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Australian Microscopy & Microanalysis Research Facility

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Camperdown, New South Wales, AU

About Australian Microscopy & Microanalysis Research Facility

The Australian Microscopy & Microanalysis Research Facility (AMMRF) is Australia's leading facility for the characterisation of matter on a fine scale. We specialise in instrumentation, methodologies and applications for characterising samples in the physical, biological and environmental sciences using... Show more »

The Australian Microscopy & Microanalysis Research Facility (AMMRF) is Australia's leading facility for the characterisation of matter on a fine scale. We specialise in instrumentation, methodologies and applications for characterising samples in the physical, biological and environmental sciences using ion and electron beams, scanned probes, X-rays as well as light and laser optics.

The six AMMRF nodes are located at the host universities in Adelaide, Brisbane, Canberra, Perth and Sydney.

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Our Services (104)


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Atomic Force Microscopy (AFM) Services

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Atomic force microscopy (AFM) is the technique of choice to provide nanoscale structural and mechanical information. It can be applied to virtually any sample and experiments can be carried out in a variety of environments. In addition to topographical information about a sample, interaction forces between the substrate and a... Show more »

Atomic force microscopy (AFM) is the technique of choice to provide nanoscale structural and mechanical information. It can be applied to virtually any sample and experiments can be carried out in a variety of environments. In addition to topographical information about a sample, interaction forces between the substrate and a probe can be measured, potentially elucidating adhesion, magnetic and electronic forces. It can also used to measure intermolecular forces, including the forces that govern lubrication, cell adhesion and colloidal interactions such as electrostatic repulsion and van der Waals adhesion. Experiments in liquid enable biological samples such as cells and proteins to be imaged giving quantitative topological data and potentially insights into interaction sites.

The AFM operates by scanning a sharp tip across a sample surface. The tip is typically a pyramid or conical in shape and is four to five microns in height with a diameter at the apex of 10 to 20 nm. It is positioned at the end of a cantilever, which is typically 100 to 200 mm long. This probe is usually made from silicon or silicon nitride with the cantilevers' spring constants ranging from 0.05 to 50 N/m depending on the AFM mode of operation being employed. The tip or surface is raster scanned using a piezoelectric control mechanism that allows the AFM to acquire an image in three dimensions. AFM can be operated in the imaging mode, which allows an image of the substrate to be collected or you can operate in the force spectroscopic mode where the deflection of the cantilever is monitored as it is moved towards and away from the surface in question. Spectroscopic mode measures what are known as force distance curves and provides information about sample material properties. This can be used to measure adhesive forces, determine bond strengths and map local elastic properties. There are numerous imaging modes for the AFM but the two main modes are contact mode and tapping mode.

Contact-mode AFM operates by scanning the tip across the sample surface while monitoring the change in cantilever deflection. By maintaining a constant cantilever deflection, the force between the tip and the sample remains constant. Spring constants usually range from 0.01 to 1.0 N/m, resulting in forces applied to the surface ranging from nN to μN in an ambient atmosphere. It is these forces that are used to collect the image and hence it is important that the right force is used for a particular sample. For example, a high force used on a soft sample will lead to damage. Operation can take place in ambient and liquid environments.

Tapping mode AFM is used for soft samples such as lipid bilayers and polymers where contact mode imaging would damage the sample. Similar information is measured but via a different imaging mechanism. Tapping mode operates by scanning a tip attached to the end of an oscillating cantilever across the sample surface. The cantilever is oscillated at or near its resonance frequency with an amplitude ranging from 20 nm to 100 nm. The frequency of oscillation can be at or on either side of the resonant frequency of the cantilever. The tip lightly 'taps' on the sample surface during scanning, contacting the surface at the bottom of each oscillation. The feedback loop maintains a constant oscillation amplitude by maintaining a constant amplitude of the oscillation signal acquired by the split photodiode detector. By maintaining a constant oscillation amplitude, a constant tip-sample interaction is maintained during imaging. Like contact mode, tapping mode can be operated in ambient and liquid environments. Tapping mode has a number of advantages compared with contact mode, including a reduction in lateral and normal forces being applied to the sample surface.

Specimen choice and preparation can be a limiting factor with any type of AFM. The AFM scanners are usually limited to a maximum vertical movement of approximately five microns with a maximum x-y range of 100 x 100 microns. Therefore any surface with more than five microns of roughness will be extremely difficult to image with most AFMs. Sample size is also restricted to a maximum area of 1 x 1 cm and with a maximum sample thickness of approximately 3 mm.

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Atom Probe Tomography (APT)

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Atom probe tomography (APT) is a new generation characterisation technique that reports the position and chemical identity of atoms within a chosen material. It achieves this through ionising and removing atoms, one at a time, from a sharpened needle-shaped specimen and directing those atoms (now ions) through an electric field to... Show more »

Atom probe tomography (APT) is a new generation characterisation technique that reports the position and chemical identity of atoms within a chosen material. It achieves this through ionising and removing atoms, one at a time, from a sharpened needle-shaped specimen and directing those atoms (now ions) through an electric field to a detector. The atom probe instruments are point-projection microscopes – the ions project in a near perpendicular direction from their original point on the surface of the specimen tip. As a result, atomic resolution may be clearly discerned, corresponding to a magnification of a few million times. The original position of an atom is determined by a position-sensitive detector and the chemical identity determined through time-of-flight mass spectroscopy. The physical mechanism of field-ionisation permits the removal of atoms. Electrically conductive specimens are typically field-ionised by high-voltage pulse, whereas semi-conducting, or non-conducting specimens, are stimulated to field-ionisation through the use of laser pulses directed at the tip. During the process of data acquisition/field-ionisation the specimen is held at a temperature of approximately 20 K and is contained in an ultra-high vacuum chamber, around 1 x 10-11 Torr. Conductive materials are commonly prepared for acquisition through electro-chemical polishing a specimen to a needle with an apex of approximately 100 nm. Non-conductive, and specimens with sights of specific interest, are prepared with a dual-beam focused-ion beam instrument. The technique of APT is typically applied to crystalline and amorphous metal alloys and semi-conductor devices. It has selective applicability to polymers and biological specimens and these sample types are at the frontier of atom probe research.

