We have modified a CTEM in order to perform Lorentz microscopy experiments at high magnification and resolution on magnetic materials. The modification consists of the adaptation of a second side entry goniometer (SEG) to the CTEM (JEOL 200CX STEM) column above the objective lens, in a region of the column where the measured residual magnetic fields are < 0.5 Gauss with the objective lens in a fully excited state (see Figure 1).

With the specimen positioned above the objective lens, it is necessary to increase the excitation of the lens by approximately 20% in order to bring the image into focus. This combination of a specimen positioned above the object pole piece, and the higher objective lens excitation, affects several changes in the optics. First, if a specimen is positioned within the pole piece gap and the objective lens is turned off for Lorentz microscopy purposes, the highest typical magnification achievable is approximately 1,000 X and the resolution is not much better than 0.1μm.

A major reason for performing microanalysis in the Field-Emission Gun Scanning Transmission Electron Microscope (FEG-STEM) is the very high spatial resolution of the information obtained. It has been estimated that in ideal cases, energy-dispersive x-ray analysis in such an instrument can provide 0.1 wt.% detection sensitivity for an element in a region about 1.5nm in diameter of a foil about 30nm thick. It is obvious that such performance requires that the instrument be properly adjusted, and hence that the probe-forming characteristics be fully understood.

There are three fundamental limits on the minimum size of an electron probe, these being i) the geometrical demagnification of the source, ii) diffraction at the beam-limiting aperture, and iii) spherical aberration in the probe-forming lens. In addition, misalignment of the beam-limiting aperture will result in probe aberrations.

Preliminary results were obtained from thick sections of biological specimens using 200 keV electron microscopes with an in-column “Omega” energy filter. Images of selectively stained specimens prepared by osmium impregnation of frog spinal ganglia, with thickness ranging from 0.5 μm to 2.5 μm, show drastic improvement in image quality with the use of the energy filter.

Two instruments with Omega filters were used. One was a non-commercial research prototype at JEOL Ltd. equipped with a LaB6 gun and the other was a commercial instrument (JEM-2010FEF) with a field-emission gun, installed at Kyushu University. We found that, although the field-emission gun offered a brightness 2 to 3 orders of magnitude higher, the LaB6 gun was more suitable for studying thick specimens, primarily due to the higher beam current of the LaB6 gun.

In the field of biological and materials sciences, the importance of energy filter transmission electron microscope(EF-TEM) is increasing. Because it is a powerful instrument for contrast enhancement and obtaining elemental mapping images. We have developed a 200kV EF-TEM equipped with a fieldemission gun and in-column spectrometer. The new EF-TEM JEM-2010FEF inherits the performance in high resolution imaging and analysis from field emission TEM. The outer view is shown in Fig.l.

Figure 2 shows the lens configuration of JEM-2010FEF. An in-column Q-type spectrometer is introduced within the imaging lens system. It was designed to have image distortion less than 1% and dispersion power 1.2p.m/eV for 200keV electrons. There is no need of compensating procedure of distortion. Imaging lens system consists of two objective lenses, three intermediate lenses and three projector lenses. The 8-stage imaging lens system enables wide range of imaging modes equal to conventional TEMs; energy spectroscopic image of magnification from ×200 to × 1,500,000, energy spectroscopic diffraction of camera length from 200mm to 2,000mm.

A new system for energy-filtered electron microscopy (EFTEM) has been developed, adapted for the study of life science specimens. Special attention has been given to integration, ease of use and the typical problems encountered while investigating unstained and cryo-specimens. Low-dose, cryocapabilities and a high-contrast objective lens have been combined with an imaging energy filter, digital image recording with phosphor scintillator and advanced image-processing capabilities. The CM120-BioFilter is an integration of the CM120-BioTWIN or the CM120TWIN with the Gatan postcolumn imaging filter GIF100. The BioTWIN objective lens (Cs=6.2 mm, f=5.9 mm) is optimized for high-contrast imaging. The TWIN lens(Cs=2mm,f=2.8mm) is optimized for high resolution in structural biology. The GIF 100 is a computer-controlled, second-order corrected energy filter with a cooled multiscan CCD for digital image recording. Improvement of the contrast can be achieved if inelastically scattered electrons are removed.

