Μm Lateral Resolution Research Articles

Volumetric HiLo microscopy employing an electrically tunable lens.

Electrically tunable lenses exhibit strong potential for fast motion-free axial scanning in a variety of microscopes. However, they also lead to a degradation of the achievable resolution because of aberrations and misalignment between illumination and detection optics that are induced by the scan itself. Additionally, the typically nonlinear relation between actuation voltage and axial displacement leads to over- or under-sampled frame acquisition in most microscopic techniques because of their static depth-of-field. To overcome these limitations, we present an Adaptive-Lens-High-and-Low-frequency (AL-HiLo) microscope that enables volumetric measurements employing an electrically tunable lens. By using speckle-patterned illumination, we ensure stability against aberrations of the electrically tunable lens. Its depth-of-field can be adjusted a-posteriori and hence enables to create flexible scans, which compensates for irregular axial measurement positions. The adaptive HiLo microscope provides an axial scanning range of 1 mm with an axial resolution of about 4 μm and sub-micron lateral resolution over the full scanning range. Proof of concept measurements at home-built specimens as well as zebrafish embryos with reporter gene-driven fluorescence in the thyroid gland are shown.

High Spatial Resolution Evaluation of Residual Stresses in Shot Peened Specimens Containing Sharp and Blunt Notches by Micro-hole Drilling, Micro-slot Cutting and Micro-X-ray Diffraction Methods

The moderately high lateral RS gradients (on the order of tens of MPa/μm) near shot peened notches in conjunction with the shallow treatment depth (some hundreds of microns) limit the application of far-field and/or high resolution synchrotron diffraction residual stress measurement techniques. Recently proposed Focused Ion Beam – Scanning Elecron Microscope – Digital Image Correlation (FIB-SEM-DIC) based micro mechanical stress relaxation methods for the measurement of residual stress at the micron scale became suitable techniques for local evaluation of residual stresses and stress gradients in shot peened specimens. In this paper ultra-high resolution (~0.5–0.8 μm depth and 5–10 μm lateral resolution) mechanical relaxation stress measurements were used to evaluate the stress variation local to individual peening dimples in ceramic (60–120 μm diameter beads) shot peened Al-7075-T651 double notched samples having 0.15, 0.5 and 2.0 mm radii using Micro-Hole Drilling (μHD), Micro-Slot Cutting (μSC) and micro X-ray Diffraction (μXRD) methods. The micron-sized sampling volumes enabled the stress to be evaluated in individual impact craters (dimples) showing significant point-to-point variation (~ +/−150 MPa) (with certain dimples even recording tensile stresses). After around 30 μm of layer removal the heavily deformed region had largely been removed and the stress profile became much more homogeneous. At this depth the μHD and μSC results were in good accord with those from μXRD measurements which sample over a much larger volume (~40 μm depth × 50 μm laterally) showing an in-plane compressive stress of around 150 MPa far from the notches with the residual stress rising to about 200 MPa at a blunt (2 mm) notch and 500 MPa for a sharp (0.15 mm) one. Further, these recorded variations of residual stresses were correlated with microstructural features, e.g. grains, networks of sub-surface cracks, intermetallics and highly deformed sub-surface regions, revealed by large volume Serial Sectioning Tomography using Plasma Xe+ Focus Ion Beam – Scanning Electron Microscope (PFIB-SEM), EDS and EBSD maps. This allowed for the first time characterize large volume (100 × 66 × 30 μm3) of shot peened regions with resolution of dozens of nanometers and correlate residual stress depth profiles with 3D microstructural features. Finally, in (Benedetti et al. 2016, Int. J. Fatigue) these RS measurements are used to reconstruct the RS field through finite element (FE) analyses.

MEMS-based handheld scanning probe with pre-shaped input signals for distortion-free images in Gabor-domain optical coherence microscopy.

High-speed scanning in optical coherence tomography (OCT) often comes with either compromises in image quality, the requirement for post-processing of the acquired images, or both. We report on distortion-free OCT volumetric imaging with a dual-axis micro-electro-mechanical system (MEMS)-based handheld imaging probe. In the context of an imaging probe with optics located between the 2D MEMS and the sample, we report in this paper on how pre-shaped open-loop input signals with tailored non-linear parts were implemented in a custom control board and, unlike the sinusoidal signals typically used for MEMS, achieved real-time distortion-free imaging without post-processing. The MEMS mirror was integrated into a compact, lightweight handheld probe. The MEMS scanner achieved a 12-fold reduction in volume and 17-fold reduction in weight over a previous dual-mirror galvanometer-based scanner. Distortion-free imaging with no post-processing with a Gabor-domain optical coherence microscope (GD-OCM) with 2 μm axial and lateral resolutions over a field of view of 1 × 1 mm2 is demonstrated experimentally through volumetric images of a regular microscopic structure, an excised human cornea, and in vivo human skin.

