detailed understanding of their excited-state deactivation pathways after two-photon excitation is beneficial, especially to assess the often-assumed presumption that the one- and two-photon excited-state dynamics are similar after excitation. Here, we showcase the breakdown of this assumption for one- and two-photon excitation of a centrosymmetric pyrrolo[3,2-b]pyrrole chromophore by combining time-resolved fluorescence and broadband femtosecond transient absorption spectroscopy. Compared to one-photon excitation, where radiative decay dominates the photodynamics, two-photon excitation leads to dynamics arising from increased nonradiative decay pathways. These different photodynamics are manifest to different quantum yields, thus highlighting the types of time-resolved studies described here to be valuable guideposts in the design of two-photon fluorophores for imaging applications.

Surface plasmon coupled nano-probe for near field scanning optical microscopy.

Near-field scanning optical microscopy (NSOM) is a powerful tool for study of the nanoscale information of objects by measuring their near-field electric field distributions. The near-field probe, which determines NSOM system performance, can be either a scattering-type or an aperture-type. Both types have strengths and weaknesses. Here we propose and study a surface plasmon-coupled type nano-probe, which works as a hybrid scheme and could potentially combine the advantages of the two NSOM probe types. The key element of the proposed probe is a nanoparticle-on-film structure designed on a tapered fiber tip. On the one hand, the probe can yield the signals scattered in the near field by a nanoparticle with a scattering mechanism; on the other hand, the scattered signals can be transmitted by the metal film and coupled into the fiber via surface plasmon coupled emission, thus providing a collection mode similar to an aperture-type NSOM. This will lead to signal enhancement, while greatly suppressing background noise. This surface plasmon-coupled nano-probe thus has great potential for near-field optical microscopy applications.

The first combined archaeological and archaeometric analyses on Bronze Age pottery from Kyrgyzstan (Uch Kurbu site)

The integration of the Bronze Age populations in Kyrgyzstan into the Andronovo sphere is largely based on the resemblance of the ceramic material discovered at the Kyrgyz sites with the pottery from various Andronovo sites, which has been explained by human migrations. However, very few detailed pottery studies have been conducted, and no archaeometric analyses have been applied to date to the material from Kyrgyzstan. We present a first investigation on Bronze Age pottery from Uch Kurbu (Kyrgyzstan) through a combined archaeological (field-based stylistic and macroscopic examinations) and archaeometric (the application of X-ray fluorescence, X-ray diffraction and thin-section Optical Microscopy) approaches. Compositional analyses on nine samples demonstrate an unexpected diversity in their provenance and a continuity in the main technological processes of pottery manufacture: raw material supply and processing to get coarse, low calcareous pastes, modelling using handmade methods and firing at low temperature in rather basic conditions. The study also highlights the combination of local peculiarities and extra-regional connections in style and technological processes compared to the pottery assemblages from other Andronovo sites. The whole integrated approach brings new information on the Bronze Age pottery production in Kyrgyzstan and contributes to the investigation of the social interactions as driving force for the diffusion of styles and techniques over central Eurasia in the Bronze Age.

Photonic-chip based free space beam shaping and steering for advanced optical microscopy application

Photonic-chip-based light illumination has recently found applications in optical microscopy and nanoscopy methodologies. The photonic chip removes the dependency on imaging objective lenses to generate the required illumination patterns for different microscopy methods. Until now, all the reported chip-based optical microscopy methods exploit the evanescent field present on top of a waveguide surface and are thus inherently limited to two-dimensional microscopy. Here, we perform systematic simulation studies to investigate different chip-based waveguide designs for static and dynamic shaping of light beams in the free-space. The simulation studies have been carefully designed considering the photo-lithography limitations and wavelength spectrum (405 nm to 660 nm) that is of interest in fluorescence based optical microscopy and nanoscopy. We first report the generation of a quasi-Bessel beam (QBB) using an on-chip axicon made at the end facet of a planar waveguide to mimic light sheet illumination. This is extended to the implementation of a counter propagating QBB for lattice light-sheet applications. The double axicon, a derivative of the axicon generates superimposed Bessel beams (SBB). Its waveguide-based implementation is proposed and analyzed. Finally, we investigate an optical phased array (OPA) approach to allow dynamic steering of the output light in the free-space. The aim of this study is to find suitable waveguide design parameters for free-space beam shaping operating in the visible spectrum opening possibilities for three-dimensional chip-based optical microscopy.