Following the acquisition of data, careful reconstruction ensures APT accurately reproduces a 3-D volume of the atoms within the specimen with sub-atomic spatial resolution and the identification of any atom in the periodic table. This data is visualised as an interactive 3-D matrix of atoms commonly 150 x 150 x 400 nm in dimension and 40 million atoms in size. Post-processing analysis reveals the richness of information available from this type of data as the quantitative metrics defining atomic clustering, geometries of structures and chemical environments is non-intuitive to visual observation. Investigating the effect of variables involved in treatment or processing parameters (thermal annealing time, composition alterations, etc.) with these metrics of atomic and chemical structuring yield unique and powerful insights into a material's behaviour and understanding of structure to property (e.g. mechanical or electrical) relationships.

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Auger Electron Spectroscopy

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Auger electron spectroscopy (AES) is a surface analytical tool that uses secondary electrons as a probe of the elemental composition of the samples’ surface. It probes the same thickness (about 0.3 nm) as other techniques that employ secondary electrons (X-ray photoelectron spectroscopy) but has the advantage of a highly focused... Show more »

Auger electron spectroscopy (AES) is a surface analytical tool that uses secondary electrons as a probe of the elemental composition of the samples’ surface. It probes the same thickness (about 0.3 nm) as other techniques that employ secondary electrons (X-ray photoelectron spectroscopy) but has the advantage of a highly focused primary electron beam of typically 10–100 nm that enables small spot analysis and elemental mapping.

It derives it surface sensitivity from a small inelastic mean free path (IMFP), which is the distance a secondary electron will travel before suffering an inelastic collision (and therefore not contribute to the characteristic peak).

It is one of a few techniques that measures true surface composition (secondary ion mass spectroscopy and its variations being another). This is also a drawback since most clean surfaces exposed to air will oxidise and have a surface composition different to that of the bulk. The surface sensitivity also prevents coating insulators with a gold layer to prevent charging since the only Auger signal will be that from the gold layer.

The technique is therefore particularly suited for studies on conductors and semiconductors and samples that can be introduced and etched within an ultra-high-vacuum chamber. The etching is done by energetic argon ions and the surface layer atoms are sputtered to expose the underlying layers. After each etching cycle, the surface concentration is measured to construct a concentration profile versus time, which is then converted into a concentration-versus-depth profile for a given etch rate. These etch rates will depend on the chemical composition and structure and may have to be measured independently. Profiles up to a depth of several micrometres can be reconstructed with a depth resolution of a few nanometres. Preferential sputtering, radiation induced diffusion and damage may complicate data analysis.

The surface concentration is calculated from the total measured intensities, using elemental sensitivities, which in turn depend on the IMFP as stated above. These IMFP’s depend on atomic structure and the calculated atomic percentage may therefore include significant uncertainties. As the Auger process involves three electron levels, it is also not particularly suited to extract chemical bonding information. Provided these limitations are heeded, AES provides an easy-to-use technique that yields the surface elemental composition on a nanometre scale with little or no sample preparation.

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Small Animal Bioluminescence Imaging

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Bioluminescent imaging is a technique for imaging living small animals such as mice, rats and guinea pigs and is commonly applied to drug development and cancer biology. The technique is non-invasive, as it measures the light emitted from the cells tagged with luciferase. It can be used to track gene expression via a... Show more »

Bioluminescent imaging is a technique for imaging living small animals such as mice, rats and guinea pigs and is commonly applied to drug development and cancer biology. The technique is non-invasive, as it measures the light emitted from the cells tagged with luciferase. It can be used to track gene expression via a luciferase-tagged protein over time, or by using a cell-specific tag that can allow cell populations to be monitored, as would be useful in the study of cancer cells and tumour progression. Tagged cells can be injected into animals and their behaviour observed.

An example would be cancer cells tagged with luciferase and injected into a specific site of the mice (e.g. tibia) and scanned using bioluminescence. The cells in the bone emit a stronger light as the cancer grows and this helps the researcher to measure tumour growth. Tracking the tumour growth enables researchers to see the beginning of bone loss (osteolysis). New drugs can be trialled on the mice, and their effect on the tumour itself and on the bone destruction, measured.

In most cases, nude mice are used or the region of interest is shaved, as the presence of hair can obscure the signal from the tumour. The animal is anaesthetised in an induction chamber and injected with the appropriate substrate for the luciferase being used. It is then placed in the anaesthesia manifold in the imaging chamber so that the animal's head is securely placed in the nose cone. The subject is then imaged. The luminescent signal level is proportional to the exposure time, and the minimum and maximum exposure times are 0.5 seconds and 5 minutes respectively. The standard image is a composite image of the photographic and luminescent images and represented as an overlay.

Advantages of this application are the use of fewer animals and shorter timelines compared to more conventional animal testing methods. Also, the response to treatment can be measured without terminal histological assessments.

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CL-SEM

Cathodoluminescence scanning electron microscopy
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Cathodoluminescence (CL) is the non-incandescent emission of light (photons) from a luminescent material excited by an electron beam. CL photons (from ultraviolet, through visible to the near-infrared) are emitted as result of electronic transitions between the conduction and valence band and may also involve electronic... Show more »

Cathodoluminescence (CL) is the non-incandescent emission of light (photons) from a luminescent material excited by an electron beam. CL photons (from ultraviolet, through visible to the near-infrared) are emitted as result of electronic transitions between the conduction and valence band and may also involve electronic transitions associated with defect levels within the band gap. The optical, electrical and mechanical properties of solids are dependent on the presence of microscopic defects (imperfections and impurities) and therefore CL microanalysis is a useful spectroscopy and imaging technique for characterising these properties with high sensitivity and spatial resolution.

Cathodoluminescence microanalysis in a scanning electron microscope (SEM) enables high-sensitivity detection of defect centers in a wide range of materials. CL image resolution can range from tens of nanometres to micrometres and is dependent on specimen configuration and interaction volume, carrier diffusion and electron beam parameters, etc. Quantitative CL microanalysis of defect and impurity concentrations is not generally achievable because of the lack of a general explanation for the complex nature of competitive recombination processes.

The preparation of a specimen for CL microanalysis in an SEM is similar to preparation for other SEM-based microanalytical techniques. Many luminescent specimens also need coating with a thin conductive film to minimise surface charging effects.