μOCT imaging using depth of focus extension by self-imaging wavefront division in a common-path fiber optic probe.

Optical coherence tomography (OCT) is an attractive medical modality due to its ability to acquire high-resolution, cross-sectional images inside the body using flexible, small-diameter, scanning fiber optic probes. Conventional, cross-sectional OCT imaging technologies have approximately 10-μm axial resolution and 30-μm lateral resolution, specifications that enable the visualization of microscopic architectural morphology. While this resolution is useful for many clinical applications, it is insufficient for resolving individual cells that characterize many diseases. To address this gap, a supercontinuum-laser-based, μm-resolution OCT (μOCT) system and a 500 μm-diameter, extended depth of focus single fiber optic probe for endoscopic and intravascular imaging were designed and fabricated. At the distal tip of the fiber optic probe, a cylindrical waveguide was used to divide the wavefront to provide multiple circular propagation modes. Once transmitted through a relatively high NA lens (NA >0.1), these modes were projected as multiple coaxial foci (~3 μm full width at half maximum (FWHM)) over a greatly extended focal depth range. The distal tip of the probe also contained a common-path reference reflectance to minimize polarization and dispersion imbalances between sample and reference arm light. Measurements showed that the probe provides a 20-fold depth of focus extension, maintaining a 3-5 µm lateral resolution (FWHM of PSF) and a 2 μm axial resolution over a depth range of approximately 1 mm. These results suggest that this new optical configuration will be useful for achieving high-resolution, cross-sectional OCT imaging in catheter/endoscope-based medical imaging devices.

High-Resolution Mapping of Thermal History in Polymer Nanocomposites: Gold Nanorods as Microscale Temperature Sensors.

A technique is reported for measuring and mapping the maximum internal temperature of a structural epoxy resin with high spatial resolution via the optically detected shape transformation of embedded gold nanorods (AuNRs). Spatially resolved absorption spectra of the nanocomposites are used to determine the frequencies of surface plasmon resonances. From these frequencies the AuNR aspect ratio is calculated using a new analytical approximation for the Mie-Gans scattering theory, which takes into account coincident changes in the local dielectric. Despite changes in the chemical environment, the calculated aspect ratio of the embedded nanorods is found to decrease over time to a steady-state value that depends linearly on the temperature over the range of 100-200 °C. Thus, the optical absorption can be used to determine the maximum temperature experienced at a particular location when exposure times exceed the temperature-dependent relaxation time. The usefulness of this approach is demonstrated by mapping the temperature of an internally heated structural epoxy resin with 10 μm lateral spatial resolution.

Selective area epitaxy of magnesium oxide thin films on gallium nitride surfaces

Abstract

Characterisation of Thin Films Using a Coherence Scanning Interferometry

Accurate measurement of film thickness and its uniformity are very important to the performance of many coated surfaces and for many applications are critical. Effective inspection of the film thickness and uniformity is the key to high performance. Conventionally, film thickness is meas- ured using a spectrophotometer/reflectometer, ellipsometer or a physical step measurement; however, these techniques all have limitations. Coherence Scanning Interferometry (CSI) is an established method to measure surface topography as this technique offers many advantages such as speed, ease of use and accuracy. The measurement of “thick” films (exceeding ~1.5 μm) which gives rise to clearly separate fringe bunches is a well-established CSI capability. However, a methodology known as the ‘helical complex field’ (HCF) [1] [2] function allows film thickness to be measured down to ~25 nm. This new method, combined with Coherence Correlation Interferometry (CCI) [3] offers film thickness measurements with sub-nanometre vertical resolution and ~1 μm lateral resolution. It is ideally suited for detailed analysis of coated optical surfaces. In this paper, the fundamentals of the techniques are described and some case studies are presented.

Analysis of Drosophila lipids by matrix-assisted laser desorption/ionization mass spectrometric imaging.

