High-resolution Objective Lenses Research Articles

High-resolution 3D fluorescent imaging of intact tissues.

Histological analysis of fluorescently labeled tissues has been a critical tool to understand molecular organization in situ. However, assessing molecular structures within large cells and in the context of human organ anatomy has been challenging because it requires penetration of staining reagents and light deep into opaque tissues, while also conforming to the spatial constraints of high-resolution objective lenses. This methodology article describes optimized sample preparation for sub-micron resolution 3D imaging in human and rodent tissues, yielding imaging depth (>100 μm) and resolution (<0.012 μm3 voxel size) that has previously been limited to whole-mount in vitro organoid systems, embryos, and small model organisms. Confocal images of adult human and rodent organs, including heart, kidney, and liver, were generated for several chemical and antibody stains in cleared tissue sections >100 μm thick. This method can be readily adopted by any lab performing routine histology and takes 3 days from the start of tissue preparation to 3D images.

High numerical aperture (NA = 0.92) objective lens for imaging and addressing of cold atoms.

We have designed, built, and characterized a high-resolution objective lens that is compatible with an ultrahigh vacuum environment. The lens system exploits the principle of the Weierstrass sphere solid immersion lens to reach a numerical aperture (NA) of 0.92. Tailored to the requirements of optical lattice experiments, the objective lens features a relatively long working distance of 150 μm. Our two-lens design is remarkably insensitive to mechanical tolerances in spite of the large NA. Additionally, we demonstrate the application of a tapered optical fiber tip, as used in scanning near-field optical microscopy, to measure the point spread function (PSF) of a high NA optical system. From the PSF, we infer the wavefront aberration for the entire field of view of about 75 μm. Pushing the NA of an optical system to its ultimate limit enables novel applications in quantum technologies such as quantum control of atoms in optical microtraps with an unprecedented spatial resolution and photon collection efficiency.

A Four-Well Dish for High-Resolution Longitudinal Imaging of the Tail and Posterior Trunk of Larval Zebrafish.

We describe the design, fabrication, and applications of a four-well dish for imaging of the trunk of larval zebrafish. The dish facilitates immobilization of anesthetized zebrafish larvae, with their tails gently pushed against a microscope cover glass, enabling longitudinal imaging at 24-72 h postfertilization using high-resolution objective lenses.

Development of TEM techniques dedicated for characterization of energy related composites and its application

In this paper, recent progress in the improvement of TEM techniques based on a 40-120 kV high resolution analytical TEM and some application to the catalysts are discussed. The spherical aberration coefficient and TEM image resolution of high resolution objective lens at the accelerating voltage of 120 kV are 1.1 mm and 0.2 nm respectively. The high resolution objective lens pole-piece has been newly designed to accommodate a high solid angle silicon drift detector of an energy dispersive X-ray analyzer. A FIB fabricated apertures with the smallest diameter of 1m are equipped as the field limiting aperture for structural analysis of individual nanometer sized crystalline. The external view of the microscope

A versatile high resolution objective for imaging quantum gases

We present a high resolution objective lens made entirely from catalog singlets that has a numerical aperture of 0.36. It corrects for aberrations introduced by a glass window and has a long working distance of 35 mm, making it suitable for imaging objects within a vacuum system. This offers simple high resolution imaging for many in the quantum gas community. The objective achieves a resolution of 1.3 μm at the design wavelength of 780 nm, and a diffraction-limited field of view of 360 μm when imaging through a 5 mm thick window. Images of a resolution target and a pinhole show quantitative agreement with the simulated lens performance. The objective is suitable for diffraction-limited monochromatic imaging on the D2 line of all the alkalis by changing only the aperture diameter, retaining numerical apertures above 0.32. The design corrects for window thicknesses of up to 15 mm if the singlet spacings are modified.