In order to reduce beam damage, biological TEM specimens are often observed at temperatures close to the boiling point of liquid nitrogen (77 K). Recently, encouraging results on single particles as well as on 2D crystals have appeared, derived from images taken near liquid helium temperature (4 K), in dedicated TEMs. At these temperatures the high resolution frequencies are much better preserved, increasing the allowable dose and thus the signal to noise ratio.4 Here we present the design of a new dedicated Philips He-TEM which combines the full functionality of a CM300 TWIN with a vacuum transfer system and a liquid helium cooled specimen holder.

A schematic overview of the Cryo-TEM is shown in figure 1. The key differences compared to a standard CM microscope are: 1) The tip of the specimen rod is cooled below 10 K and the rod itself cannot be taken out of the goniometer (CompuStage). 2) The specimen enters the column on the opposite side.

Three-dimensional electron microscopic imaging, or electron tomography, involves the reconstruction of individual objects from projection data collected over a large range of specimen tilts. Practical limitations of tomography are set by the range of specimen tilt available to generate projection data. In terms of tilt range, current data sets typically cover a range of up to +/-700° of tilt.

When used for applications requiring high tilt such as tomography, problems with specimen holder designs which use circular 3mm diameter grids include restricted field of view created by the standard mesh pattern of commercially available specimen grids. The standard square or hexagonally patterned support grids provide minimal area between grid bars to view a sample at high tilt. The width of the specimen clamping device and holder tip blade also limit the useable grid area to the center of the specimen when the holder is at high tilt positions.

For microstructure analysis of magnetic materials, in particular, for a new class of Nd-Fe-B hard magnets, a number of magnetic imaging techniques can be applied to gain different microstructure information. For instance, SEM magnetic imaging can be successfully used for very thick samples from a bulk ones down to a sample thickness of 10-20 μ. Below this limit the resolution of SEM magnetic images generally is too low to visualize fine details of magnetic structure. For very thin samples (less then 0.5 μm) magnetic Lorentz microscopy methods in conventional TEM are very useful tools to reveal magnetic domains and their structure. Therefore, it seems that relatively thick films of 0.5-20 μm in thickness are left out of the experimental limits of the methods discussed.

We report on a novel, indirect TEM method that allows us to extend the limits of magnetic imaging for TEM non-transparent magnetic samples by viewing the surface stray fields and fringing fields generated by magnetic domains at specimen edge.

Semiconductor production using 8-inches wafers seems to be widely carried out in semiconductor factories. Many FE-SEMs are coming to be used for their particle/defect inspection. As semiconductor processing turns precise, the need of a high resolution FE-SEM has been rising all the more.

Now, JEOL announces a high resolution FE-SEM (8"FE-SEM) for 8-inches wafer inspection, which takes over the strong point of the semi-in-lens field emission scanning electron microscope (FE-SEM) JSM-6340F which has been getting expensive evaluation with its high resolution performance. The 8"FE-SEM possesses a resolution of 3.0nm at lkV of its acceleration voltage and 1.2nm at 15kV, as its semi-in-lens type object lens makes it possible to minimize the aberration.

High resolution observation and inclined wafer observation can be selected properly by changing the wafer holder, as such a holder exchange system has been usually used for transmission electron microscopes.

At the University of Melbourne we have been pursuing an ongoing program of investigation into the recovery of phase information from intensity measurements [1-9]. In this paper we consider the implications of this work in optical and electron microscopy.

Many objects of interest to biologists are phase objects which means that light is slowed and refracted in the object but not absorbed. Techniques such as phase-contrast microscopy and Nomarski differential interference contrast (DIC) microscopy are traditionally used to render the phase structure visible but do not directly map the phase distribution and are unable to produce quantitative data. These techniques also entangle the phase and amplitude information and so have limitations where both phase and amplitude information is required. In this paper, we demonstrate quantitative non-interferometric recovery of microscopic phase structure using incoherent illumination of the type commonly found in optical microscopes, and show that our results correlate with structure observed using Nomarski DIC techniques.