Controlling the Transverse Magneto-Optical Kerr Effect in Cr/NiFe Bilayer Thin Films by Changing the Thicknesses of the Cr Layer.

Here, we demonstrate the impact of ferromagnetic layer coating on controlling the magneto-optical response. We found that the transverse magneto-optical Kerr effect (TMOKE) signal and TMOKE hysteresis loops of Ni80Fe20 thin layers coated with a Cr layer show a strong dependence on the thickness of the Cr layer and the incidence angle of the light. The transmission and reflection spectra were measured over a range of incidence angles and with different wavelengths so as to determine the layers’ optical parameters and to explain the TMOKE behavior. The generalized magneto-optical and ellipsometry (GMOE) model based on modified Abeles characteristic matrices was used to examine the agreement between the experimental and theoretical results. A comprehensive theoretical and experimental analysis reveals the possibility to create a TMOKE suppression/enhancement coating at specific controllable incidence angles. This has potential for applications in optical microscopy and sensors.

On the application of digital optical microscopy in the study of materials structure and deformation

Digital optical microscopy (DOM) has revolutionised many aspects of industrial inspection and metrology through its ability to acquire and manipulate accurate images of samples using the power of computers. We report the application of HokusAI®, a suite of techniques for digital optical microscopy developed at the Hierarchically Structured Materials (HSM) lab at the Skolkovo Institute of Science and Technology (Skoltech), to elasto-plastic deformation and fracture mechanics analysis of samples of stainless steel AISI 304. Focus Stacking Optical Microscopy (FSOM) is combined with image stitching and Digital Image Correlation (DIC) to collect exhaustive and precise description of sample geometry, surface roughness, fracture surface morphology, and the evolution of deformation during in situ tensile testing. The implications of these advances for the analysis of deformation and fracture of advanced engineering materials are discussed.

Simultaneous Two-Angle Axial Ratiometry for Fast Live and Long-Term Three-Dimensional Super-Resolution Fluorescence Imaging.

The application of optical microscopy in four-dimensional (spatial and temporal) super-resolution imaging poses challenges because of the requirement of a long acquisition time or high illumination intensity. In this paper, we introduce simultaneous two-angle axial ratiometry (STARII) for <20 nm axial super-resolution imaging and for fast and long-term imaging of live cells up to hundreds of frames per second. This method involves recording two raw images in two incident angle channels in the context of evanescent wave illumination and obtaining the corresponding intensity ratio. Furthermore, we demonstrate the combination of STARII with the lateral super-resolution method to resolve three-dimensional nanoscale structures of microtubules and to visualize the long-term dynamical plasma membrane curvature and fast remodeling of endoplasmic reticulum tubule meshwork and three-way junctions. These demonstrations indicate an important potential application of STARII in investigating nanoscale cellular complex processes in the native state.

Background free imaging in stimulated emission fluorescence microscopy

Recently, the pump-probe technique has found highly valued developments and applications in optical microscopy to study both labeled (fluorescence) and non-labeled (vibrational) for observation of cells and tissues. In this work, we use the mechanism of stimulated emission (SE) with dual frequency modulation to remove the large background signal that is originated from spontaneous emission. We have applied a high performance digital signal processing based lock-in detection for stimulated emission fluorescence microscopy. Critically, the pump beam is modulated at f1 and the probe beam is at a different frequency, f2, with the demodulation carried out at the sum of the two frequencies, (f1 + f2). In this way, the DC background is effectively removed. Note that the background is mainly attributed to the spontaneous emission of the pump beam, with modulated frequency f1.

