Integration of a high-NA light microscope in a scanning electron microscope.

We present an integrated light-electron microscope in which an inverted high-NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high-resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub-10 µm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum-compatible immersion oil. For a 40-nm-diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.

In multi electron beam systems, "Neighbours Matter".

In the Multi beam source (MBS) of our Multi Beam Scanning Electron Microscope (MBSEM), an aperture lens array (ALA) splits the emission cone of the Schottky field emitter into multiple beamlets. When the apertures in the ALA are close to each other, the ALA can introduce aberrations to these beamlets through the electrostatic interaction of neighbouring apertures with each aperture's lens field. When the apertures are arranged in a square grid pattern, the aberration causes fourfold astigmatism. The effect on the beam spot is analyzed through a combination of 3D simulations and experimental validation. To counterbalance the fourfold astigmatism, a correction scheme is proposed in which a slightly non-round profile is applied to the aperture lenses.

Principles of electron wave front modulation with two miniature electron mirrors.

We have analyzed the possibilities of wave front shaping with miniature patterned electron mirrors through the WKB approximation. Based on this, we propose a microscopy scheme that uses two miniature electron mirrors on an auxiliary optical axis that is in parallel with the microscope axis. A design for this microscopy scheme is presented for which the two axes can be spatially separated by as little as 1 mm. We first provide a mathematical relationship between the electric potential and the accumulated phase modulation of the reflected electron wave front using the WKB approximation. Next, we derive the electric field in front of the mirror, as a function of a topographic or pixel wise excited mirror pattern. With this, we can relate the effect of a mirror pattern onto the near-field phase, or far field intensity distribution and use this to provide a first optical insight into the functioning of the patterned mirror. The equations can only be applied numerically, for which we provide a description of the relevant numerical methods. Finally, these methods are applied to find mirror patterns for controlled beam diffraction efficiency, beam mode conversion, and an arbitrary phase and amplitude distribution. The successful realization of the proposed methods would enable arbitrary shaping of the wave front without electron-matter interaction, and hence we coin the term virtual phase plate for this design. The design may also enable the experimental realization of a Mach-Zehnder interferometer for electrons, as well as interaction-free measurements of radiation sensitive specimen.

Flat electron mirror.

Electron beams can be reflected by an electrode that is at a more negative potential than the cathode from which the beam is emitted. We want to design a mirror with a flat mirror electrode where the electrons are reflected at a plane very close to the electrode. The wave front of an electron can then be shaped when the mirror contains a surface topography or modulated potential. However, electron beams reflected by flat electron mirrors are usually characterized by high coefficients of chromatic and spherical aberration. When the mirror is combined with an electrostatic lens to form a tetrode mirror system, the situation deteriorates even further. This places a restrictive limit on the maximum aperture angle of the beam, and consequently also limits the attainable resolution at the image plane. We have numerically studied the dependence of these aberrations as a function of design parameters of the tetrode mirror consisting of a ground, lens, cap, and mirror electrode, and limited ourselves to only using flat electrodes with round apertures, at fixed electrode spacing. It turns out that the third order spherical aberration can be made negative. The negative third order aberration is then used to partially compensate the positive fifth order aberration. This way, a system configuration is obtained that, at 2 keV beam energy, provides a diffraction limited resolution of 7.6 nm at an image plane 25 mm from the mirror at beam semi-angles of 2.3 mrad, enabling an illumination radius of 40 µm at the mirror. The presented tetrode mirror design could spark innovative use of patterned electron mirrors as phase plates for electron microscopy in general, and for use as coherent beam splitters in Quantum Electron Microscopy in particular. An appendix presents a method to calculate the spot size of a focused beam in the presence of both third and fifth order spherical aberration coefficients, which is also applicable to Scanning (Transmission) Electron Microscopes with aberration correctors.

Pulse length, energy spread, and temporal evolution of electron pulses generated with an ultrafast beam blanker.