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Tomography Services

Computed tomography services
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Confocal Microscopy

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A confocal microscope scans the sample with a spot of light in a regular ‘raster’ pattern and so acquires an image point by point. The end result, on this basis, is no different from the image given by a regular microscope. But because only one point is acquired at a time, we can place a pinhole where the spot is imaged, and so... Show more »

A confocal microscope scans the sample with a spot of light in a regular ‘raster’ pattern and so acquires an image point by point. The end result, on this basis, is no different from the image given by a regular microscope. But because only one point is acquired at a time, we can place a pinhole where the spot is imaged, and so eliminate out of focus light. Light that is in focus will be a small spot and will pass through the pinhole, but out of focus light will be a fuzzy blob and will mostly be blocked. This enables true three-dimensional imaging. To get enough light in one point, a laser is usually the illumination source.

Confocal microscopes can be operated in reflection mode – typically for surface profiling, but in the life sciences they are more often used in fluorescence mode, where out of focus glare can be a major problem. Typically, one uses a confocal microscope either to collect a three-dimensional set of slice images, or to image a single layer in cases where strong surrounding fluorescence would spoil the image in a conventional fluorescence microscope.

In confocal microscopy one needs to bear in mind that there are only a few, discrete, laser lines for illumination and the stain must be chosen to suit. Excitation (except in some special cases) is only in the visible range, with 405 nm (deep violet) the shortest wavelength available. Also, correct matching of mounting medium, coverslip and objective becomes critically important for 3-D imaging.

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Cryo-Electron Microscopy

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Cryo scanning electron microscopy (cryo-SEM)
Samples for conventional scanning electron microscopy (SEM) must be prepared for the high-vacuum environment of the microscope by being fixed, dehydrated and dried. In some cases, this processing technique cannot be used because it would either destroy the sample or cause unacceptable... Show more »

Cryo scanning electron microscopy (cryo-SEM)
Samples for conventional scanning electron microscopy (SEM) must be prepared for the high-vacuum environment of the microscope by being fixed, dehydrated and dried. In some cases, this processing technique cannot be used because it would either destroy the sample or cause unacceptable artifacts.

An excellent alternative in such situations is cryo scanning electron microscopy (cryo-SEM). This technique allows wet or liquid samples to be snap frozen, prepared and held at very low temperature while being examined in the microscope. Sample preparation time is relatively short, so can be readily repeated if necessary. An added advantage is that the frozen sample can be fractured during preparation to reveal internal structures. For example, a leaf can be fractured to reveal the internal detail, or a suspension of nanoparticles can be examined to assess the dispersion. Sublimation of some of the ice at the surface of the fracture will help to enhance detail. After cooling below sublimation temperature, the sample is coated with a conductive metal to prevent electrical charging during examination. Freeze-fracture can also be extremely valuable in working with lipid bilayers and membranes as fracturing can split the bilayers. Such preparations can also be visualised by transmission electron microscopy (TEM) for high-resolution imaging of these structures.

Samples for cryo-SEM are generally not prepared at all beforehand; the best results are obtained from samples that are as close to their natural state as possible. The technique has proved its effectiveness for samples such as plant and animal tissues, biofilms, food, pharmaceuticals and nanoparticles in suspensions, among numerous others.

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DIC Microscopy

Differential interference contrast microscopy
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Transmission optical microscopy illuminates the sample from one side and images it from the other. Optimal illumination is vital for a good quality image. Some samples are naturally dark, or coloured, but most biological specimens will require staining. There is a huge range of stains available, many of them highly specific,... Show more »

Transmission optical microscopy illuminates the sample from one side and images it from the other. Optimal illumination is vital for a good quality image. Some samples are naturally dark, or coloured, but most biological specimens will require staining. There is a huge range of stains available, many of them highly specific, though certain formulations such as haematoxylin and eosin, or Masson’s trichrome, are quite routine in histology. Typically samples will be fixed, embedded in paraffin wax or a water-miscible plastic resin, and sectioned. Paraffin sections are then washed with a solvent such as xylene to remove the wax before staining. Resin sections are stained without removal of the resin. A coverslip is affixed with a suitable mounting medium, which should match the refractive index of glass.
Optical contrast techniques provide a method of observing detail in material, such as living cells, which cannot be stained. Phase contrast introduces contrast based on refractive index, while differential interference contrast (DIC) gives contrast based on the local rate of change in refractive index, which gives an artificial relief appearance to the image. Typically phase contrast is better for thin samples such as cell monolayers or bacteria, while DIC is better for thicker samples such as embryos or protozoa. Living cells will need to be in an aqueous medium and for high resolution it is therefore necessary to use water immersion lenses, which are designed for the refractive index of water.

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Dynamic Light Scattering

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DLS particle sizing

DLS particle sizing

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Electron Backscatter Diffraction Microscopy

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Electron backscatter diffraction (EBSD) is a relatively new analytical technique that is increasingly common on scanning electron microscopes (SEMs). Inside the SEM chamber, flat and polished samples are tilted to a high angle (typically 70°) and the electron beam is rastered across a regular grid of points on the sample surface.... Show more »

Electron backscatter diffraction (EBSD) is a relatively new analytical technique that is increasingly common on scanning electron microscopes (SEMs). Inside the SEM chamber, flat and polished samples are tilted to a high angle (typically 70°) and the electron beam is rastered across a regular grid of points on the sample surface. At each point a diffraction pattern is projected onto an EBSD detector; the pattern is indexed by the EBSD software and the phase and the crystallographic orientation are stored. The whole process is fully automated and very fast (nowadays >100 points per second is commonplace), and can provide information on a range of scales varying from <100 nm to several centimeters.

Additionally, EBSD can be combined with energy dispersive X-ray spectroscopy (EDS) to allow identification of unknown phases in a sample. The chemical measurements using EDS are used to find a list of matching candidate phases from a phase database, and the corresponding diffraction pattern is then indexed using these candidate phases in order to find the phase that matches both the chemistry and crystallography. It is a simple and fast approach to phase identification, but the results are only as good as the phase database(s) that are used.

EBSD only works on crystalline materials, and so is generally limited to minerals, metals and ceramics. It is impossible to measure amorphous materials such as glass, plastics, wood and most biological materials, and the relatively high beam current needed for good diffraction patterns makes the analysis of beam sensitive samples (such as polymers) practically impossible. The measurement of the orientations is typically with <1° error, and the spatial resolution of the technique is in the range of 20–100 nm (dependent on the sample and the SEM). This relatively high resolution means that the preparation of the sample is very important, as standard mechanical polishing techniques general introduce damage into the crystal lattice close to the surface, degrading the quality of the EBSD data. It is usually necessary to have a final stage of polishing using one of the following four approaches:

Mechanical-chemical polishing using colloidal silica or colloidal alumina.
Electropolishing.
Chemical etching.
Ion polishing (either using broad ion beam or focused ion beam techniques).
Automated EBSD measurements can generate datasets with many millions of analysis points, so the processing of the data is an important part of the technique. The datasets can be reconstructed into maps of the surface of the sample showing orientations, phase distributions, grain and sub-grain boundaries, localised deformation, grains and so on. Quantitative measurements can be made of the local and global textures (the degree and nature of alignment of the crystal lattices), grain sizes and boundary populations. This wide range of measurements makes EBSD an ideal technique to study the microstructures in many different materials and across many fields of research, including metallurgy, microelectronics and geology.

