FIB/SEM for Biological Data Mining

Freeze-cracked taste buds from rabbit foliate papillaes were studied using both the secondary electron (SE) image and the back-scattered electron (BSE) image. Zinc-Iodide-Osmium (ZIO) and Ruthenium red (RR) staining were used for SEM observation of the BSE image.With the SE image, the three-dimensional architecture of taste buds was relatively easy to visualize. However, individual cells could not be identified clearly. Basal cells were recognized easily from their position in the taste bud. The dense substance (DS) in the taste pore and the dark granules (DG) of the type I taste cell could be seen in the SE image. However, it was found that the BSE image produced by the ZIO method provided a strong contrast for both DS and DG that was superior to that of the conventional technique. Also a distinct BSE image could be obtained from the Golgi vesicles and DG near the Golgi apparatus at the perinuclear area in only the type I cell. It is of interest that DS and DG exhibit the same strong BSE image. It could be strongly suggested that the DG originated from the Golgi apparatus. RR, Which has an affinity for acid glycoconjugate, strongly stained the substance present along the membrane of cytoplasmic processes and some of the pore vesicles but DS and DG were only faintly stained or not at all in the BSE image. This implies that DS and DG contains only a small amount of polysaccharides. It appears then, that utilizing the BSE image in conjunction with the SE image is very useful in determinating the three-dimensional localization of histocytochemical components in taste buds.

High resolution secondary electron (SE) images can be obtained in a scanning transmission electron microscope (STEM) by collecting the type I secondary electrons generated directly by the incident electron beam. The high resolution SE signal is, in general, localized to about 1 nm in the generation process. The escape depth of the secondary electrons, however, varies from about 1 nm for most metals to about 10 nm for some insulating materials. Surface steps can be imaged by collecting high resolution secondary electrons Among other possible contrast mechanisms for SE imaging of surface steps, topographic contrast plays an important role. The topographic contrast of surface steps depends on: (1) the incident electron probe size; (2) step height to SE escape depth ratio; (3) localization of the generated SE signals and (4) the incident beam angle relative to the specimen surface.In our UHV STEM (MIDAS) instrument, both the entrance and exit surfaces of the sample can be imaged by collecting secondary electrons. For thin samples, the resolution is < lnm for SE images of entrance and exit surfaces but for thicker samples the resolution of the SE image obtained from the exit surface will be degraded by the beam broadening effects. For studying steps on thick or bulk crystal surfaces, secondary electrons emitted from the entrance surfaces are, in general, collected to form images with high contrast and high resolution. In this report, all the SE images are obtained, in MIDAS, by collecting secondary electrons emitted from the entrance surfaces of the samples. Figure 1 is a SE image of a MgO smoke crystal revealing the surface growth steps with high contrast. The parallel straight steps, along <100> directions, constitute the edges of unfinished atomic planes on the MgO {100} crystal surfaces. This image indicates that the {100} surfaces of MgO smoke crystals are flat surfaces. Figure 2 shows a SE image of a MoO3 crystal with surface growth steps spiraling from an end-on screw dislocation (the black dots in the SE image represent electron beam induced reduction products). The dark lines (indicated by D) represent up-steps and the bright lines (indicated by B) represent down-steps. It is interesting to note that the two-dimensional crystal growth around the dislocation is anisotropic, forming terraces with long edges parallel to [010] direction.

Surface organic residues inhibit the extraordinary electronic properties of graphene, hindering the development of graphene electronics. However, fundamental understanding of the residue morphology is still absent due to a lack of high-throughput and high-resolution surface characterization methods. Here, we demonstrate that secondary electron (SE) imaging in the scanning electron microscope (SEM) and helium ion microscope (HIM) can provide sub-nanometer information of a graphene surface and reveal the morphology of surface contaminants. Nanoscale polymethyl methacrylate (PMMA) residues are visible in the SE imaging, but their contrast, i.e. the apparent lateral dimension, varies with the imaging conditions. We have demonstrated a quantitative approach to readily obtain the physical size of the surface features regardless of the contrast variation. The fidelity of SE imaging is ultimately determined by the probe size of the primary beam. HIM is thus evaluated to be a superior SE imaging technique in terms of surface sensitivity and image fidelity. A highly efficient method to reveal the residues on a graphene surface has therefore been established.

The low-loss electron (LLE) image in the scanning electron microscope (SEM) is useful for the study of uncoated photoresist and some other poorly conducting specimens because it is less sensitive to specimen charging than is the secondary electron (SE) image. A second advantage can arise from a significant reduction in the width of the “penetration fringe” close to a sharp edge. Although both of these problems can also be solved by operating with a beam energy of about 1 keV, the LLE image has the advantage that it permits the use of a higher beam energy and therefore (for a given SEM) a smaller beam diameter. It is an additional attraction of the LLE image that it can be obtained simultaneously with the SE image, and this gives additional information in many cases. This paper shows the reduction in penetration effects given by the use of the LLE image.