Crucial for the field of ultrafast electron microscopy is the creation of sub-picosecond, high brightness electron pulses. The use of a blanker to chop the beam that originates from a high brightness Schottky source may provide an attractive alternative to direct pulsed laser illumination of the source. We have recently presented the concept of a laser-triggered ultrafast beam blanker and argued that generation of 100 fs pulses could be possible [Weppelman et al., Ultramicroscopy 184, 8-17 (2017)]. However, a detailed analysis of the influence of a deflection field changing sign on sub-picoseconds time scale on the quality of the resulting electron pulses has so far been lacking. Here, we present such an analysis using time-dependent, three-dimensional numerical simulations to evaluate the time-evolution of deflection fields in and around a micrometers-scale deflector connected to a photo-conductive switch. Further particle tracing through the time-dependent fields allows us to evaluate beam quality parameters such as energy spread and temporal broadening. We show that with a shielded, "tunnel-type" design of the beam blanker limiting the spatial extent of fringe fields outside the blanker, the blanker-induced energy spread can be limited to 0.5 eV. Moreover, our results confirm that it could be possible to bring laser-triggered 100 fs focused electron pulses on the sample using a miniaturized ultrafast beam blanker. This would enable us to resolve ultrafast dynamics using focused electron pulses in an SEM or STEM.

Concept and design of a beam blanker with integrated photoconductive switch for ultrafast electron microscopy.

We present a new method to create ultrashort electron pulses by integrating a photoconductive switch with an electrostatic deflector. This paper discusses the feasibility of such a system by analytical and numerical calculations. We argue that ultrafast electron pulses can be achieved for micrometer scale dimensions of the blanker, which are feasible with MEMS-based fabrication technology. According to basic models, the design presented in this paper is capable of generating 100 fs electron pulses with spatial resolutions of less than 10 nm. Our concept for an ultrafast beam blanker (UFB) may provide an attractive alternative to perform ultrafast electron microscopy, as it does not require modification of the microscope nor realignment between DC and pulsed mode of operation. Moreover, only low laser pulse energies are required. Due to its small dimensions the UFB can be inserted in the beam line of a commercial microscope via standard entry ports for blankers or variable apertures. The use of a photoconductive switch ensures minimal jitter between laser and electron pulses.

Shot noise in electron-beam lithography and line-width measurements.

Electron-beam lithography is used extensively in nanoscience and technology for making masks for the semiconductor industry and, on a limited scale, for maskless lithography: that is, writing the patterns directly on the chip. We expect the latter application to extend in the years to come. Control of the dimensions of the written structures is essential in the semiconductor industry. For 45 nm generation, which is presently under development and should reach production at the end of the decade, the required control over the line width is between 1.5 and 5 nm, depending on the application. One of the factors of influence on the line-width control is the statistics in the number of electrons illuminating the resist. This effect gives line edge roughness, or in other words a lack of control over the local position of a resist edge. This has long been recognized and often discussed. Recently, we developed an analytic model for the line edge position variation, which we shall illustrate and expand in this paper. The model, supported by Monte Carlo simulations, demonstrates that the line-width variation is inversely proportional to the dose used for the illumination of the resist. This makes it impossible to increase the lithography throughput by developing ultrasensitive resists. For 45 nm features written with a typical resolution of 30 nm, a 30 microC/cm2 resist gives 3 nm line-width variation over line segments of 45 nm long. The line width is usually measured in an adapted critical dimension scanning electron microscope (CD-SEM). This measurement needs to be more precise than the result of the lithography step, so the requirements are typically sub-nm. Apart from all the problems to avoid systematic errors, this measurement also suffers from statistical variations, resulting from the finite number of electrons used for the measurement. In this paper we shall derive an estimate for that variation with a similar model as used for the shot noise effect in the lithography step. One of the conclusions is that for the most precise measurements of the line width it is not advisable to tune the CD-SEM for the best resolution. It is better to allow a larger probe size of the electron beam because that can be accompanied by a much larger current and thus a decrease in the noise level.

Aberration model of a multibeam scanning microscope for electron beam-induced deposition.