A final, recent development of EBSD has been 3-D EBSD. This is a technique in which a series of automated EBSD datasets (orientation maps) are collected from serial sections through the volume of a sample; the resulting orientation and phase maps are reconstructed to form a 3-D dataset. A focused ion beam (FIB) SEM is usually used to section through the sample, limiting the technique to relatively small volumes (i.e. <20 x 20 x 20 mm) but providing high resolution in the z-direction (i.e. <50 nm between successive slices). 3-D EBSD can now be fully automated, although the collection of data can take a long time (>20 hours is typical) due to the relatively slow rate of material removal in the FIB-SEM.

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Electron Beam Lithography (EBL)

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Electron beam (or e-beam) lithography (EBL) is a technique that enables the user to create extremely small structures on the surface of a sample, often down to the 10s of nm scale. It is usually applied in the field of microelectronics, often to develop semiconductor components on the sub-micrometre scale. The principle of the... Show more »

Electron beam (or e-beam) lithography (EBL) is a technique that enables the user to create extremely small structures on the surface of a sample, often down to the 10s of nm scale. It is usually applied in the field of microelectronics, often to develop semiconductor components on the sub-micrometre scale. The principle of the technique involves scanning an electron beam in a predetermined pattern across the surface covered with a thin film (normally referred to as the “resist”). The electron beam selectively removes the resist creating patterns that can subsequently be transferred to the substrate, such as Si wafers. During the exposure process, secondary electrons are generated by the forward scattering and back scattering of the incident electron beam, causing the exposed regions of the resist material to change its solubility and hence allow for its selective removal (or the selective removal of unexposed areas) in the subsequent development processes. Because EBL has much higher resolution than conventional optical lithography, it is often used for photomask fabrication and nanoscale device prototyping in research. It is possible to achieve sub 100 nm patterning of silicon wafers.

The throughput and the sensitivity of EBL exposure are mainly affected by the frequency of beam blanking and beam settling of the control system, where a beam blanker and pattern generator controls the movement and dwelling of the beam on the polymer surface.

The substrates can be metallic, semiconducting or insulating wafers, but must be flat and with maximum dimensions of 20 x 20 mm. These substrates should be spin-coated with an electron-sensitive polymer resist e.g. PMMA (positive tone), HSQ (negative tone) or a variety of other commercial resist formulations. The samples should be prepared at low contamination level and annealed in vacuum oven.

Patterns designs should be created in GDS-II or BMP formats and the dimensions should not exceed the exposure field. Commercial software and freeware exists for creating pattern designs. A maximum field of 1 x 1 mm can be achieved although there is generally a loss of resolution for larger write fields in order to maintain reasonable write times. Field stacking is possible however.

Depending on the type of polymer resist used, a post-exposure bake may be required. The development normally involves the dipping of sample in aqueous or solvent-based developers followed by rinsing with deionised water or IPA, and drying in N2. Determination of the optimum protocols and developers may require experimentation, although parameters can often be obtained from the literature or resist manufacturers.

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Electron Probe Microanalysis

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Typically you would choose to do electron probe microanalysis (EPMA) when accurate quantified chemical compositions are required. It is commonly used to determine the compositions of mineral phases for geological samples but is also used in cases where quantified compositional information is required e.g. metal phases within... Show more »

Typically you would choose to do electron probe microanalysis (EPMA) when accurate quantified chemical compositions are required. It is commonly used to determine the compositions of mineral phases for geological samples but is also used in cases where quantified compositional information is required e.g. metal phases within welds, zoning or compositional variations across minerals or within mineral phases.

EPMA is a powerful analytical tool of electron microscopy, unique in its mode of non-destructive X-ray analysis of elements from boron to plutonium. It uses wavelength dispersive spectroscopy (WDS) to count X-ray peak intensities and their associated background counts with superior X-ray energy resolution than the commonly used energy dispersive spectroscopy (EDS). Certified standards of materials, minerals and metals are used in EPMA to calibrate the measurement of X-ray intensities for comparison with the sample of interest. The raw data is then processed with corrections associated with atomic number, X-ray absorption and fluorescence to produce highly accurate quantitative compositions of the sample.

Typical EPMA analyses of oxide minerals will acquire raw data of cation element concentrations and subsequently calculate oxygen concentration based on the valency of those cations analysed. Detection limits for cations can be as low as 100 ppm depending on the specific element of interest, the instrument, the acquisition times and calibration.

In contrast to the normalised standard-less X-ray analysis most commonly used in EDS on the scanning electron microscope, EPMA provides highly quantitative compositional data as it incorporates a fully calibrated analytical technique. EDS has X-ray energy resolution in the order of 120–150 eV whereas WDS used on the electron microprobe has X-ray energy resolutions in the order of 10 eV. This improved X-ray energy resolution allows you to resolve complex overlaps in the X-ray energy spectrum using the EPMA, which is not possible to resolve using EDS. An example would be in the analysis of rare-earth elements where common peak overlaps are observed with lead and sulphur.

For each analysis EMPA provides a sum total of element concentrations. The value of this sum provides additional information about the quality of the data, e.g. are all possible elements included in the analysis, is the mineral hydrated, what is the true the valency of each cation and are there possible overlaps in the X-ray spectrum?

Sample preparation for EPMA is critical for the quality of data. Since the X-ray intensities generated on the unknown sample are being compared to that of highly polished certified calibration standards it is important that the unknown sample be prepared and polished as well as the standards. Poor sample polish will increase X-ray scattering from the sample and result in poor analyses. Samples that are not easily prepared as flat polished samples can be difficult to analyse by EPMA. Due to the higher beam currents used in this technique, those samples that degrade easily under higher beam currents may not be possible to analyse.

