Wednesday, June 29, 2011

Elemantal Mapping: Inclusion in Dinosaur Bone

This image shows an SEM image of the cross-section of an Archaeopteryx femur bone. The bone was embedded in a polymer and then lapped smooth. The image is taken in compositional BEI mode where the total backscattered intensity is mapped. Since the backscattering coefficient is dependent upon atomic number, the BEI intensity is a measure of the average density of the sample.

The area surrounding the bone is dark because the embedding material is largely carbon based-- phenolic. The medullary cavity of bone is darker because it is of lower density than the bone itself. A fracture of the bone is present at the bottom of the image. In the vicinity of this fracture are regions of very high backscattered intensity-- much brighter than the bone-- indicating some material of very high density. There are similar high density inclusions on the margins of the medullary cavity as well.

The compositional BEI image was used to locate a high density inclusion in the medullary cavity of the bone. This region was used for an elemental map using energy dispersive X-ray spectroscopy (EDS). In an elemental map an entire EDS spectrum is taken for each pixel in the SEM image. Spectroscopic regions of interest (ROI's) can be selected and mapped.

In this image the blue color indicates the intensity of Ca Ka X-rays, yellow P Ka X-rays, and red Fe Ka X-rays. This spectroscopic information is overlayed on the compositional BEI image.

The medullary cavity is calcium rich due to the presence of calcium carbonate-- thus the solid blue color of that part of the specimen. Bone is largely calcium hydroxyapatite which is a calcium phosphate mineral. As the blue from Ca Ka and yellow from P Ka mix on the image a green color is formed. The inclusion is red as it is iron rich. By performing additional maps on elements associated with clay minerals-- e.g. Si and Al-- it was determined that this iron rich inclusion was associated with a clay mineral that entered the medullary cavity through the bone fracture. (Thanks to Dr. Greg Erickson, FSU Department of Biology.)

High Tilt: Surface Topography in Profile

This is an image of the surface of a Ti filament from a Ti sublimation pump for ultra high vacuum. When viewed from above a fairly unremarkable granular structure was observed. What was of interest was the organization of grains into domains with raised edges-- little walls- between them.

A nice feature of SEM's is the ability to tilt samples at very high tilt angles. At large working distances the maximum tilt angle is nearly 90 degrees allowing one to examine surface morphology in profile. At a large tilt angle these raised domain walls were shown to consist of columnar grains of very high aspect ratio. As seen in the foreground of this image, if these structures were imaged solely from above, they would have appeared like roundish globular grains. It is thought that this remarkable structure occurs when the layer of molten Ti on the surface of the TSP filament forms fluid convection cells and then suddenly cools when the heating current is abruptly removed.

Tuesday, June 28, 2011

Length Calibration

In the SEM vertical distances can not be directly measured from secondary electron intensity because there are many factors that impact the generation and efficient collection of secondaries. Vertical distances can be only determined using stereoscopic pairs or shadowing techniques.

Lateral distances can be measured directly from SEM images. While the SEM scan amplifiers are calibrated, an imaging resolution standard can be used to check the accuracy of the magnification. This image shows a series of horizontal and vertical 10 um pitch lines that can be used to check the lateral length calibration in two orthogonal dimensions. Many such structures are available on a test pattern at CMMP for the purpose of length calibration. These include longer "ruler" types of patterns that can be used to calibrate very long distance measurements.

Stigmators

This is an image of three concentric rings lithographed inside a circular hole at 35,000X magnification. The bright edges are edge effects-- enhanced secondary collection near the edges of features. Above a few 10's kX fine focusing is generally not enough to produce a high resolution image. The condenser lens can focus the probe to a point-- but only if the intrinsic shape of the probe is point-like. Because of aberrations in the column, and because of the spatial extension of the tungsten emitter, the spot is often extended in shape. Such a distortion of the probe leads to a loss of resolution as the probe shape is essentially convoluted with the morphology of the surface. While this generally critical at higher magnifications (over 10-20 kX), it is good practice to deal with this issue even for lower mag images as it will add to the image crispness and definition.

This distortion of the probe shape is compensated by stigmators. The stigmators are a set of octapole lenses in the X- and Y-directions that act together to adjust the shape of the probe. They quite literally tweek the shape of the beam to condition it to its optimal shape. In practice one adjusts the fine focus, then one stigmator (X or Y), and then the other stigmator-- to return and adjust the fine focus.

This image is of the same three rings except with the Y-stigmator slightly misadjusted. Notice that the rings are blurry at the top and bottom of the image, but not on the right and left sides. This is because the misadjustment of the stigmator has not elongated the SEM electron probe in the vertical plane of this image thus blurring the image vertically.

Nannofossils

This is an SEM image in SEI mode of a nanofossil collected from marine sediment. Some marine algae form structures of calcium carbonate called coccoliths. These structures are unique signatures of the species of microorganism. After the death of the organism, these calcium carbonate structures sink through the water column and become part of the marine sediment.