A multibeam electron beam-induced deposition (EBID) system is presented, which aims at the fabrication of sub-10 nm structures with EBID. This system consists of a multibeam source (MBS) module, delivering 100 virtual sources and a standard scanning electron microscope (SEM) column to image the 100 sources onto a wafer. In the proposed concept, beamlets are traveling off-axis through the projection lenses, introducing off-axis aberrations. An analytical description of the projection lens aberrations is derived and the system is optimized by tuning a field lens, which only affects the direction of the beamlets as they enter the projection system. It is found that this lens must be excited such that all beamlets go through the center of the last projection lens. This is an important design rule for the total system.

Resolution limit for electron beam-induced deposition on thick substrates.

Recently, the fabrication resolution in electron beam-induced deposition (EBID) has improved significantly. Dots with an average diameter of 1 nm have been made. These results were all obtained in transmission electron microscopes on thin samples. As one may think that such resolution can be achieved on thin samples only, it is the objective of this paper to show that this should also be possible on thick samples. For that purpose we use Monte Carlo simulations of the electron-sample interaction and determine the surface area where secondary electrons are emitted. Assuming that these electrons cause the deposition in EBID, a comparison can be made between deposition on a thin and a thick sample. The Monte Carlo code we developed will be described and applied to the deposition induced by a 200 keV primary electron beam on an ultra-thin (10 nm) and a bulk-like (1,000 nm) Cu sample. Near the point of incidence of the primary beam, the deposit size is independent of the substrate thickness, such that a 1-nm resolution should be possible to achieve on a thick substrate as well. Thicker substrates only affect the tails of the deposit distribution which contain more mass than thin substrate deposit tails.

Designs for a quantum electron microscope.

One of the astounding consequences of quantum mechanics is that it allows the detection of a target using an incident probe, with only a low probability of interaction of the probe and the target. This 'quantum weirdness' could be applied in the field of electron microscopy to generate images of beam-sensitive specimens with substantially reduced damage to the specimen. A reduction of beam-induced damage to specimens is especially of great importance if it can enable imaging of biological specimens with atomic resolution. Following a recent suggestion that interaction-free measurements are possible with electrons, we now analyze the difficulties of actually building an atomic resolution interaction-free electron microscope, or "quantum electron microscope". A quantum electron microscope would require a number of unique components not found in conventional transmission electron microscopes. These components include a coherent electron beam-splitter or two-state-coupler, and a resonator structure to allow each electron to interrogate the specimen multiple times, thus supporting high success probabilities for interaction-free detection of the specimen. Different system designs are presented here, which are based on four different choices of two-state-couplers: a thin crystal, a grating mirror, a standing light wave and an electro-dynamical pseudopotential. Challenges for the detailed electron optical design are identified as future directions for development. While it is concluded that it should be possible to build an atomic resolution quantum electron microscope, we have also identified a number of hurdles to the development of such a microscope and further theoretical investigations that will be required to enable a complete interpretation of the images produced by such a microscope.

Circulating antibodies to corneal epithelium in patients with uveitis.

We investigated the incidence of circulating corneal epithelium antibodies in patients with uveitis. A high percentage of the patients (42%) were positive, whereas only 4% of controls had these antibodies in their serum. Significantly more patients with anterior and diffuse uveitis had corneal epithelium antibodies than did those with posterior uveitis. Subdivision of anterior uveitis into HLA-B27 positive versus negative patients showed a higher incidence of the antibodies in the HLA-B27 positive group. A previous history of uveitis may play a role in the pathogenesis of peripheral corneal thinning diseases, for which an autoimmune aetiology has been suggested.

Serology in ocular toxoplasmosis.