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Ellipsometry

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Energy-Filtered Transmission Electron Microscopy (EFTEM)

Energy filtered transmission electron microscopy
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ESEM

Environmental scanning electron microscopy
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Scanning electron microscopes can be operated in a variety of vacuum states (or modes): high vacuum for conventional SEM and low vacuum (reduced vacuum or variable pressure) and environmental SEM (ESEM) for viewing samples in their natural state without the need for desiccation.

For viewing samples using a high vacuum mode,... Show more »

Scanning electron microscopes can be operated in a variety of vacuum states (or modes): high vacuum for conventional SEM and low vacuum (reduced vacuum or variable pressure) and environmental SEM (ESEM) for viewing samples in their natural state without the need for desiccation.

For viewing samples using a high vacuum mode, samples must be completely dry and conductive. Nonconductive samples are usually coated with a thin layer of gold, platinum or carbon. However this can limit further use of the sample. In the low vacuum mode and in ESEM, samples can be viewed without being conductive. They do not need to be coated or to have extensive sample preparation such as fixation and dehydration. The absence of coating is ideal when compositional analysis using spectroscopic techniques is required as it can be undertaken on the native sample without being compromised by the coating material.

An advantage of using the low vacuum (LV) mode in SEM is that the pressure can be adjusted in the sample chamber until the artefact of “electron charging” is removed from images. This charging artefact occurs when electrons from the electron beam build up in a nonconductive sample. The extra electrons then discharge from the sample unpredictably, causing lines and streaks on the generating image, or they repel the beam, causing jumps in the image or the appearance of black patches.

In LV mode samples are imaged using backscattered electron imaging (BSE) and therefore, nonconductive, uncoated samples viewed in this way can provide information about composition via the contrast of the image: whiter regions have a higher average atomic number than darker regions.

The LV mode can also be used to freeze-dry samples. The sample is placed on a mount, plunged into liquid nitrogen and then placed on the SEM machine stage. The chamber is then pumped free from air (evacuated). It takes about 10 minutes to remove the water from the frozen sample, as it warms up. It is then ready for viewing. This technique works best on samples that have some basic structural integrity, such as plant tissue.

A wide variety of samples can be viewed and analysed using the LV mode; for example insect tissues, plant material, biological and non-biological polymers such as hydrogels, particulate samples, and geological materials.

It should be noted that while LV mode allows adjustment of the pressure within the sample chamberthis is not to the degree achieved in an ESEM.ESEM, like LV SEM, has the advantage over conventional SEM of being able to image samples in their natural state. It can be used in what is termed the wet mode where samples containing water can be viewed without the need for desiccation. Relative humidity (RH) can be controlled within the chamber by adjusting the temperature of the conventional stage (±20º C) along with the pressure. For example a relative humidity of 100% can be achieved by combination of low temperature (e.g. 4º C) and high water vapour pressure (e.g. 6.1 Torr). The advantage of using 100% RH is that the sample is not being dehydrated as it is being imaged. Water can also be condensed on the samples by going above 100% RH.

Dynamic experiments can also be carried out on wet samples in real time, involving heating on a specialised hot-stage, anywhere up to 1500º C, cooling, wetting and drying. The samples can be imaged while these dynamic processes are occurring. Some examples of experiments that can be undertaken in the ESEM include the determination and imaging of melting dynamics for physical science materials; determination of crystallisation dynamics; and imaging of biological processes, for example pollen tube growth in real time through wetting of pollen.

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Fluorescence Activated Cell Sorting (FACS)

Fluorescence Activated Cell Sorting
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FRET

Fluorescence resonance energy transfer
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Fluorescence resonance energy transfer (FRET) is an interaction between the electronic excited states of two dye molecules. Excitation energy is transferred from a donor molecule to an acceptor molecule without emission of a photon from the donor. FRET depends on the inverse sixth power of the intermolecular separation, making it... Show more »

Fluorescence resonance energy transfer (FRET) is an interaction between the electronic excited states of two dye molecules. Excitation energy is transferred from a donor molecule to an acceptor molecule without emission of a photon from the donor. FRET depends on the inverse sixth power of the intermolecular separation, making it useful over distances comparable with the dimensions of biological macromolecules, which are typically 2 nm to 50 nm. The efficiency with which this energy transfer is conducted can be used as a “molecular ruler” to determine distances between molecules. This powerful technique allows us to study molecular interactions within cells in live and fixed conditions. There are several conditions that need to be met before FRET will occur:

donor and acceptor molecules must be in close proximity (typically 1–10 nm).
absorption (excitation) spectrum of the acceptor must overlap fluorescence emission spectrum of the donor.
donor and acceptor transition dipole orientations must be approximately parallel.
When FRET occurs the donor fluorescence is diminished and the acceptor fluorescence is increased. The amount of these intensity changes is determined by the FRET efficiency which is directly proportional to the distance between any pair of fluorophores. While this is an incredibly powerful technique care must be taken in setting up the experiment.

This technique can be used on both wide-field and confocal microscopes for examining either fixed or living tissue.
Selection of efficient dye pairs is critical.
When calculating FRET results, excitation cross talk, bleed-through emission, background fluorescence and the possibility of bleaching must all be taken into account.
Appropriate control preparations are essential.

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FT-IR

Fourier transform infrared spectroscopy
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Field Ion Microscopy

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ield ion microscopy (FIM) permits inspection of the location of atoms on the surface of a needle-shaped specimen tip with a radius of around 50 nm. It was the first technique, developed in 1951, that permitted clear observation of atoms. The imaging is achieved by backfilling an ultra-high vacuum analysis chamber with an imaging... Show more »

ield ion microscopy (FIM) permits inspection of the location of atoms on the surface of a needle-shaped specimen tip with a radius of around 50 nm. It was the first technique, developed in 1951, that permitted clear observation of atoms. The imaging is achieved by backfilling an ultra-high vacuum analysis chamber with an imaging gas, such as helium or neon, which adsorbs to the specimen tip, and then desorbs through field-ionisation and is directed to a detector by an electric field. The specimen is kept at a temperature of between 20 and 80 K during observation depending on field-ionisation conditions. The FIM instrument is a point-projection microscope – the desorbing gas atoms project in a near perpendicular direction from their original point upon the surface of the specimen tip. As a result, atomic resolution may be clearly discerned, corresponding to a magnification of a few million times. FIM is typically applied to crystalline and amorphous metal alloys and semi-conductor devices. It has selective applicability to polymers and biological specimens and these sample types are at the frontier of research in this area.