Broadband integrated metalens for creating super-oscillation 3D hollow spot by independent control of azimuthally and radially polarized waves

Three-dimensional hollow spots have attracted significant interest for a wide variety of optical applications including optical trapping, simulated emission depletion microscopy, and optical tweezers. In this paper, an integrated superoscillatory metalens is proposed based on all-dielectric metasurfaces for independent control of azimuthally and radially polarized waves for broadband operation. The reported metalens integrates the functions of two independent lenses on single substrate, whose phase profiles are overlapped on the same plane and are optimized for independent modulation of azimuthal and radial components in vector cylindrically polarized waves. The independent manipulation of the two orthogonal polarizations allows for significantly reducing the transverse size of the 3D hollow spot. Numerical simulation shows the formation of a 3D hollow spot with inner sizes of 0.33 λ and 1.32 λ in the transverse and axial directions respectively. More importantly, the broadband performance of the proposed superoscillatory lens was also demonstrated with a bandwidth of 40 nm (from 612.8 nm to 652.8 nm). Such a superoscillatory lens is promising for applications of optical trapping, optical tweezers, and super-resolution microscopy.

Mid-Infrared Spectral Compression of Soliton Pulse in an Adiabatically Suspended Silicon Waveguide Taper

Spectral compression (SPC) can be used for generating narrow bandwidth and wavelength-tunable light sources, which have important applications in optical communication system, spectroscopy, and nonlinear microscopy. In this paper, we numerically demonstrate the high-degree SPC of the chirp-free femtosecond pulse at wavelength 2.4 μm in a 6-cm long adiabatically suspended silicon waveguide taper. The silicon waveguide taper is designed with a dispersion-increasing profile along the propagation distance z . Simulation results show that the SPC factor can be up to 10.9, along with the brightness-enhanced factor of 8.0 and negligible sidelobe. The impacts of the higher order dispersion, higher order nonlinearity, losses (including linear and nonlinear loss), and variation of Kerr nonlinear coefficient along z on the SPC are also investigated. It is found that variation of Kerr nonlinear coefficient γ ( z ) and linear loss are the dominant perturbation to the degradation of the SPC performance.

Subsynaptic Domains in Super-Resolution Microscopy: The Treachery of Images.

The application of super-resolution optical microscopy to investigating synaptic structures has revealed a highly heterogeneous and variable intra-synaptic organization. Dense subsynaptic protein assemblies named subsynaptic domains or SSDs have been proposed as structural units that regulate the efficacy of neuronal transmission. However, an in-depth characterization of SSDs has been hampered by technical limitations of super-resolution microscopy of synapses, namely the stochasticity of the signals during the imaging procedures and the variability of the synaptic structures. Here, we synthetize the available evidence for the existence of SSDs at central synapses, as well as the possible functional relevance of SSDs. In particular, we discuss the possible regulation of co-transmission at mixed inhibitory synapses as a consequence of the subsynaptic distribution of glycine receptors (GlyRs) and GABAA receptors (GABAARs).LAY ABSTRACTSuper-resolution imaging strategies bypass the resolution limit of conventional optical microscopy and have given new insights into the distribution of proteins at synapses in the central nervous system. Neurotransmitter receptors and scaffold proteins appear to occupy specialized locations within synapses that we refer to as subsynaptic domains or SSDs. Interestingly, these SSDs are highly dynamic and their formation seems to be related to the remodeling of synapses during synaptic plasticity. It was also shown that SSDs of pre-and post-synaptic proteins are aligned in so-called nanocolumns, highlighting the role of SSDs in the regulation of synaptic transmission. Despite recent advances, however, the detection of SSDs with super-resolution microscopy remains difficult due to the inherent technical limitations of these approaches that are discussed in this review article.