The diagnostic value of toxoplasma serology in ocular disease was evaluated in the following groups of patients: (I) uveitis cases of various causes (n = 291); (II) consecutive posterior and panuveitis patients (n = 60); (III) patients with definite congenital and ocular toxoplasmosis (n = 8); (IV) cases of clinical ocular toxoplasmosis (n = 25); and control patients with uveitis of non-toxoplasma origin (n = 12). No relation was observed between the level of the dye test titres and the diagnosis of ocular toxoplasmosis (groups I and II). During the active stages of the disease no typical change of the titres occurred in several longitudinally studied patients with toxoplasmosis. In group III one case was discovered to be negative by the dye test despite active ocular disease; however, IgG antibodies against toxoplasma were detected by the ELISA technique. In group IV, which was investigated by the ELISA technique, 100% of the toxoplasmosis patients were positive for IgG versus 58% of the control patients. Circulating immune complexes containing IgG and toxoplasma antigen were detected in seven of 25 toxoplasmosis patients (28%) and in two of 12 control patients (16%). Our study shows that the definite diagnosis of ocular toxoplasmosis or its exclusion by serological means only is not yet feasible. The possible superiority of the ELISA test to the dye test warrants further investigation.

Genes on chromosome 14q and their role in the pathogenesis of HLA-B27 associated diseases.

Genetic factors other than HLA-B27 may play a role in the pathogenesis of ankylosing spondylitis (AS), acute anterior uveitis (AAU) and Reiter's syndrome (RS). Studies by Brewerton et al. and Kijlstra et al. showed associations between the MZ phenotype of alpha 1-antitrypsin and the Gm phenotype zafngb of IgG in patients with AAU, who developed AS. The loci for alpha 1-antitrypsin (PI) and Gm allotypes (IGH) are situated on the tip of the long arm of chromosome 14. In the present study we tried to clarify and extend the above studies. In 41 B27+ AAU patients with AS the alpha 1-antitrypsin and Gm phenotype and allotype frequencies were not statistically different from those in B27+ AS patients developing AAU and in B27+ AAU patients without AS, in B27+ AS patients without AAU, B27+ patients with Reiter's syndrome, B27+ patients with low back pain, B27- AAU patients and normal controls. It is therefore unlikely that genes on the tip of chromosome 14 play a role in the pathogenesis of B27 associated diseases. A hypothesis was formed suggesting that a bacterial-derived modifying factor may replace the position of beta 2 microglobulin in the HLA-B27 molecule resulting in an impaired cytotoxic T cell reactivity.

A double-blind, randomized study of Healon GV compared with Healon in penetrating keratoplasty.

A double-blind, parallel, randomized study was conducted to compare the efficacy of Healon GV versus Healon in penetrating keratoplasty (PK). Its objective was to investigate whether Healon GV maintains space in the open anterior chamber better than Healon does during removal of the corneal button. Excluding those patients with an anterior chamber lens implant or a known history of glaucoma, 35 patients scheduled for PK entered the study. After penetration of the cornea with the trephine, a specific amount (0.2 ml) of Healon GV or Healon was injected into the anterior chamber. The corneal button was removed and the distance (mm) between iris and cornea (epithelial side) was measured with a digital slide gauge mounted between the permanent and mobile stand of the microscope. A significant difference between the two groups was shown in the measurements of the distance between the iris and the outer side of the cornea. The mean distance for Healon GV was 1.5 +/- 0.6 mm and for Healon it was 1.1 +/- 0.4 mm (p = 0.038). The removal of the corneal button was significantly facilitated when Healon GV was used (p = 0.021). In conclusion, Healon GV was found to be superior in the ability to maintain space in the open anterior chamber.

The influence of objective lens aberrations in energy-loss spectrometry.

The conditions under which the energy resolution and collection efficiency in electron energy-loss spectrometry are limited by the spherical and chromatic aberrations of the objective lens have been studied. It is shown that, for the optimum settings of the pre-spectrometer optics, the energy resolution will be of the same order in diffraction mode as it is in magnification mode. The influence of the aberrations on the practice of energy-loss spectroscopy is discussed, and it is demonstrated that the chromatic aberration can act as a broadband filter for energy-loss electrons.

Detection of X-rays and electron energy loss events in time coincidence.

Experiments are described in which characteristic X-rays are detected in coincidence with the electron energy losses that are responsible for these X-rays. The possibility to use this technique for improving the detection limits of microanalysis is evaluated. It is concluded that, because of the occurrence of false coincidences, better than the state-of-the-art instrumentation will be required for most practical applications.

Low-energy foil aberration corrector.