A confocal, all-reflecting, infinity corrected microscope was developed to improve infrared microspectrometer (IMS) systems. This system consists of a Fourier transform spectrometer, an infinity corrected all-reflecting microscope, a viewing system for visible light microscopy, a single remote confocal mask for aperturing in either a transmission or reflection mode and an infrared detector. The radiant energy passes through a confocal aperture mask as it travels from the source to the sample and through the same confocal aperture mask as it travels from the sample to the radiant energy detector. A single confocal mask insures that the sample defining mask's size, geometrical shape and orientation are always matched. Mask alignment is assured and simplified since only one adjustment is needed. An aperture beam-splitter is used for reflection measurements. This system simplifies the operation of the microspectrometer, permits the use of standard commercial infinity corrected microscope optical components and reduces the cost of IMS accessory microscopes.

Isotopic measurements via Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) have generally not been considered as practical because of the low duty cycle at which ToF-SIMS instruments operate and the corresponding low data rate. The recent discovery of pre-solar material in meteorites has shown that large variations in isotopic ratios (several orders of magnitude for some elements) exist in small (∼1 μm), refractory meteoritic grains. These grains are ideal candidates for ToF-SIMS, which consumes little sample material, compared to dynamic, magneticsector SIMS. ToF-SIMS also allows for parallel detection of all species present in the sample; thus, multiple isotopic systems can be studied in one measurement. As a prerequisite to studying the isotopic composition of meteoritic materials, preliminary determinations of ratios for a number of elements have been made on materials of known isotopic composition. This allows us to investigate problems that may be unique to ToF-SIMS for the measurement of isotopic ratios.

In recent years there has been considerable interest in two and three-photon excited fluorescence in laser scanning optical microscopy. Because absorption is confined tot he focal plane of the objective, these techniques provide intrinsic optical sectioning without the use of a confocal aperture. In addition, photobleaching and phototoxicity are greatly reduced above and below the focal plane. We have adapted a two-photon microscope to utilize surface second harmonic generation (SHG) as a new contrast mechanism for nonlinear optical biological imaging.

Surface SHG was first described by Shen [1] and arises from the second order nonlinear susceptibility, χ(2). Signal will only arise from a non-centrosymmetric environment such as an interfacial region. Thus this technique has the potential to probe cellular membranes at high specificity. Further, since SHG results from an induced polarization and not absorption, photobleaching considerations are greatly reduced over fluorescence based methods.

The two photon microscope provides optical sectioning of fluorescent specimens with a resolution comparable to that obtained in confocal microscopy (see refs 2,3). However, the excited volume in 2-photon microscopy is limited to the focal volume (unlike conventional fluorescence microscopy where excitation occurs throughout the specimen). This means that photodamage is limited to the plane of section being examined. Thus, the light emitted from each point in the specimen depends on the amount of fluorochrome present without the problem of prior illumination (of other planes within the specimen) reducing the photon yield so a better signal to noise ratio can be obtained when examination of multiple image planes is needed. Since 2-photon excitation spectra are wide and chromatic aberration is eliminated (because the emitted light does not have to be focused on a pinhole), it is possible to excite several fluorochromes simultaneously and map their positions with high accuracy.

The laser confocal microscope (LCM) is now an established research tool in biology and materials science. In biological applications, it is usually employed to detect the location of fluorescent marker molecules and, under these conditions, detected signal levels from bright areas often represent <20 photons/pixel (assuming a standard 1.6 μs pixel time) while those from dark areas are likely to average <1 photon/pixel. Although this data rate limits the speed at which information can be derived from the specimen, saturation of the fluorophor, photobleaching of the dye, and phototoxicity often prevent it being increased by simply using more laser power. Over the past 10 years, the optical photon efficiency of commercial confocal instruments has improved significantly and it is now reaching the point where further improvement is becoming very expensive. The only component is which a significant improvement is still possible is the photodetector.