Aberration-insensitive microscopy using optical field-correlation imaging

The possibility to reduce the effect of optical aberrations has been proposed in several publications on classical ghost imaging. The two-armed ghost-imaging systems make use of spatially incoherent illumination and point-by-point scanned intensity-correlation measurements in the arms. In this work, we introduce a novel ghostlike imaging method that uses a Mach-Zehnder interferometer and is based on optical-field interference instead of intensity correlations. The method allows us to obtain sharp images of microscopic objects even in the presence of severe aberrations that completely destroy the intensity-based image. Furthermore, pure phase objects can be imaged with micrometer-scale resolution in the presence of strong aberrations, which has not been demonstrated previously with a correlation-based imaging technique. In the setup, we use a light-emitting diode source and an ordinary camera as the only light detector. The imaging approach that we put forward in this work may find significant applications in advanced optical microscopy, optical coherence tomography, and a variety of interferometric sensors and detectors.

Dynamic response of large tilt-angle flexoelectro-optic liquid crystal modulators.

We present here the first time-resolved tilt-angle and retardance measurements for large-tilt (>45°) flexoelectro-optic liquid crystal modulators. These devices have potential for next generation fast switching (>1 kHz), 0-2π analog phase spatial light modulators (SLMs), with applications in optical beamsteering, microscopy and micromachining. The chiral nematic device used consisted of a mixture of CBC7CB and the chiral dopant R5011 in a nominally 5 µm-thick cell, aligned in the uniform lying helix mode. As the device is dynamically switched over angles of ± 54°, retardance changes of up to 0.17λ are observed. Furthermore, the time-resolved measurements reveal an asymmetry in the tilt in the optic-axis depending on the polarity of the applied electric field. The change in the optic-axis exhibits a pattern dependence, whereby it is determined by both the pulse history and the applied field. This pattern dependence results in tilt-angle errors of up to 8.8°, which could manifest as phase errors as large as 35.2° in potential SLMs. These time domain measurements may allow correction of these deterministic errors, to realize practical devices.

A Comparison between Anodizing and EBSD Techniques for Primary Particle Size Measurement

Proper understanding and knowledge of primary particle or grain size is of paramount importance in manufacturing processes as it directly affects various properties including mechanical behavior. Application of optical microscopy coupled with etching techniques has been used conventionally and in conjunction with color metallography (polarized microscopy) has been the preferred method for grain size measurement. An advanced technique as an alternative to light microscopy is using electron backscatter diffraction (EBSD). A comparison is made between these two techniques using Al-7Si alloy produced with various casting techniques to highlight the cost and time of the sample preparation and analysis for both techniques. Results showed that color metallography is certainly a faster technique with great accuracy and a much cheaper alternative in comparison with EBSD.

Huygens' dipole for polarization-controlled nanoscale light routing

Structured illumination allows for satisfying the first Kerker condition of in-phase perpendicular electric and magnetic dipole moments in any isotropic scatterer that supports electric and magnetic dipolar resonances. The induced Huygens' dipole may be utilized for unidirectional coupling to waveguide modes that propagate transverse to the excitation beam. We study two configurations of a Huygens' dipole -- longitudinal electric and transverse magnetic dipole moments or vice versa. We experimentally show that only the radially polarized emission of the first and azimuthally polarized emission of the second configuration are directional in the far-field. This polarization selectivity implies that directional excitation of either TM or TE waveguide modes is possible. Applying this concept to a single nanoantenna excited with structured light, we are able to experimentally achieve scattering directivities of around 23 dB and 18 dB in TM and TE modes, respectively. This strong directivity paves the way for tunable polarization-controlled nanoscale light routing and applications in optical metrology, localization microscopy and on-chip optical devices.