The projected image from FIM can qualitatively communicate atomic structure and chemical environments. Select in-situ observations may also be made, for example, of corrosion or catalysis chemical reactions. Field-ionisation of the specimen surface atoms may be performed in parallel to observing the surface using the imaging gas. Voltage or laser pulses are used to field-ionise the specimen atoms for conductive or semi-/non-conductive materials, respectively. The ionisation of the specimen's surface atoms reveals more of the internal structure of the material for inspection. Applications for FIM typically include investigating the variables involved in treatment or processing parameters (thermal annealing time, composition alterations, etc.) and identifying the physical and chemical structures that relate to a material's behaviour (e.g. mechanical or electrical).

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Flow Cytometry

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Flow cytometry and cell sorting

Flow cytometry allows rapid multiparametric analysis of fluorescently labeled cells and particles in suspension. The suspension is passed in front of a laser causing excitation of the attached fluorophores. The emitted light is then split into different wavelengths (colours) and collected using a... Show more »

Flow cytometry and cell sorting

Flow cytometry allows rapid multiparametric analysis of fluorescently labeled cells and particles in suspension. The suspension is passed in front of a laser causing excitation of the attached fluorophores. The emitted light is then split into different wavelengths (colours) and collected using a number of photomultiplier tubes. High-speed analysis allows fast and accurate quantitation of population statistics and fluorescence intensity changes. As well as population analysis of the suspended cells or particles, cell sorters allow cells to separated and isolated on the basis of the attached fluorophores. These techniques can analyse and sort both living and fixed cells.

Flow cytometry is routinely used in clinical diagnostics, especially leukaemia and AIDS, but has many applications in research. These include:

immunophenotyping, where cells are labeled with fluorescently tagged antibodies
DNA and RNA analysis
cell viability
cytokine expression
kinetics and proliferation
intracellular calcium
oxidative burst
intracellular pH
phagocytic activity
mitochondrial membrane potential
identification of phytoplankton populations
Flow cytometry analysisgenerates bivariate dot-plots and histograms that can be used to calculate population statistics and changes in mean fluorescence intensity within the sample. Further information on these techniques is listed below, but this list is by no means exhaustive.

Immunophenotyping

Cells can be rapidly identified and quantified by staining with fluorescently tagged antibodies to both extracellular and intracellular antigens. A large number of fluorophores are available and, depending on the instrument, up to eight different colours can be run simultaneously. High-speed instruments can run up to 10,000 cells per second allowing identification of rare populations.

DNA analysis and ploidy determination

Using fluorescent dyes that bind stoichiometrically to DNA (fluorescence is directly proportional to DNA content) researchers can perform a number of assays including measurement of cell cycle, cell kinetics and proliferation. A further application is determining ploidy (the number of chromosomes in a cell), which is particularly useful in plant breeding programs.

Cell viability and apoptosis

Flow cytometry offers numerous assays for the study of apoptosis and cell death. These include DNA degradation, mitochondrial membrane potential, staining with antibodies specific for caspase proteins, and the commonly used Annexin V, 7AAD kits for detection of early apoptosis and cell death. Viability markers such as Propidium Iodide (PI) or 7AAD are only able to enter cells with a compromised membrane and are commonly included in flow cytometry assays to ensure only viable cells are analysed.

Cell sorting

Fluorescence activated cell sorting (FACS) allows the simultaneous separation of up to four pure populations from a heterogeneous suspension sample of cells, nuclei, bacteria or phytoplankton. Particles varying from 200 nm to 60 µm in size, can be sorted at a speed of up to 90,000 cells per second. The collected cells or particles may then be used for subsequent analysis, tissue culture or injection into experimental animal models.

Aquatic pico phytoplankton

Rapid and accurate identification of vast numbers of phytoplankton cells can be achieved by taking advantage of inherent autofluorescent properties of different phytoplankton species. Detection of cell pigments such as chlorophyll and phycoerythrin together with side scatter populations can provide “fingerprints” for different phytoplankton cells and therefore allow researchers to quantify these populations in marine and freshwater samples.

Soluble protein quantitation

Bead-based assays allow detection and quantification ofoligonucleotidesand soluble proteins from serum, cell supernatant and tissue homogenates. The benefits offered over traditional ELISA assays include much lower sample volume, analysis of multiple analytes in the same sample (multiplexing) and a higher dynamic range of sensitivity. Applications include the detection of cytokines (replacing conventional ELISA), nucleic acid assays, serology isotyping and enzyme-ligand research. Samples are run in 96-well plates with sample volumes as low as 25 µl capable of multiplexing of up to 100 analytes per well. Software is able to create standard curves using a number of fitting methods and can automatically calculate analyte concentrations.

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Fluorescence Correlation Spectroscopy

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FCS is a highly sensitive technique that measures fluctuations in the intensity of fluorescence over time. It can be used to study the interactions and relative movements of fluorescent or fluorescently-labeled molecules due to diffusion, or physical or chemical interactions. It provides extremely detailed time-resolved data from... Show more »

FCS is a highly sensitive technique that measures fluctuations in the intensity of fluorescence over time. It can be used to study the interactions and relative movements of fluorescent or fluorescently-labeled molecules due to diffusion, or physical or chemical interactions. It provides extremely detailed time-resolved data from a given area of interest in the nanosecond- to millisecond-range with single-molecule sensitivity. It therefore allows the behaviour of molecules to be observed in living cells. It is usually used on a confocal or multiphoton microscope.

Because fluctuations in the signal are measured, the technique works best when the numbers of labeled molecules in the sample area are small. In this way, changes in the number of molecules in the target area are significant as a proportion of the total fluorescence present. This problem needs to be balanced with the opposite situation, where too few molecules enter or exit from the target area to give any significant fluctuations and therefore, meaningful results. By using differently coloured fluorescent markers, several species of molecule can be observed simultaneously.

When optimised, FCS can be used to obtain quantitative information on properties such as:

diffusion coefficients
hydrodynamic radii
average concentrations
kinetic chemical reaction rates
singlet-triplet dynamics

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FLIM

Fluorescence lifetime imaging microscopy
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Fluorescence lifetime imaging microscopy (FLIM) is a technique in which the mean fluorescence excitation lifetime of a fluorophore is measured at each position of a microscope image. Most biologically-active fluorophores typically have excitation lifetimes in the nanosecond timescale and this lifetime is independent of probe... Show more »

Fluorescence lifetime imaging microscopy (FLIM) is a technique in which the mean fluorescence excitation lifetime of a fluorophore is measured at each position of a microscope image. Most biologically-active fluorophores typically have excitation lifetimes in the nanosecond timescale and this lifetime is independent of probe concentration and photobleaching.The factors that do affect fluorescence lifetimes include ion intensity, hydrophobic properties, oxygen concentration, molecular binding, and molecular interaction. Fluorescence resonance energy transfer (FRET), which is the energy transfer when two fluorescent proteins approach each other, affects the excitation lifetime. Therefore FLIM can be used to measure FRET. These variables mean fluorescence lifetime imaging is a particularly valuable technique to obtain information about the molecular environment of labeled macromolecules at each microscopically resolvable locationin living cells.