Drosophila melanogaster is a major model organism for numerous lipid-related diseases. While comprehensive lipidomic profiles have been generated for D. melanogaster, little information is available on the localization of individual lipid classes and species. Here, we show the use of matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) to profile lipids in D. melanogaster tissue sections. The preparation of intact cryosections from whole insects presents a challenge due to the brittle hydrophobic cuticle surrounding the body and heterogeneous tissue types beneath the cuticle. However, the introduction of a novel sucrose infiltration step and gelatin as an embedding media greatly improved the quality of tissue sections. We generated MS image profiles of six major lipid classes: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and triacylglycerides. In addition, signals corresponding to two male-specific sex pheromones were detected in the ejaculatory bulb, a specialized site of pheromone production. MSI performed with 35 μm lateral resolution provided high sensitivity detection of at least 92 different lipid species, based on exact mass. In contrast, MSI with 10 μm lateral resolution enabled the detection of 36 lipid species but allowed lipid profiling of individual organs. The ability to localize lipid classes in intact sections from whole Drosophila provides a powerful tool for characterizing the effects of diet, age, stress, and environment on lipid production and distribution.

Fully motorized optical-resolution photoacoustic microscopy.

We have developed fully motorized optical-resolution photoacoustic microscopy (OR-PAM), which integrates five complementary scanning modes and simultaneously provides a high imaging speed and a wide field of view (FOV) with 2.6 μm lateral resolution. With one-dimensional (1D) motion-mode mechanical scanning, we measured the blood flow through a cross section of a blood vessel in vivo. With two-dimensional (2D) optical scanning at a laser repetition rate of 40 kHz, we achieved a 2 kHz B-scan rate over a range of 50 μm with 20 A-lines and 50 Hz volumetric-scan rate over a FOV of 50 μm × 50 μm with 400 A-lines, which enabled real-time tracking of cellular dynamics in vivo. With synchronized 1D optical and 2D mechanical hybrid scanning, we imaged a 10 mm × 8 mm FOV within three minutes, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechanical contour scanning, we maintained the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects with uneven surfaces, which is essential for quantitative studies.

Special multicolor illumination and numerical tilt correction in volumetric digital holographic microscopy

We introduce a color imaging method in our digital holographic microscope system (DHM). This DHM can create color images of freely floating, or moving objects inside a large volume by simultaneously capturing three holograms using three different illumination wavelengths. In this DHM a new light source assembly is applied, where we use single mode fibers according to the corresponding wavelengths that are tightly and randomly arranged into a small array in a single FC/PC connector. This design has significant advantages over the earlier approaches, where all the used illuminations are coupled in the same fiber. It avoids the coupling losses and provides a cost effective, compact solution for multicolor coherent illumination. We explain how to determine and correct the different fiber end positions caused tilt aberration during the hologram reconstruction process. To demonstrate the performance of the device, color hologram reconstructions are presented that can achieve at least 1 µm lateral resolution.

Video-rate structured illumination microscopy for high-throughput imaging of large tissue areas

We report the development of a structured illumination microscopy instrument specifically designed for the requirements for high-area-throughput, optically-sectioned imaging of large, fluorescently-stained tissue specimens. The system achieves optical sectioning frame-rates of up to 33 Hz (and pixel sampling rates of up to 138.4 MHz), by combining a fast, ferroelectric spatial light modulator for pattern generation with the latest large-format, high frame-rate scientific CMOS camera technology. Using a 10X 0.45 NA objective and a 7 mm/sec scan stage, we demonstrate 4.4 cm(2)/min area-throughput rates in bright tissue-simulating phantoms, and 2 cm(2)/min area-throughput rates in thick, highly-absorbing, fluorescently-stained muscle tissue, with 1.3 μm lateral resolution. We demonstrate high-contrast, high-resolution imaging of a fluorescently-stained 30.4 cm(2) bovine muscle specimen in 15 minutes comprising 7.55 gigapixels, demonstrating the feasibility of the approach for gigapixel imaging of large tissues in short timeframes, such as would be needed for intraoperative imaging of tumor resection specimens.

Chemically assisted femtosecond laser machining for applications in LiNbO3 and LiTaO3

We introduce and optimize a fabrication procedure that employs both femtosecond laser machining and hydrofluoric acid etching for cutting holes or voids in slabs of lithium niobate and lithium tantalate. The fabricated structures have 3 μm lateral resolution, a lateral extent of at least several millimeters, and cut depths of up to 100 μm. Excellent surface quality is achieved by initially protecting the optical surface with a sacrificial silicon dioxide layer that is later removed during chemical etching. To optimize cut quality and machining speed, we explored various laser-machining parameters, including laser polarization, repetition rate, pulse duration, pulse energy, exposure time, and focusing, as well as scanning, protective coating, and etching procedures. The resulting structures significantly broaden the capabilities of terahertz polaritonics, in which lithium niobate and lithium tantalate are used for terahertz wave generation, imaging, and control. The approach should be applicable to a wide range of materials that are difficult to process by conventional methods.