Of microscopes and microbes; novel applications of optical microscopy to microbiology

The development of optical microscopy has been traditionally driven by the needs of eukaryotic cell biology. Therefore, there are a number of unmet requirements for the application of advanced microscopy techniques to the field of microbiology. Conventional techniques, such as widefield epi-fluorescence and confocal laser scanning microscopy, are common-place in most laboratories, however these methods have trade-offs in terms of their attainable resolution and limited imaging volume. Here we present the application of several optical microscopy methods with the aim addressing the unmet needs of the field; increasing spatial resolution and sampling volume. We demonstrate the use of Interference Reflection Microscopy (IRM) for investigating the morphology and gliding motility of Myxococcus xanthus. This label-free technique provides super-resolution in the axial plane where changes in cell shape on the order of 100 nm can be detected in live cells. Using IRM we show novel insights into the gliding behaviour of these bacteria. We also present the application of the Mesolens to microbiology. The Mesolens is a large optical microscope with the unique combination of a low magnification and a high numerical aperture which results in an imaging volume &gt;100 mm 3 with isotropic sub-cellular resolution. We demonstrate the use of the Mesolens to image live bacterial communities at multiple spatial scales simultaneously and offer new insights for bacterial community dynamics and biofilm architecture. Our work details novel applications of advanced microscopy to the field, and in doing so fills the technology gap which has previously restricted the study of complex microbial behaviours.

Effective iterative method for accurate amplitude modulation in complex optical field generation

We experimentally demonstrate an iterative method for a vectorial optical field generator (VOF-Gen) to achieve accurate amplitude modulation in creating arbitrary complex beams. The method could converge rapidly in several steps and is effective to optimize the patterns applied to the liquid crystal spatial light modulators on a pixel-by-pixel basis to obtain arbitrary desired amplitude distribution. Meanwhile, this method could also mitigate the speckles caused by the defects of the optical components used in the VOF-Gen system and calibrate the wavefront related to the amplitude distribution. Several kinds of optical fields with different intensity distributions are successfully generated to verify the capability and versatility of the presented technique. This effective method may find many important applications in optical tweezers, microscopy, and unidirectional coupling.

Label-Free Assessment of Premalignant Gastric Lesions Using Multimodal Nonlinear Optical Microscopy

In this paper, a nonlinear optical microscopy employing two-photon excited fluorescence and second-harmonic generation was used for the detection of premalignant gastric lesions. It was found that gland morphology and collagen structure in mucosa will change with the progression of gastric diseases from normal to intestinal metaplasia, to low-grade intraepithelial neoplasia, and to high-grade intraepithelial neoplasia, and this microscopy was able to directly distinguish these warning symptoms. Furthermore, two features were quantified from nonlinear optical images to demonstrate the changes of gland size and collagen content during the development process of preneoplastic lesions. These results clearly show that nonlinear optical microscopy can effectively differentiate normal and precancerous gastric tissues without contrast agents, which would be helpful for early diagnosis and treatment of gastric diseases. This study may provide the groundwork for further application of nonlinear optical microscopy in clinical practice.

超分辨光学显微的成像原理及应用进展

超分辨光学显微的成像原理及应用进展

Phase and intensity analysis of non-diffracting beams via digital holography

This work presents the intensity and phase analysis of the special non-diffracting beams via digital holography. The experimental realizations of non-diffracting beams (Bessel, Mathieu, Airy and superposition of co-propagating Bessel beams) are made through computer-generated holograms (CGHs) reproduced in spatial light modulators (SLMs). And, the intensity and phase analysis of these non-diffracting beams by digital holography (DH). A holographic setup that combines the experimental generation process of the non-diffracting beams, via CGHs and SLMs, and the analysis through digital holography is proposed. The results are in agreement with the theoretical predictions, where measurements of intensity and phase were made in some particular cases of Bessel, Mathieu, Airy and special beams generated by the superposition of co-propagating Bessel beams. The understanding of the phase and intensity properties of non-diffracting beams is very important because of their potential applications in optical micromanipulation, optical microscopy and optics communications; and this technique are presenting excellent prospects for analysis of non-diffracting beams and structured light.