Two-Photon Excitation (TPE) microscopy is a powerful tool for the imaging of biological specimen. The most important advantages compared with conventional confocal microscopy are: intrinsic optical sectioning, reduced phototoxcicity and photobleaching, and an increased penetration into the specimen. The increased penetration arises from the use of the longer wavelength excitation light, in the red or near infra-red.

Here, a quantitative in-depth TPE imaging study is presented on dental biofilm. The biofilm contains a mixed culture of a large number of oral bacteria with a typical size of ≈ 1 μm diameter. They exhibit a complex microstructure and heterogeneous physiological and biochemical properties. The high density of the biofilms limits the depth range over which confocal images can be acquired to 25-40 μm.

Using a two-photon excitation microscope fluorescence intensity images could be recorded over the whole thickness (100 μm) of the biofilm sample.

Research in our lab is aimed at examining how signaling molecules affect the differentiation of neurons in the developing cortex. Most morphological studies rely on single time point images of cells within a population. It is obviously advantageous to examine changes in individual living cells over time. By combining 2-photon imaging with co-transfection of genes for green fluorescent protein (GFP) and neurotrophins in living neurons, we can asses the effects of these on individual living cells.

Individual neurons in brain slices from ferret visual cortex were transfected with GFP using particle mediated gene-transfer, or Biolistics. Using the Helios Gene Gun (Bio-Rad), DNA-coated gold particles are blasted into living brain slices. Neurons are transfected when a gold particle lands in the nucleus. Within 24 to 36 hours individual cells are well-filled with GFP allowing visualization of dendrites, spines, and axons.

A video-rate scanning two-photon excitation microscope (TPEM) has been successfully constructed and tested. The TPEM, based on a Nikon RCM-8000, incorporates a femtosecond pulsed laser, a pre-chirper, and a non-confocal detection box for ratio imaging. Fig. 1 shows the schematic layout of the main components of the instrument, each of which is briefly discussed below.

Laser System: A Tsunami Ti: Sapphire laser (from Spectra-Physics) is optically pumped by a 5 W green laser (Millennia from Spectra-Physics) and is capable of generating 100 fs pulses at a repetition rate of 82 MHz and an average power of 0.8 W. The output wavelength is tunable from 690 to 1050 nm with three optical sets, each covering part of the spectrum with some overlapping.

Pre-chirper: After leaving the Tsunami, the laser beam enters an optic unit known as a pre-chirper which pre-chirps laser pulses to compensate for the group velocity dispersion which will result when the laser beam goes through the microscope optics.

Hair cells, the mechanotransducers of the inner ears and lateral lines of vertebrates offer an excellent preparation for the study of presynaptic mechanisms at fast chemical synapses. They offer the compact, almost cylindrical shape of an epithelial cell without entangling dendrites and axons, and make afferent synapses directly from the cell body. The biology of hair cells demands an unusually high performance of the output synapse, requiring continuous, and very quickly modulated transmitter exocytosis. Release follows the small receptor potential generated by the transduction apparatus; more transmitter is released upon depolarization, less after hyperpolarization. In some hair cells, transmission can phase-lock to kilohertz stimulus frequencies, preserving temporal information across the synapse.

We have exploited the synaptic terminal-like features of these cells to explore both the physiology and ultrastructure of the machinery underlying neurotransmitter release.

Electron microscope tomography is a useful method for deriving three-dimensional (3-D) structures from tilt series. We used an intermediate, high-voltage electron microscope operated at 400 kV to study the 3-D structure of selectively stained or labeled mitochondria from diverse sources. We have examined the structure of neuronal mitochondria and mitochondria from brown adipose tissue (BAT) employing either single-axis or double-axis techniques over a tilt range of ±60° with 1° or 2° angular increments (Fig. 1). Although BAT mitochondria contains only lamellar cristae with no tubular cristae as we have observed in neuronal mitochondria, we found that the inner membrane of BAT mitochondria invaginates to form cristae only through narrow, tubular openings or crista junctions (Fig. 2).

Recent interest in the possibility of structural artifacts in chemically fixed mitochondria imaged in situ motivated us to perform electron tomography on cryofixed brain and brown fat tissue.