Ultrahigh speed endoscopic optical coherence tomography using micromotor imaging catheter and VCSEL technology

We developed a micromotor based miniature catheter with an outer diameter of 3.2 mm for ultrahigh speed endoscopic swept source optical coherence tomography (OCT) using a vertical cavity surface-emitting laser (VCSEL) at a 1 MHz axial scan rate. The micromotor can rotate a micro-prism at several hundred frames per second with less than 5 V drive voltage to provide fast and stable scanning, which is not sensitive to the bending of the catheter. The side-viewing probe can be pulled back to acquire a three-dimensional (3D) data set covering a large area on the specimen. The VCSEL provides a high axial scan rate to support dense sampling under high frame rate operation. Using a high speed data acquisition system, in vivo 3D-OCT imaging in the rabbit GI tract and ex vivo imaging of a human colon specimen with 8 μm axial resolution, 8 μm lateral resolution and 1.2 mm depth range in tissue at a frame rate of 400 fps was demonstrated.

Reflection-mode multifocal optical-resolution photoacoustic microscopy

Compared with single-focus optical-resolution photoacoustic microscopy (OR-PAM), multifocal OR-PAM utilizes both multifocal optical illumination and an ultrasonic array transducer, significantly increasing the imaging speed. A reflection-mode multifocal OR-PAM system based on a microlens array that provides multiple foci as well as an ultrasonic array transducer that receives the excited photoacoustic waves from all foci simultaneously is presented. Using a customized microprism to reflect the incident laser beam to the microlens array, the multiple optical foci are aligned confocally with the focal zone of the ultrasonic array transducer. Experiments show the reflection-mode multifocal OR-PAM is capable of imaging microvessels in vivo, and it can image a 6×5×2.5 mm³ volume at 16 μm lateral resolution in ∼2.5 min, which was limited by the signal multiplexing ratio and laser pulse repetition rate.

On the stress characteristics of graphite anode in commercial pouch lithium-ion battery

The stress of graphite anode in commercial pouch lithium-ion battery is characterized ex-situ by using micro-Raman mapping technique at 2 μm lateral resolution. The results show that the stress in graphite anode is always compressive and its distribution is inhomogeneous both in the manufacturing process and in cycling process. The stress mainly generates and accumulates in the early stage of cycling process, which causes not only big anode thickness swelling but also graphite particles cracking or even degrading. The deterioration of electrical contact of graphite particles from current collector caused by anode volume expansion and formation of solid electrolyte interface (SEI) in fresh surface of cracked graphite particle is responsible for the faster capacity fading of pouch battery in this early stage.

Robot-assisted transvaginal peritoneoscopy using confocal endomicroscopy: a feasibility study in a porcine model

Optical biopsy methods such as probe-based confocal laser endomicroscopy (pCLE) provide useful intraoperative real-time information, especially during minimally invasive surgery with flexible endoscopic or robotic platforms. By translating the probe at constant pressure across the target tissue, undistorted "mosaics" can be produced. However, this poses ergonomic challenges with a conventional flexible endoscope. A 100 μm confocal depth pCLE probe was integrated into a previously described seven degrees-of-freedom articulated endoscopic robot. After estimating the average workspace created by a female pneumoperitoneum, the accessibility of the peritoneal cavity by the device for robot-assisted pCLE peritoneoscopy was calculated. To demonstrate its in vivo feasibility, the robot was inserted transvaginally in a pig, under laparoscopic vision. Optical biopsy was performed of several targets within the peritoneal cavity. The workspace analysis calculated that 88 % of the surface of an estimated average female pneumoperitoneum could be contacted by the probe using the robot transvaginally. In vivo, the robot was manoeuvred to provide views of all abdominal and pelvic organs. At each target there was robotic acquisition of still pCLE images, and slowly translating images for the construction of increased field-of-view mosaics up to 2 mm in length. Optical biopsies took 1-2 min per target, and at 3.5 μm lateral resolution, the mosaic images showed characteristic features of anterior abdominal wall, liver, and spleen. In the porcine model, the robotically actuated method of performing peritoneoscopy and pCLE mosaicked optical biopsy is safe and provides a consistent means of acquiring near-histological grade images of submesothelial tissue. Clinical translation is likely to provide sufficient accessibility of the peritoneal cavity.

