Aperture Objective Research Articles

Optimizing focus: switchable modes and sub-diffraction spots in inverse circular Airy beams.

We report an experimental investigation into the tight-focusing characteristics of linearly polarized inverse circular Airy beams (ICABs). Our study reveals that tightly focused ICABs exhibit Bessel-like, needle-like, or dual foci profiles depending on whether the main ring's radius is smaller than, equal to, or larger than the critical radius. The emergence of the dual foci structure is attributed to the constrained entrance aperture of the microscope objective (MO). In contrast to traditional Gaussian beams (GBs), ICABs demonstrate remarkable advantages in terms of focal spot size. Notably, we observe a focal spot with a size of 245 nm, representing a 26.4% reduction compared to the diffraction limit. These unique properties open up promising avenues for potential applications in optical multi-plane particle trapping, conveying, and super-resolution optical imaging.

Full-aperture extended-depth oblique plane microscopy through dynamic remote focusing.

The reprojection setup typical of oblique plane microscopy (OPM) limits the effective aperture of the imaging system, and therefore its efficiency and resolution. Large aperture system is only possible through the use of custom specialized optics. A full-aperture OPM made with off the shelf components would both improve the performance of the method and encourage its widespread adoption. To prove the feasibility of an OPM without a conventional reprojection setup, retaining the full aperture of the primary objective employed. A deformable lens based remote focusing setup synchronized with the rolling shutter of a complementary metal-oxide semiconductor detector is used instead of a traditional reprojection system. The system was tested on microbeads, prepared slides, and zebrafish embryos. Resolution and pixel throughput were superior to conventional OPM with cropped apertures, and comparable with OPM implementations with custom made optical components. An easily reproducible approach to OPM imaging is presented, eliminating the conventional reprojection setup and exploiting the full aperture of the employed objective.

Simplification of selective imaging of dislocation loops: diffraction-selected on-zone STEM

ABSTRACT Contrary to the conventional belief that, in transmission electron microscopy (TEM), selective and sharp imaging of dislocation loops can be realized only by accurate tilting of a specimen from the condition that the symmetrical axis of incident electron beam distribution is parallel to a zone axis of the TEM specimen (on-zone condition), we demonstrate that selective dark-field (DF) imaging of dislocation loops at on-zone condition is possible with a scanning TEM (STEM) mode that uses an objective lens aperture to select a diffraction disk of interest. Diffraction-selected on-zone STEM (DsoZ-STEM) has been applied to selective DF imaging of dislocation loops with a short axis length of <2 nm in a single-crystal aluminum irradiated by argon ions and an electron beam at room temperature. It was found that a Kikuchi line enhances the contrast among the dislocation loops and the matrix of DsoZ-STEM images. DsoZ-STEM obeyed g·b invisibility criterion and showed good agreement with a typical visibility change of a dislocation line and a loop in conventional DF images with a specific pair of ± g. In addition, dislocation loops always showed much higher brightness in the inner side compared to the outer side in DsoZ-STEM images, simplifying the distinction of dislocation loops with apparently the same long-axis direction but different b. Thus, DsoZ-STEM can simplify the selective DF imaging for the determination of the number and the character of dislocation loops.

Transmission electron microscopy of thick polymer and biological specimens

We explore the properties of elastic and inelastic scattering in a thick organic specimen, together with the mechanisms that provide contrast in a transmission electron microscope (TEM) and scanning-transmission electron microscope (STEM). Experimental data recorded from amorphous carbon are used to predict the bright-field image intensity, mass-thickness contrast and dose-limited resolution as a function of thickness, objective-aperture size, and primary-electron energy E0. Combining this information with estimates of chromatic aberration, objective-aperture diffraction and beam broadening in the specimen, we calculate the achievable TEM and STEM resolution to be around 4 nm at E0 = 300 keV (or below 3 nm at MeV energies) for a 10 µm-diameter objective aperture and 1 – 2 µm thickness of hydrated biological tissue. The 3 MeV resolution for a 10-μm tissue sample is probably closer to 10 nm. We also comment on the error involved in quadrature addition of resolution factors, when one or more of the point-spread functions are non-Gaussian.

Super-resolution SRS microscopy with A-PoD.