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Fluorescence-Based Microscopy

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Fluorescence microscopy illuminates the sample through the objective lens, with short wavelength light, which will excite a fluorescent dye, or fluorochrome. The fluorochrome will emit light at a longer wavelength and a wavelength-selective dichroic mirror in the optical path will let this light travel to the eyepieces or camera.... Show more »

Fluorescence microscopy illuminates the sample through the objective lens, with short wavelength light, which will excite a fluorescent dye, or fluorochrome. The fluorochrome will emit light at a longer wavelength and a wavelength-selective dichroic mirror in the optical path will let this light travel to the eyepieces or camera. Typically excitation is with near-ultraviolet, blue or green light, and emission is respectively in the blue, green or red. Some samples are naturally fluorescent but most will need to be stained. There are three approaches to fluorescent staining:

Fluorescent dyes
Some dyes are designed to target specific cellular structures and chemical entities. The best known is DAPI, which stains DNA, but stains are available for most cell structures and organelles. Some (vital stains) will work on living cells, others require the cells to be fixed. Calcium movement within and between cells is commonly studied using calcium-specific fluorescent dyes in living cells.
Immunolabeling
This targets specific proteins in the cell with antibodies raised in some other animal (rat, mouse, goat or rabbit). The antibody is coupled to a fluorescent dye. The cells will normally be fixed to allow penetration of the antibody. After staining the cells are usually mounted in a medium (often glycerol-based) containing an anti-fade agent, since fluorescent dyes do fade under continued exposure to light.
Fluorescent proteins
The original fluorescent protein, green fluorescent protein (GFP) was isolated from a jellyfish. Now a range of other colours is available, some genetically modified from GFP, others derived from related organisms (corals and sea anemones). These are genetically encoded proteins, which are expressed within the cell and therefore allow us to observe living material. Typically a construct is made which will code for the FP attached to a target molecule, for example tubulin. This is either incorporated into the genome (permanent transfection) or introduced into the cell (temporary transfection). Then the targeted structure (microtubules in this example) will be fluorescent, and can be observed dynamically in a living cell. The sample will be in an aqueous medium, so the same caveats about correct objectives apply as for other living cells. Since this technique involves genetically modified organisms.

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FRAP

Fluorescence recovery after photobleaching
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Fluorescence recovery after photobleaching (FRAP) involves bleaching a defined area of a sample with high-intensity light and then recording the recovery of fluorescence into the bleached area using a lower light intensity (low enough to record a signal, but not to induce further bleaching). The recovery of fluorescence into the... Show more »

Fluorescence recovery after photobleaching (FRAP) involves bleaching a defined area of a sample with high-intensity light and then recording the recovery of fluorescence into the bleached area using a lower light intensity (low enough to record a signal, but not to induce further bleaching). The recovery of fluorescence into the bleached area then results from movement of unbleached fluorophores from surrounding areas moving into the bleached region. The rate of this recovery can be used to estimate the mobility of molecules and to determine, diffusion, active transport, synthesis or natural turnover of those molecules. An alternative technique measures the decrease in fluorescence in surrounding areas to determine these rates. This is called fluorescence loss in photobleaching or FLIP.

FRAP can be used to determine whether there is transport between different cellular compartments within a cell. For example, FRAP may be able to determine:

if molecules are moving from or between organelles, or from the cytoplasm into the nucleus.
if molecules are relocating within a cellular membrane.
if molecules are being incorporated into other molecules.
the speed at which molecules are moving.
As FRAP is measuring an active process, experiments are usually carried out on live samples. Special care must be taken the ensure samples are prepared in such a way that measurements most accurately represent activity in the normal cellular environment.

Confocal microscopes are usually used for FRAP experiments as they allow very accurate definition of the region of interest which is to be bleached. Additionally, all modern confocal microscopes have very precise control over input powers and, therefore, bleaching and subsequent measurements can be accurately controlled. This is very important since excessive bleaching can cause molecular alteration and cellular damage resulting in meaningless data.

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Focused Ion Beam Tomography

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A focused ion beam (FIB) instrument uses an ion beam (typically gallium ions), which is focused to an extremely fine probe size (<10 nm) onto the surface of a specimen. This beam is very powerful and can cut through very hard specimens. As such it is used to selectively mill precise areas of material from the specimen surface.... Show more »

A focused ion beam (FIB) instrument uses an ion beam (typically gallium ions), which is focused to an extremely fine probe size (<10 nm) onto the surface of a specimen. This beam is very powerful and can cut through very hard specimens. As such it is used to selectively mill precise areas of material from the specimen surface. This technique can be used for cross-sectioning and imaging, along with the fabrication of specimens for other microscopy and microanalysis techniques such as transmission electron microscopy (TEM) and atom probe tomography (APT). It can cut ultra-thin sections of very hard materials that conventional microtomy cannot manage. This allows transmission electron microscopy of these samples where it would otherwise not be possible. FIB can shape materials for a variety of purposes such as the needle-shaped samples for use in an atom probe instrument. Many current-generation focused ion beam platforms consist of both ion and electron columns on a single instrument (called dual beam instruments), allowing the specimen to be imaged in detail using the electron beam, without damaging the surface of the specimen with the ion beam.

Additionally, various conducting, non-conducting and semiconducting materials may also be deposited onto selected areas of the surface (of a diameter of approximately 100 nm) by applying specific gases to the surface, which are decomposed onto the surface only in areas to which the ion beam is applied.

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ICP-MS

Inductively Coupled Plasma Mass Spectrometry
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Request a quote for more information about this service.