A spherical and chromatic aberration corrector for electron microscopes is proposed, consisting of a thin foil sandwiched between two apertures. The electrons are retarded at the foil to almost zero energy, so that they can travel ballistically through the foil. It is shown that such a low-voltage corrector has a negative spherical aberration for not too large distances between aperture and foil, as well as a negative chromatic aberration. For various distances the third- and fifth-order spherical aberration coefficients and the first- and second-order chromatic aberration coefficients are calculated using ray tracing. Provided that the foils have sufficient electron transmission the corrector is able to correct the third-order spherical aberration and the first-order chromatic aberration of a typical low-voltage scanning electron microscope. Preliminary results show that the fifth-order spherical aberration and the second-order chromatic aberration can be kept sufficiently low.

Autoimmunity against corneal antigens. I. Isolation of a soluble 54 Kd corneal epithelium antigen.

Corneal epithelium antibodies were detected in patients with corneal melting disease and uveitis using an immunofluorescence technique with cryostat sections of corneas obtained from various species (man, guinea pig, rabbit, mouse, rat, cow, pig). No differences in results were found using these various substrates, indicating that the autoimmune response is directed against common non-species specific corneal epithelium antigens. The serum of a patient with corneal melting disease, containing a high antibody titer against corneal epithelium was used to identify and isolate one of the bovine corneal antigens. A 54,000 dalton protein was isolated, which was shown to be the major protein present in the corneal epithelium. Absorption studies with other tissues taken from human eyes showed that cornea epithelium, cornea devoid of epithelium, ciliary body and retina contained material which cross-reacted with the isolated bovine corneal epithelium antigen, whereas iris and sclera showed no detectable cross-reaction. The incidence of autoantibodies directed against this antigen was investigated in patients with corneal melting disease, corneal transplantion and in uveitis patients using an ELISA and comparing the results with those obtained with the immunofluorescence assay on rabbit cornea sections. A positive ELISA was always associated with a positive immunofluorescence test. The presence of antibodies against the 54 Kd antigen as detected by the ELISA could be confirmed by immunoblotting in 7 out of 9 positive sera tested. A large number of sera showed a positive immunofluorescence test but a negative ELISA against the 54 Kd corneal epithelium antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoimmunity against corneal antigens. II. Accessibility of the 54 kD corneal antigen for circulating antibodies.

Autoantibodies against corneal epithelium antigens are frequently found in patients with corneal diseases, but also in patients with uveitis. To investigate whether these autoantibodies play a primary role in the pathogenesis of corneal disease we studied the distribution of a 54 kD corneal antigen (isolated in a previous study) within the eye. Animal experiments were performed to determine the accessibility of this antigen by its corresponding antibody. Immunohistochemistry showed that the 54 kD corneal antigen was abundantly present in the rat corneal and conjunctival epithelium. A high concentration of this antigen was seen just above the nuclei in the basal cell layer of the corneal epithelium. The antigen could also be detected in the keratocytes of the corneal stroma, the corneal endothelium and the lens epithelium. The 54 kD corneal antigen was not detected in other parts of the eye, nor could it be found in the rat liver, kidney, spleen, lung or skeletal muscle. Incubation of intact rat eyes with antiserum against the 54 kD corneal antigen in vitro, resulted in a weak binding of immunoglobulins to the corneal surface epithelium cells. Passive transfer experiments, whereby rats received an intravenous injection of antiserum against the 54 kD corneal antigen resulted in a weak deposition of immunoglobulins in the corneal stroma and sclera. However, no antibodies were bound to the corneal epithelium. These observations show that although antibody production to corneal epithelium antigens is easily triggered, these antibodies do not reach the corneal epithelium.

Cyclosporine A treatment in four cases with corneal melting syndrome.

Four patients with progressive corneal melting were treated with CsA. In all cases of corneal melting a healing of the ulceration was observed. In one case an accompanying scleral melting responded poorly to this therapy. Hypertension as a side effect was noted in one case. It could be concluded that CsA has a beneficial effect on corneal melting but not on scleral melting.