Stimulated Raman scattering (SRS) offers the ability to image metabolic dynamics with high signal-to-noise ratio. However, its spatial resolution is limited by the numerical aperture of the imaging objective and the scattering cross-section of molecules. To achieve super-resolved SRS imaging, we developed a deconvolution algorithm, adaptive moment estimation (Adam) optimization-based pointillism deconvolution (A-PoD) and demonstrated a spatial resolution of lower than 59 nm on the membrane of a single lipid droplet (LD). We applied A-PoD to spatially correlated multiphoton fluorescence imaging and deuterium oxide (D2O)-probed SRS (DO-SRS) imaging from diverse samples to compare nanoscopic distributions of proteins and lipids in cells and subcellular organelles. We successfully differentiated newly synthesized lipids in LDs using A-PoD-coupled DO-SRS. The A-PoD-enhanced DO-SRS imaging method was also applied to reveal metabolic changes in brain samples from Drosophila on different diets. This new approach allows us to quantitatively measure the nanoscopic colocalization of biomolecules and metabolic dynamics in organelles.

Design of a Hybrid Refractive/Diffractive Lens System for Broadband UV.

Traditional broadband UV optical designs often have complex structural problems and cannot meet the current requirements of light and miniaturization. In this study, we first design the substrate material of double-layer diffractive optical elements (DOEs) in the 0.23-0.8 μm band, optimize the diffraction efficiency and analyze the effect of the angle of incidence on the diffraction efficiency of double-layer DOEs. Second, we design a refractive lens system and a refractive/diffractive hybrid lens system with double-layer DOEs designed for a wide UV wavelength range of 0.23-0.8 μm, a half field of view of 15 mm, an objective aperture of 0.1 and a magnification of 1. The refractive/diffractive hybrid lens system designed with seven lenses produces a higher image quality than the refractive lens system. The novel design is an effective solution to the problem of the low transmission rate of traditional UV refractive lens systems.

An Interactive Simulation and Visualization Tool for Conventional and Aberration-Corrected Transmission Electron Microscopy

Abstract: Contrast transfer function (CTF) is a vital function in transmission electron microscopy (TEM). It expresses to what extent amplitudes converted from the phase changes of the diffracted waves contribute to the TEM image, including the effects of lens aberrations. Simulation is very helpful to understand the application of the function thoroughly. In this work, we develop the CTFscope as a component in the Landyne software suite, to calculate the CTF with temporal and spatial dumping envelopes for conventional TEM and to extend it to various aberrations (up to fifth order) for aberration-corrected (AC)-TEM. It also includes effects on the CTF and imaging due to the objective aperture and image drift for tutorial purposes. The CTFscope has a user-friendly graphical interface for simulation, visualization, saving, and loading of the microscopy conditions and results. It can be used to explore instrumental performance information due to optical aberrations, effects on the resolution of a TEM and AC-TEM, and for teaching electron microscopy courses.

Wall Shear Stress Measurements Using A MTV-Based Plenoptic Microscope

In this contribution, we describe the first ever microscopic Molecular Tagging Velocimetry (MTV) measurements using Integral Imaging. This technique permits the measurement of the 3D-2C velocity field in an axi-symmetric stagnation jet and observed profiles agree well with analytical predictions. Standard reconstruction algorithms are compared against more advanced deconvolution algorithms and impressive improvements in reconstructed probe resolution are obtained. Integral imaging is compared with a scanning confocal microscope which serves as a basis of comparison and good agreement is achieved. The flow facility was designed for a low Reynolds number, but with an adjustable viscous length scale comparable with higher Reynolds number flows. Our instrument proves capable of resolving viscous length scales on the order of 30 micrometers which can be improved by increasing the numerical aperture of the microscope objective.

Prism-based tri-aperture laparoscopic objective for multi-view acquisition.

This paper presents the design and prototype of a novel tri-aperture monocular laparoscopic objective that can acquire both stereoscopic views for depth information and a wide field of view (FOV) for situational awareness. The stereoscopic views are simultaneously captured via a shared objective with two displaced apertures and a custom prism. Overlapping crosstalk between the stereoscopic views is diminished by incorporating a strategically placed vignetting aperture. Meanwhile, the wide FOV is captured via a central third aperture of the same objective and provides a 2D view of the surgical field 2x as large as the area imaged by the stereoscopic views. We also demonstrate how the wide FOV provides a reference data set for stereo calibration, which enables absolute depth mapping in our experimental prototype.

Adaptive coherence volume in full-field optical coherence tomography

Optical sectioning is instrumental for the observation of extended biological samples. It allows the observation of only a slice of the sample while rejecting contributions from out of focus depths. The acquisition of the whole volume then requires an axial displacement of the sample or the focus. To satisfy Nyquist sampling, this axial displacement has to be equal to half the axial resolution. As lateral and axial resolutions are coupled by the numerical aperture of the microscope objective in most imaging techniques, high-resolution imaging of a volume is a time-consuming task, especially caused by the slow axial scanning. Here, we propose to adapt the axial resolution, or axial extent of the coherence volume, by filtering the spectrum of the illumination of an interferometric imaging technique. We applied our approach on full-field optical coherence tomography and show a tuning of this axial extent from 1.5 to 15 μm, allowing to adapt both the acquisition time and the amount of data. We finally demonstrate that the method is especially suited to image large biological samples such as millimetric engineered tissues.