Development of a specimen heating holder with an evaporator and gas injector and its application for catalyst

A specimen heating holder equipped with a gas injector and an evaporator has been developed for use with conventional transmission electron microscopes (TEMs). The developed specimen holder allows both synthesis of metal oxide support and deposition of catalyst nano-particles in situ. Since the holder is designed to be used in small gapped high-resolution objective lens pole-piece, all the procedure from the synthesis of support material to the deposition of catalyst as well as the behavior of the catalyst nano-particles on the support can be observed at near atomic resolution. The developed specimen holder was applied to the study of AuPd catalyst. First, air was injected onto heated aluminum particles via a gas injector to synthesize Al(2)O(3) support. Then, nano-particles of AuPd were deposited on the Al(2)O(3) support. After the deposition, the synthesized Al(2)O(3) support was heated and air was injected again to observe behaviors of the deposited AuPd nano-particles at elevated temperatures in the aerial environment. Behaviors of the AuPd nano-particles such as coalescence, segmentation and diffusion to the Al(2)O(3) support were dynamically observed at atomic level high resolution.

Aberration-Corrected STEM: the Present and the Future

Surprising as it may seem, aberration correction for the scanning transmission electron microscope (STEM) is now a practical proposition. The first-ever commercial spherical aberration corrector for a STEM was delivered by Nion to IBM Research Center in June 2000, and other deliveries have taken place since or are imminent. At the same time, the development of corrector hardware and software is still proceeding at full speed, and our understanding of what are the most important factors for the successful operation of a corrector is deepening continuously.Fig. 1 shows two high-angle dark field (HADF) images of [110] Si obtained with the IBM VG HB501 STEM operating at 120 kV, about 2 weeks after we fitted a quadrupole-octupole corrector into it. Fig. 1(a) shows the best HADF image that could be obtained with the corrector's quadrupoles on but its octupoles off. Sample structures were captured down to about 2.5 Å detail, easily possible in a STEM with a high resolution objective lens with a spherical aberration coefficient (Cs) of 1.3 mm. Fig. 1(b) shows a HADF image obtained after the Cs-correcting octupoles were turned on and the corrector tuned up. The resolution has now improved to 1.36 Å. This is sufficient to resolve the correct separation of the closely-spaced Si columns.

Evolution and ecology: The pace of life

We consider the influence of physics on the development of the microscope, using the unusual approach of working backwards in time from the present day to the earliest times. Modern microscopes use techniques including stimulated emission depletion microscopy and near-field scanning optical microscopy that relies on the properties of evanescent waves. These instruments allow optical instruments greatly to exceed conventional concepts of resolution, and they rely on computers to drive the system. High-resolution objective lenses, meanwhile, are now being made at very high cost using aspheric lenses. These are not used by microscopists, but are essential for the systems of photollithography used in the printing of microcircuits on computer chips. Apochromatic objectives resulted from the theoretical physics elaborated by Ernst Abbe in the nineteenth century, yet before the development of the color-corrected lens, simple (i.e., single-lens) microscopes were capable of resolving surprisingly fine details. Microscopes made by Bancks of London were used by Darwin, Brown, Bentham, and Hooker and—with careful adjustment—can reveal remarkably fine detail that seems to defy the constraints of the theoretician. Simple microscopes were used by Leeuwenhoek in the Netherlands in the seventeenth century, and here we will see that the design—widely described as a “Leeuwenhoek microscope”—was actually a concept of the London philosopher, Robert Hooke. The chapter will conclude at the earliest days of the microscope, throwing new light on the earliest microscopic descriptions in the literature of science. We learn how the theoretical constraints of physics can be challenged by the use of personal preference and experience.