This image is of a coccosphere, a spherical arrangement of coccoliths. The complicated shapes are the result of cellular processes within the microorganism. The biological function is unclear and hypotheses suggest that they might aid in photosynthesis and/or assist in the bouyancy of the organism. Despite the underlying biology, these coccoliths are of profound interest to earth scientists as they give clues to oceanic conditions.

The bottom image is also a coccosphere. In this case the sphere consists of umbrella like structures packed into a spherical arrangement. Some of the coccoliths have come apart and can be seen to the bottom left of the image.

Thanks to Dr. Woody Wise, FSU Department of Earth, Ocean and Atmospheric Science.


Thursday, June 23, 2011

Crystal Habits: Crystallography in the SEM

The ability to image crystal habits can be of great utility in identifying phases. The top BEI image shows SnO2 deposited on Al2O3 using PVD with Ar as a carrier gas. EDS determined these structures to all be either Sn or SnOx. SnO2 is a tetragonal system that tends towards prismatic. acicular and botryoidal crystals. Toward the center of the image is what appears to be perfect bipyramids consistent with a tetragonal system. The spherical objects have what appear to be hints of facets. (Thanks to Dr. Peng Xiong, FSU Department of Physics.)

The the bottom SEI image shows a cluster of pyrite pyritohedra-- irregular dodecahedra-- clustered together in a clay mineral sample. The phase identification was immediate upon observation and confirmed using EDS spectroscopy.
(Thanks to Dr. Woody Wise, FSU Department of Earth, Ocean and Atmospheric Science.)

Light Element Sensitivity

The CMMP EDS detector is what is known as a light element EDS detector. The sensitivity of the EDS detector is entirely determined by the thickness and composition of the detector window. The older "thick window" systems are generally sensitive to Al and higher Z elements. The light element systems are sensitive to C and above.

Even with a light element system one should be careful with the quantitation of light elements. One has no idea what the effective window thickness is and thus it is difficult to model and subsequently compensate for window absorption. The EDS detector is cooled to LN2 temperatures which causes the EDS detector window to be the coolest part of the SEM chamber. Diffusion pump fluids and volatiles evolving from samples in the SEM vacuum are cryopumped on the EDS detector window. This can result in a severe reduction in light element signal.

This EDS spectrum of quartz at 30 kV shows no sign of the O Ka peak. Quantitation without standards yields 99% silicon by weight. One could attempt to use standards to quantitate light elements such as C, N and O with a heavily contaminated EDS window like this-- but this leads to the problem of very high statistical uncertainty in both the standard and specimen data. Since the k-ratios used in quantitation are ratios of specimen and standard data, quantitative uncertainties are enormous.

This EDS spectrum of quartz is taken immediately after cleaning the light element window. The EDS detector was completely warmed, the electron trap removed, and the window cleaned by flowing a fluorohydrocarbon solvent across the window using a dropper. Standardless quant yielded 66% Si and 34% O by weight. This still underestimates the oxygen content which should be ~ 53% weight, but the O Ka peak is sufficiently strong to perform meaningful analysis. The use of standards would account for the remaining systematic error at low energies due to attenuation in the EDS detector window.

Thursday, June 16, 2011

Directionality of SEM Imaging Signals

When performing SEM imaging it is important to have an intuitive sense of the directionality-- or lack thereof-- of the signals being used. All SEM images are maps of position, which is generated by the rastering of the SEM beam by the scan coils, and some signal related to the interaction of the electron beam in the sample. The most common of these signals is secondary electrons which are detected by the Everhart-Thornley or "ET" detector. The ET-detector uses a grid biased at positive voltage to suck the low energy secondary electrons towards a scintillator where they are converted into light and then detected by a photomultiplier. The secondary electron detection efficiency of the ET-detector is very high, allowing it to detect secondary electrons emitted from virtually every portion of complex surface morphology hit by the primary beam. While there may be some changes in detection efficiency with topography, there is never any portion of the structure where the ET-detector is masked or blocked. This image of a TEM grid is a good example. Even though the ET-detector is oriented towards the top of the image, the topographical contrast of the grid structure does not mask the detection of the secondary electrons. The brighter than expected edges are also a signature of the excellent detection efficiency of the ET-detector. These are edge effects due to the ET-detector being able to collect secondaries that are emitted not only at the top of the sample, but at the edges of the sample.

In this X-ray map of the Cu Ka line, it is immediately obvious that the TEM grid morphology is masking the Cu Ka X-rays generated by the primary electron beam. The EDS detector comes in near the top left quadrant of the image-- masking is absent in the top left quadrant of the TEM grid and present in each of the three other quadrants. Only X-rays generated in direct line of sight to the EDS detector are visible without attenuation. This is of concern in the interpretation of EDS X-ray maps, but also in the EDS point analysis on samples with non-flat morphology.

Imaging Plastics at 1 kV

Low voltage imaging is useful in circumstances where coating with graphite or AuPd might destroy a component or device. Even though coating for SEM aims at depositing only ~ 10-12 nm of material, such a coating should be considered permanent. Also such energetic deposition as sputtering and flash coating can permanently damage delicate materials and devices.