Development and optimisation of focused ion beam/scanning electron microscopy as a technique to investigate cross-sections of organic coatings

Secondary electron (SE) imaging techniques, such as scanning electron microscopy and helium ion microscopy (HIM), use electrons emitted by a sample in response to a focused beam of charged particles incident at a grid of raster scan positions. Spot size—the diameter of the incident beam's spatial profile—is one of the limiting factors for resolution, along with various sources of noise in the SE signal. The effect of the beam spatial profile is commonly understood as convolutional. We show that under a simple and plausible physical abstraction for the beam, though convolution describes the mean of the SE counts, the full distribution of SE counts is a mixture. We demonstrate that this more detailed modeling can enable resolution improvements over conventional estimators through a stylized application inspired by semiconductor inspection: localizing the edge in a two-valued sample. We derive Fisher information about edge location in conventional and time-resolved measurements (TRM) and also derive the maximum likelihood estimate (MLE) from the latter. Empirically, the MLE computed from TRM is approximately efficient except at very low beam diameter, so Fisher information comparisons are predictive of performance and can be used to optimize the beam diameter relative to the raster scan spacing. Monte Carlo simulations provide an example of the MLE giving a 5-fold reduction in root mean-squared error (RMSE) of edge localization as compared to conventional interpolation-based estimation. The RMSE is substantially below both the beam diameter and the raster scan spacing and thus sub-pixel localization is demonstrated. Applied to three real HIM datasets, the average RMSE reduction factor is 5.4.

Secondary electron (SE) imaging in a scanning electron microscope is used to map electrically active dopant distributions of B-doped superlattices in Si. By comparing SE contrast profiles with secondary ion mass spectroscopy data, it is shown that B is electrically deactivated when the damage caused during Si implantation falls onto a doped region. Following a 450 °C anneal, the effect of the implantation damage is severely reduced in the SE profiles and the B is partially reactivated. An 815 °C anneal results in transient enhanced diffusion of some of the B with the remainder trapped in an inactive immobile peak.

Extract Extended abstract of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007

Platinum nanoparticles finely dispersed in activated carbon powders have been observed with high contrast in secondary electron (SE) and backscattered electron (BE) images. The factors that contribute to the visibility of small metal particles in high-resolution BE and SE images are discussed. Monte Carlo simulations provided insight into the scattering of electrons by small, heavy-element particles located on, or embedded in, light-element supports. The visibility of Pt nanoparticles in BE images depends on many factors including the size and the location of the Pt particles, the energy of the incident electrons, and the size of the electron probe. The SE signals generated by backscattered electrons may not significantly contribute to the visibility of small Pt particles in high-resolution SE images of carbon supported Pt catalysts. Only those small Pt particles that are located on or very close to the surface of the carbon support can be revealed in high-resolution SE images.

Biological ultrastructures have been extensively studied with the scanning electron microscope (SEM) for the past 12 years mainly because this instrument offers accurate and reproducible high resolution images of cell shapes, provided the cells are dried in ways which will spare them the damage which would be caused by air drying. This can be achieved by several techniques among which the critical point drying technique of T. Anderson has been, by far, the most reproducibly successful. Many biologists, however, have been interpreting SEM micrographs in terms of an exclusive secondary electron imaging (SEI) process in which the resolution is primarily limited by the spot size of the primary incident beam. in fact, this is not the case since it appears that high resolution, even on uncoated samples, is probably compromised by the emission of secondary electrons of much more complex origin.When an incident primary electron beam interacts with the surface of most biological samples, a large percentage of the electrons penetrate below the surface of the exposed cells.

A focused ion beam (Ga+-FIB) drawing on a non-conductive ceramic substrate was investigated for the formation of finely electrified images. The electrified images were formed by using a 30kV of Ga+-FIB with a drawing pattern equipment. The region irradiated with Ga+ ions on the substrates showed positive surface potential and the decay rate of the surface potential was less than 10% for 2000s in air. The electrified images were in-situ observed with a scanning electron microscope (SEM) at 2.5kV. The secondary electron images, namely the voltage contrast images, showed dark contrast. This fact indicates that the dark image corresponds to the positively electrified regions on the substrate. The width of the electrified image was 8μm. Finely electrified images formed by Ga+-FIB can be applied to the electrostatics processes.

In this study, the authors present a new method for electrical potential observation in which helium ion microscope (HIM) observations can be performed while applying a selected voltage (up to approximately +5 V) to a sample. The in situ voltage-application system is operated in a high vacuum HIM chamber and consists of a transfer rod, tilt joint, x-, y-, and z-axis mechanisms, and two probes at the end of the transfer rod. The new system was used to acquire a secondary electron (SE) images of the cross-sectional surfaces of a multilayer ceramic capacitor (MLCC) with voltages (from 0.5 to 5 V) applied to the internal electrodes of the MLCC. The active voltage contrast corresponding to the electrical potential in the SE images could be observed when the applied voltage was less than or equal to 1.5 V. This technique enables any voltage to be applied to the samples from outside the chamber and can be used to measure the nanometer-scale electrical potential distribution of Li-ion rechargeable batteries, solar cells, etc.