Exploring the hidden depth by confocal Raman experiments with variable objective aperture and magnification

The article analyzes experimentally and theoretically the influence of microscope parameters on the pinhole-assisted Raman depth profiles in uniform and composite refractive media. The main objective is the reliable mapping of deep sample regions. The easiest to interpret results are found with low magnification, low aperture, and small pinholes. Here, the intensities and shapes of the Raman signals are independent of the location of the emitter relative to the sample surface. Theoretically, the results can be well described with a simple analytical equation containing the axial depth resolution of the microscope and the position of the emitter. The lower determinable object size is limited to 2–4 μm. If sub-micrometer resolution is desired, high magnification, mostly combined with high aperture, becomes necessary. The signal intensities and shapes depend now in refractive media on the position relative to the sample surface. This aspect is investigated on a number of uniform and stacked polymer layers, 2–160 μm thick, with the best available transparency. The experimental depth profiles are numerically fitted with excellent accuracy by inserting a Gaussian excitation beam of variable waist and fill fraction through the focusing lens area, and by treating the Raman emission with geometric optics as spontaneous isotropic process through the lens and the variable pinhole, respectively. The intersectional area of these two solid angles yields the leading factor in understanding confocal (pinhole-assisted) Raman depth profiles.Graphical abstractSpearfishing is a well-known example of the effects of refraction at the boundary between two index-mismatched media. The object Greal is seen, due to refraction, as Gvir from the angle β (without knowing the depth position). The real position is obtained under the angle α. In a microscope (see inset), index mismatch deforms the image point of Greal into an image line. The pinhole substantially reduces deformations and allows the determination of the position of the point emitter G. (Cartoon designed by Sofia Anker)

Enhancing Efficiency of Multi-Objective Neural-Network-Assisted Nonlinear Dynamics Lattice Optimization via 1-D Aperture Objectives & Objective Focusing

Enhancing Efficiency of Multi-Objective Neural-Network-Assisted Nonlinear Dynamics Lattice Optimization via 1-D Aperture Objectives & Objective Focusing

Three-dimensional reconstruction of super-resolved white-light interferograms based on deep learning

White-light scanning interferometry is an effective and widely used technology for measuring the microscopic three-dimensional morphology of an object. Its vertical resolution can reach the sub-nanometer level, and its lateral resolution reaches submicron level. However, for the samples containing complex structure or high-density periodic distribution structural units, the measurement results are strongly restricted by magnification and numerical aperture (NA) of the microscopic objective. In this paper, we proposed a three-dimensional reconstruction algorithm for white-light interferograms after super resolution processing, using fast super-resolution convolutional neural networks (FSRCNN) to improve the detailed information of the interferograms, and then we used centroid method combined with the five-step phase-shift method to extract the zero optical path difference (ZOPD) position of the interference signal after super resolution processing. After processed by the proposed method, the interferograms collected by the 10X microscope objective (NA=0.3) recovered the 3D surface is the same as that measured by the 100X microscope objective (NA=0.7), which is proved by the experiment results.

Interference-based wide-range dynamic tuning of the plasmonic color of single gold nanoparticles.

Dynamic tuning of nanoscale coloration by exploiting localized surface plasmon resonance of gold nanoparticles (AuNPs) combined with an interference coloration mechanism is demonstrated experimentally. When interference between the scattering field from AuNPs and the reflected field from the substrate is observed under back-scattering white-light microscopy, the AuNPs exhibit various colors depending on their distance to the substrate. When the numerical aperture of the microscope objective is optimized, much greater coverage of the color space than was achieved with previously reported plasmon-based approaches is attained. Also, color tunability is examined by exploiting the temperature-induced volume change of a temperature-responsive hydrogel with embedded AuNPs to dynamically modify the distance to the substrate.

Multiscale modeling and analysis for high-fidelity interferometric scattering microscopy

Interferometric scattering microscopy (iSCAT), as an ultrasensitive fluorescence-free imaging modality, has recently gained enormous attention and has been rapidly developing from demonstration of principle to quantitative sensing and bioanalytics. Here we report on a theoretical framework of multiscale modeling and analysis for iSCAT with samples of arbitrary shapes under any types of illumination and detection schemes. We theoretically predict and experimentally confirm different evolution behaviors of the interference contrast as a function of the axial defocusing for dielectric and metallic nanoparticles. We provide a transparent understanding of the origin of the interference phenomenon in terms of plane wave components and explain how the interference contrast changes with the size and material of the nanoprobe and the numerical aperture of the microscope objective. Moreover, we investigate a sample system mimicking a gold nanoparticle in a simplified cell environment and show the position-dependent and asymmetric point spread function of the nanoparticle.