Practical Correction of Three Fold Astigmatism in the Philips CM TEM

With the improvement of the optical properties of the modern TEM objective lenses the point resolution is pushed beyond 0.2 nm. The objective lens of the CM300 UltraTwin combines a Cs of 0. 65 mm with a Cc of 1.4 mm. At 300 kV this results in a point resolution of 0.17 nm. Together with a high-brightness field-emission gun with an energy spread of 0.8 eV the information limit is pushed down to 0.1 nm. The rotationally symmetric part of the phase contrast transfer function (pctf), whose first zero at Scherzer focus determines the point resolution, is mainly determined by the Cs and defocus. Apart from the rotationally symmetric part there is also the non-rotationally symmetric part of the pctf. Here the main contributors are not only two-fold astigmatism and beam tilt but also three-fold astigmatism. The two-fold astigmatism together with the beam tilt can be corrected in a straight-forward way using the coma-free alignment and the objective stigmator. However, this only works well when the coefficient of three-fold astigmatism is negligible compared to the other aberration coefficients. Unfortunately this is not generally the case with the modern high-resolution objective lenses. Measurements done at a CM300 SuperTwin FEG showed a three fold-astigmatism of 1100 nm which is consistent with measurements done by others. A three-fold astigmatism of 1000 nm already sinificantly influences the image at a spatial frequency corresponding to 0.2 nm which is even above the point resolution of the objective lens. In principle it is possible to correct for the three-fold astigmatism a posteriori when through-focus series are taken or when off-axis holography is employed. This is, however not possible for single images. The only possibility is then to correct for the three-fold astigmatism in the microscope by the addition of a hexapole corrector near the objective lens.

Electron-optical design of a high-resolution objective lens for low-voltage Scanning Electron Microscope of biological applications

By providing higher image contrast and reduced charging artifacts, the low voltage scanning electron microscope (LVSEM) is a valuable tool for surface characterization, of particular importance on nonconductive material such as biological specimens. Several SEM designs optimized for use at low voltage have been proposed.Recently, we have designed a new high resolution LVSEM using a field emission gun. The key problem is to decrease both the spherical and chromatic aberration coefficients by using a magnetic lens of small bore diameters(5mm and 10mm) and a narrow gap (7.5mm) (FIG. 1). In our first design, the magnetic lens is built around the side-entry stage of a Philips 300kV TEM and performs as well as that in the present Hitachi SEM H-900 or H-900S. Its simple design has been chosen for reliability and flexibility in farbrication.

Design and development of an ultrahigh vacuum high-resolution transmission electron microscope

A 200 kV ultrahigh vacuum high-resolution transmission electron microscope (UHV-HRTEM) for in-situ surface observation has been developed. With use of metal sealings, the vacuum at the specimen position was 2.6×10 −7 Pa without any cooling traps, and less than 1.5×10 −7 Pa with the built-in cooling traps cooled by LN 2. A cantilever-type goniometer, a specimen holder and high-resolution objective lens were newly developed for high-resolution transmission (HRTEM) and reflection (HRREM) to observe surfaces with a point-to-point resolution of 0.21 nm. The specimen tilt is up to ±20°. Crucible-type and direct-heating-type specimen holders were constructed for in-situ experiments on thin films and small particles, and on bulk crystalline surfaces, respectively. The usefulness of the microscope for surface studies is shown by images of clean surfaces of Au and Si prepared in the microscope.

The twin lens system - A high resolution objective lens at 300 kV

Point resolution of better than 0.2 nm is today achievable in medium voltage electron microscopes. Crucial for this achievement is the objective lens design which has to provide sufficiently low spherical and chromatic aberration figures. In addition to the lens itself, its integration into the overall microscope optics has to ensure a reproducible and accurate alignment.Cleaver has shown that symmetrical lenses have lower aberrations than their asymmetrical counterparts by a factor of 1.5 or 2. Theoretical optimization of this lens at 300 kV can be carried out using a first-order finite element program, as developed by Munro. The symmetrical single-field condenser objective with saturated pole-piece tips produces particularly good values with Cs = 1.0 mm and Cc = 1.4 mm at 300 kV. In this lens the specimen is placed in the centre of the magnetic field.