In this work, an innovative cathodoluminescence (CL) system is coupled to a scanning electron microscope and synchronized with a Raspberry Pi computer integrated with an innovative processing signal. The post-processing signal is based on a Python algorithm that correlates the CL and secondary electron (SE) images with a precise dwell time correction. For CL imaging, the emission signal is collected through an optical fiber and transduced to an electrical signal via a photomultiplier tube (PMT). CL Images are registered in a panchromatic mode and can be filtered using a monochromator connected between the optical fiber and the PMT to produce monochromatic CL images. The designed system has been employed to study ZnO samples prepared by electrical arc discharge and microwave methods. CL images are compared with SE images and chemical elemental mapping images to correlate the emission regions of the sample.

Platinum nanoparticles finely dispersed in activated carbon powders have been observed with high contrast in secondary electron (SE) and backscattered electron (BE) images. The factors that contribute to the visibility of small metal particles in high-resolution BE and SE images are discussed. Monte Carlo simulations provided insight into the scattering of electrons by small, heavy-element particles located on, or embedded in, light-element supports. The visibility of Pt nanoparticles in BE images depends on many factors including the size and the location of the Pt particles, the energy of the incident electrons, and the size of the electron probe. The SE signals generated by backscattered electrons may not significantly contribute to the visibility of small Pt particles in high-resolution SE images of carbon supported Pt catalysts. Only those small Pt particles that are located on or very close to the surface of the carbon support can be revealed in high-resolution SE images.

The scanning electron microscope is a specialized closed-circuit television system, which must be understood before the properties of thin films can be monitored using it. Therefore, the first part of this paper discusses fundamentals of scanning electron microscopy, with particular emphasis on signal generation caused by primary electron beam interaction with the object, collection, and amplification of the generated signal, and optimum display. Secondary electrons are the most frequently used information signal in conventional scanning electron microscopes. Images produced by the secondary electron signal show great depth of field, good shadow detail on very rough objects, are capable of high resolution (100 Å), and are unexcelled for showing topographical features of rough samples, particularly when stereo pairs are used. Secondary electron images can also show voltage contrast and magnetic contrast. Backscattered electron images show somewhat higher contrast, poorer resolution, poor shadow detail on rough objects, but can show contrast between areas of different atomic number. Images produced by electron-beam-induced currents in the sample can be used to locate p-n junctions (even when they lay beneath the surface), and to find crystallographic defects near the surface. Characteristic x-rays and Auger electrons can be used for chemical identification; the efficiency of generation of these signals is low, requiring larger beam currents for useful signals. Cathodoluminescent signals also promise to provide vital chemical information about samples, but these signals are weak, and often decrease as the exposure of the sample to electrons increases. The detailed understanding of scanning electron micrographs obtained using different information signals from the sample is not a trivial problem. The scientist must always remember that lighter parts of the image correspond to higher values of collected information signal, whatever that signal may be. Some pictures show contrast because the amount of information signal varies from place to place on the sample when the fraction of the signal that is collected remains constant, while other micrographs show contrast because although the amount of information signal is constant across the sample area of interest, the fraction of collected signal varies from place to place. Both of these effects can be illustrated with a micrograph of an integrated circuit, with bias applied between different electrodes. Sometimes more information is available in the video signal than can be displayed by a conventional television image, and improved display techniques are advisable. Deflection modulation (or Y-modulation) allows small signals superimposed on large signals to be seen clearly. Differentiation of the video signal is useful in many applications. Nonlinear operations on the signal, such as gamma correction and contrast expansion, can improve the presentation of desired information on the micrograph also. The scanning electron microscope can be quite useful in the study of thin films and thin-film structures. General surface topography can be determined, including surface roughness, voids, cracks, and often grain size. When voltage is applied across a region, electrical barriers such as p-n junctions, high-resistance regions, etc., can be detected readily. By pulsing the electron beam, carrier generation and recombination can be studied. Domains on magnetic samples and piezoelectric samples have also been studied. As the electron beam interacts with the sample, the properties of the sample may change. For example, bombardment of metal-oxide-semiconductor devices with voltage applied between metal and semiconductor can result in a stored charge in the oxide. Florescent light output from many materials decreases with bombardment. Resist materials are polymerized by electron bombardment, and very fine patterns can be defined by correctly steering the beam of a scanning electron microscope. Devices having dimensional tolerances considerably shorter than a wavelength of visible light have already been produced in this manner. In some applications, it may be desirable to monitor the size and orientation of single crystal formed during thin film formation. A recent method of producing Kikuchi-like patterns using a scanning electron beam may be useful in this regard. At present, the method is useful on large single crystals, but modifications of the method should make it useful for crystallites down to a few microns in size. The patterns show a wealth of fine detail, and should be useful for accurate measurements. The application of scanning electron microscopy to thin-film problems is in its infancy. Future work will use not only the techniques cited above, but also new techniques devised by thin-film scientists to produce the information they desire about the submicron structures they fabricate.