Focus on lasers, imaging, microscopy, and nanoscience

Focus on lasers, imaging, microscopy, and nanoscience

Fluorescence Correlation Spectroscopy Combined with Multiphoton Laser Scanning Microscopy—A Practical Guideline

Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue—being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research.

Lattice resolution of vibrational modes in the electron microscope

The combination of aberration correction and ultra high energy resolution with monochromators has made it possible to record images showing lattice resolution in phonon modes, both with a displaced collection aperture and more recently with an on –axis collection aperture. In practice the objective aperture has to include Bragg reflections that correspond to the observed lattice image spacings, and the specimen has to be sufficiently thick for adequate phonon scattered intensity. There has been controversy as to whether the images with the on axis detector are really a consequence of lattice resolution in a phonon mode or just a transfer of information from an image that was formed by elastically scattered electrons. We present results of calculations based on a theory that includes the possibility of dynamical electron diffraction for both incident and scattered electrons and the full phonon dispersion relation. We show that Umklapp scattering from the second Brillouin Zone back to the first Brillouin Zone is necessary for lattice resolution with the on axis detector and that it is therefore reasonable to attribute the lattice resolution to the phonon scattering.

Оценка регенеративного потенциала плазмы, обогащенной тромбоцитами, при уретропластике в экспериментальной модели

Reconstructive plastic surgery is the gold standard in the treatment of primary urethral strictures, but the effectiveness of these methods does not reach 100%. In cases of recurrent urethral strictures, the effectiveness of standard operations is lower than with primary strictures, which requires a search for methods to improve the results of surgical treatment. To evaluate the structure of the intercellular matrix, the cellular composition and regenerative potential of a plasma enriched in platelets after performing urethroplasty on a biological model. Experiment was carried out on male rabbits ("Burgundy" breed) weighting 3.0-4.5 kg (18 individuals). 18 animals were divided into two groups: an experimental one (contained 9 individuals) and a control one (contained 9 individuals). All animals in each group were subjected to end-to-end plastic surgery of the urethral bulbous region using standard procedures. In the control group 4 ml of 0.9% NaCl isotonic solution was injected along the perimeter of the suture into anastomotic zone of the spongy body. Autologous plasma was injected to the animals from the experimental group. Histopathological examination was made by using routine pathological assessment with hematoxylin-eosin staining. The study also assessed the distribution and orientation of collagen fibers with Van Gieson stain. In order to objectively detail inflammatory and regenerative changes an additional immunohistochemical analysis was performed for the following antibodie groups: CD79a, CD43, CD31 (PECAM1), MMP1, MMP9. Quantitative analysis of structural changes was carried out by counting B- and T-lymphocytes having a positive membrane reaction with CD79a and CD43, respectively, in 10 representative sites in view (HPF) with a lens aperture of 0.65 in the highest concentration areas ("hot spots"). The expression level of MMP1, MMP9 was estimated by counting positive cells in 10 representative sites in view with a lens aperture of 0.65 in the submucosal and muscle layers. The level of angiogenesis in micropreparations was evaluated by counting the number of vessels in 10 sites in view (objective aperture of 0.65), reliable endothelial visualization was performed using CD31 expression (PECAM1). In the analysis of histological material use of platelet-rich plasma in the suture zone helps to reduce the area of necrotic changes and the inflammation severity, accelerated migration of macrophage-histiocytic cells to the alteration site and increased blood supply due to enhanced angiogenesis. In experimental samples a higher expression of metalloproteinases (collagenases) types 1 and 9, decreased collagen production and the correct orientation of collagen fibers during repair processes were noticed. The use of platelet-rich plasma helps to accelerate the reparative processes in the spongy body after urethroplasty. Another important positive effect of platelet-rich plasma is an increase in the expression of metalloproteinases, which leads to a decrease in collagen production and the correct orientation of collagen fibers. This allows to reduce the amount of pathological fibro-scar tissue in the operation area.

Nanoscale ion implantation using focussed highly charged ions

We introduce a focussed ion beam (FIB) based ion implanter equipped with an electron beam ion source (EBIS), able to produce highly charged ions. As an example of its utilisation, we demonstrate the direct writing of nitrogen-vacancy centres in diamond using focussed, mask-less irradiation with Ar8+ ions with sub-micron three dimensional placement accuracy. The ion optical system was optimised and is characterised via secondary electron imaging. The smallest measured foci are below 200 nm, using objective aperture diameters of 5 and 10 µm, showing that nanoscale ion implantation using an EBIS is feasible.