In this case we had a novel Viton seal for a high vacuum application that was suspected of leaking. It was imaged at a beam voltage of 1 kV allowing us to see a break. If this part had been found to be undamaged it could have been returned to service with no concerns of it being damaged by conductive coating for SEM imaging.

Resolution Test

Every SEM and TEM has a resolution benchmark. This is a recipe that combines a specific resolution standard sample with a standard instrumentation configuration. In electron microscopy a common resolution standard sample is Au clusters on HOPG. Such a sample is produced by evaporating Au on HOPG at such a temperature that mobility is limited and Au nucleates into clusters. Au/HOPG is an ideal resolution standard because gold has very good secondary electron emission efficiency while carbon very poorly emits secondaries.

In the case of the JEOL 5900 the test configuration is a 30 kV beam energy with a 10 mm working distance. We use a sample that has Au clusters with gaps of ~ 10 nm between them. It should be noted that Au/HOPG samples with different cluster gap distances can be obtained. After aligning the column and objective aperture, clearing the lenses and optimizing focus and stigmation one should be able to easily observe gaps of ~ 10 nm between these gold clusters.

It should be noted that there are some horizontal lines on the image. This is periodic in time and will manifest differently with different acquisition times. It is due to vibration in the building. It can also be mitigated using frame averaging.

Generally speaking, for work requiring resolution below a few 10's nm I would recommend a field emission SEM (FE-SEM). BSIR, The Biological Science Imaging Resource, has a FEI Nova 400 nanoSEM that is available to the research community. It provides 1.0 nm resolution at 15 kV and 1.8 nm resolution at 1 kV.

Charging & Low Voltage SEM

When insulating samples are imaged at high beam energies in the SEM they are subject to charging. Samples don't charge when the current into the sample as incident beam current (I0) equals the current leaving the sample as secondary electrons (ISE) and backscattered electrons (IBE):

I0 = ISE + IBE

At high beam energies secondary production is very efficient due to an increased number of energy loss channels, and thus samples tend towards a positive charge. The result is a slow and steady increase of charge at the sample surface resulting in electrostatic fields that deflect the incident beam and the trajectories of secondary electrons and backscattered electrons leaving the sample. In an SEM this manifests as time varying changes in contrast and brightness and sudden changes in the image position or image quality. This image of uncoated cellulose taken at 20 kV shows these signs of charging. At the top right portion of the image one can see a cellulose fiber that has been physically deflected by electrostatic charge during imaging. Note the very bright "flaring" portions of the image.

By imaging at very low beam energies one can generally find a beam energy where there is no charging. Because of this the JEOL 5900 low voltage SEM (LV-SEM) can adjust its beam energy in 100 V steps from 300 V to 2500 V. Finding the ideal beam energy to avoid charging can take some finessing, but 1500 to 2000 V is a good starting point. One should keep in mind that if one does manage to induce charging in the sample with too high of a beam voltage that this charging will only very slowly dissipate over time. As such, try optimizing the beam voltage with insulating samples on an edge of the sample. If one charges this area, one can move to another part of the sample margin and so on until one has optimized the beam voltage. Then one can move to the center of the sample and image it without sample charging and without coating.

This is an image of teflon tape (the type used in plumbing) imaged at a beam voltage of 2500 V without coating. Note the absence of charging and the presence of domains connected by fibers. While the ability to image at low voltages without sample coating is nice, it does provide decreased resolution in the JEOL 5900. As the beam voltage decreases the effects of astigmatism are greatly enhanced. Imaging at low voltages will require not only readjustment of stigmators but ideally a column realignment.

Tuesday, June 14, 2011

JEOL 5900 SEM

The CMMP group at the Florida State University Department of Physics has a JEOL 5900 scanning electron microscope (SEM) that is available to the local research community. A small fee of $40/hour is assessed for usage of this instrument.

The JEOL 5900 has a maximum beam voltage of 30 kV. It is also a low voltage SEM, allowing one to control the beam voltage in increments of 100 V from 300 V to 2500 V. This is useful in imaging insulating materials without coating. The resolution at 30 kV and at a 10mm working distance is ~ 10 nm. In addition to imaging with secondary electrons (SEI) and backscattered electrons (BEI), an energy dispersive X-ray spectrometer (EDS) is available for quantitative elemental analysis and elemental maps.

SEM Advantages: fast; huge depth of field; large field of view; ability to image in SEI and BEI modes; low voltage imaging of insulating materials.

SEM Disadvantages: special sample preparation for biological and other "wet" samples; while lateral distances can be measured directly, vertical ones can not; no digital image processing tools to get Ra, bearings, feature depths, etc.

EDS Advantages: fast; elemental mapping; good accuracy with standards; good for defect analysis.

EDS Disadvantages: insensitive to light elements; poor energy resolution; spectral overlaps.