Monolithic integration of binary-phase fresnel zone plate objectives on 2-axis scanning micromirrors for compact microscopes

We demonstrated a unique monolithic integration of Fresnel elliptical zone plate (EZP) objective on a 2-axis staggered vertical comb-drive micromirror with 500 μm by 800 μm surface area via direct patterning of reflective binary phase modulation elements on a silicon chip. The need for focusing optics is thus obviated, simplifying the micro-endoscope assembly and improving its form factor. The design of binary phase EZP was guided by simulations based on FFT based Rayleigh-Sommerfeld diffraction model. For dual-axis scanning angles up to 9º by 9º at the image plane, the simulated diffracted Airy disks on a spatial map have been demonstrated to vary from 10.5 μm to 28.6 μm. Micromirrors scanning ±9º (optical) about both axes are patterned with elliptical zones designed for 7 mm focal length and 20þ off-axis 635-nm illumination using 635 nm laser. Videos of samples acquired with ~15 μm lateral resolution over 1mm × 0.35 mm field of view (FOV) at 5.0 frames/second using the device in both transmission and reflectance modes bench-top single-fiber laser scanning confocal microscope confirmed the applicability of the device to micro-endoscopy.

Infrared Laser Ablation Atmospheric Pressure Photoionization Mass Spectrometry

In this paper we introduce laser ablation atmospheric pressure photoionization (LAAPPI), a novel atmospheric pressure ion source for mass spectrometry. In LAAPPI the analytes are ablated from water-rich solid samples or from aqueous solutions with an infrared (IR) laser running at 2.94 μm wavelength. Approximately 12 mm above the sample surface, the ablation plume is intercepted with an orthogonal hot solvent (e.g., toluene or anisole) jet, which is generated by a heated nebulizer microchip and directed toward the mass spectrometer inlet. The ablated analytes are desolvated and ionized in the gas-phase by atmospheric pressure photoionization using a 10 eV vacuum ultraviolet krypton discharge lamp. The effect of operational parameters and spray solvent on the performance of LAAPPI is studied. LAAPPI offers ~300 μm lateral resolution comparable to, e.g., matrix-assisted laser desorption ionization. In addition to polar compounds, LAAPPI efficiently ionizes neutral and nonpolar compounds. The bioanalytical application of the method is demonstrated by the direct LAAPPI analysis of rat brain tissue sections and sour orange (Citrus aurantium) leaves.

Feasibility of simultaneous surface topography and XRF mapping using Shear Force Microscopy

Marketed sources equipped with polycapillary optics allow now laboratory X–Ray Fluorescence (XRF) analysis with 5–10 µm lateral resolution. To improve it, we had the idea to use a thin cylindrical X–ray capillary fitted to the XRF detector. The combination with near–field microscopy would then lead to a simultaneous record of both topography and XRF from a sample at µm lateral resolution. For this purpose, we have built a home–made Shear Force Microscope to carry out this experiment in the future.In a first step, we have validated the microscope, operating in SNOM configuration, using test sample consisting in ZnO clusters deposited on a Si3N4 grating. Second, the feasibility of XRF collection through a thin X–ray cylindrical capillary on Co/Ti sample is shown in this work. The results suggest that sub–1 µm in–lab XRF analysis is possible, replacing the optical fibre of our SNOM apparatus by an X–ray capillary. On the basis of modelling our results, we then further discuss the possibility to reach 100 nm XRF resolution, combined to surface topography, working in synchrotron environment.

Details of 1π sr wide acceptance angle electrostatic lens for electron energy and two-dimensional angular distribution analysis combined with real space imaging

Abstract We propose a new 1π sr Wide Acceptance Angle Electrostatic Lens (WAAEL), which works as a photoemission electron microscope (PEEM), a highly sensitive display-type electron energy and two-dimensional angular distribution analyzer. It can display two-dimensional angular distributions of charged particles within the acceptance angle of ±60° that is much larger than the largest acceptance angle range so far and comparable to the display-type spherical mirror analyzer developed by Daimon et al. [1] . It has good focusing capabilities with 5-times magnification and 27(4) μm lateral-resolution. The relative energy resolution is typically from 2 to 5×10–3 depending on the diameter of energy aperture and the emission area on the sample. Although, the lateral resolution of the presented lens is far from those are available nowadays, but this is the first working model that can form images using charged particles collected from 1π sr wide acceptance angle. The realization of such lens system is one of the first possible steps towards reaching the field of imaging type atomic resolution electron microscopy Feynman et al. [2] . Here some preliminary results are shown.