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Infrared Spectroscopy Services

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Infrared mass spectroscopy (IRMS)

Infrared mass spectroscopy (IRMS)

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LAICP-MS

Laser Ablation Inductively Coupled Plasma Mass Spectrometry
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LAICPMS is used to quantify trace elements in a sample. This involves focussing a high-energy, pulsed-UV laser onto the surface of the sample, releasing (or ablating) material that is then transported into the argon plasma where it is broken down into constituent atoms and ionised. It then passes into the mass spectrometer where... Show more »

LAICPMS is used to quantify trace elements in a sample. This involves focussing a high-energy, pulsed-UV laser onto the surface of the sample, releasing (or ablating) material that is then transported into the argon plasma where it is broken down into constituent atoms and ionised. It then passes into the mass spectrometer where the elements of interest are analysed. It can analyse individual spots or, in some instruments, the focussed laser beam can be rastered the across the sample so that intensity and spatial distribution of elements can be recorded. Post acquisition processing then allows an image of elemental intensity to be compiled.
Existing techniques used to investigate trace element distribution in mineral and biological samples include electron probe microanalysis, transmission electron microscopy, scanning electron microscopy, ion microprobe, proton microprobe, synchrotron X-ray fluorescence and X-ray diffraction analysis. These all have their benefits, but also their inherent limitations such as high detection limits (>0.1–1.0 wt%), very small analytical areas, complex sample preparation, and often prohibitively expensive instrumentation. Although its spatial resolution will never compare to electron and proton beam techniques, imaging with LAICPMS offers the benefits of superior detection limits (ppb) over a wide elemental range from 7Li to 238U and the capacity to analyse a wide variety of materials with minimal sample preparation.
The success of mapping with LAICPMS depends on very fast signal response from ablation to detection, along with fast washout times in the ablation cell. Although standard cells do not offer this feature, more advanced cells are available in some of our locations. Performance is being optimised on a wide variety of samples including, minerals, plant material, and other biological tissue.

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Laser Capture Microdissection

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Laser microdissection (LMD) is a technique used to isolate specimens for biomedical research. A fundamental step in preparation for LMD is to develop a specimen preparation protocol that optimally balances the dissection of the sample and downstream analyses. LMD is often used to collect samples from which RNA is to be isolated.... Show more »

Laser microdissection (LMD) is a technique used to isolate specimens for biomedical research. A fundamental step in preparation for LMD is to develop a specimen preparation protocol that optimally balances the dissection of the sample and downstream analyses. LMD is often used to collect samples from which RNA is to be isolated. As RNA is particularly sensitive to degradation, the following description emphasises precautions required for this end use.

To maintain and preserve RNA integrity it is best to avoid formalin fixation as it has an adverse effect on nucleic acids and consequently downstream analysis. It is recommended that alternative fixatives for paraffin-embedded tissues are used, e.g. methacarn, DSP or HOPE.

All materials used for RNA work need to be prepared RNAase free by treating with RNase ZAP or RNase AWAY. All solutions (including staining solutions) are prepared in DEPC-treated water. Alternative treatments to remove RNases are autoclaving and UV treatment. For UV irradiation treatment of slides, place them in a UV crosslink chamber and deliver at least 1 joule of energy (maximum power for 30–45 minutes). For autoclave treatment of slides, place slides into a steel basket or slide holder and place the basket into a beaker or jar and cover tightly and autoclave at 121°C for 20 minutes. Note that sterilising slides by autoclaving or UV treatment does not guarantee complete destruction of RNases, therefore it is recommended to purchase RNase-free certified slides.

Fresh frozen tissues are preferred for isolating RNA and proteins. Best quality RNA is achieved from tissues frozen immediately after surgery, which minimises RNA degradation over time by endogenous RNases. Tissue is placed in a disposable cryomould in OCT (optimum cutting temperature) and immersed in pre-cooled isopentane (e.g. Pyrex beaker) in liquid nitrogen. The samples can then be stored at -70°C until ready for cryosectioning.

Before sectioning, the cryostat should be cleaned with 70% ethanol and RNase Zap (including the disposable microtome blade). A sheet of aluminium foil should be placed down in the cryostat to keep an RNase free area.

Sections of between 5 and 20 um in thickness can be used, however 7-10 um achieves the best balance between good quality sections and laser microdissection. In general terms, the thicker the section, the greater the laser 'power' required to dissect cells.

Specially coated slides are required for LMD applications to enable the dissected regions to be readily released from the slide. The tissue sections are mounted on these foiled membrane slides (2–3 sections per slide depending on the size of the sample).

Once the frozen sections have been mounted on the appropriate slides, there are three approaches. They can either be fixed and stained immediately for LMD or stored in sealed slide boxes (seal with masking tape) at -70°C until required. They can also be fixed and then stored at -70°C for LMD at a later date. Prior to LMD, the slides should be thawed and slowly adjusted to room temperature e.g. from -70°C to -20°C freezer for 30 mins. The slides are then taken out to thaw at room temperature in a slide box with silica gel desiccant. This helps to avoid formation of water condensation. This precaution is critical for RNA quality, especially when working with small numbers of cells, since water (moisture) activates RNases. The presence of water also affects the efficiency of the laser. If moisture is still present, place slides in 40°C oven for 5 mins.

Sections can be fixed with ice-cold acetone for 2-3 mins. 70% or 100% ethanol for 30 secs or a mixture of ethanol: acetic acid (19:1) to increase the adhesion of the tissue to the membrane slides. LMD can be performed on stained or unstained cells. Suggested stains are toluidine blue, haematoxylin and eosin or methyl-green.

To perform the dissection, the cell or groups of cells to be isolated are marked out on an image of the sample on a computer screen. The focused laser then cuts along the pre-marked path with very high precision. During excision, the membrane is vaporised which releases the dissected region from the slide. This is then either catapulted into the cap of a microcentrifuge tube for subsequent analyses, or the excised part falls into the collection cap by means of gravity. These methods depend on the instrument being used.

0.2 ml and 0.5 ml microcentrifuge tubes are best for collection and the caps may be filled with lysis buffer to prevent the degradation of nucleic acids. The instruments allow viewing of the caps to confirm the collection of dissectates.

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Live Cell Imaging

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To study many functions of cells it is desirable to observe cells in their living state. There are many ways to do this with various levels of sophistication. For very quick imaging (seconds to a few minutes) you can study cell cultures on a slide with buffered media. For longer-term experiments it is adequate to use a heated... Show more »

To study many functions of cells it is desirable to observe cells in their living state. There are many ways to do this with various levels of sophistication. For very quick imaging (seconds to a few minutes) you can study cell cultures on a slide with buffered media. For longer-term experiments it is adequate to use a heated stage and objective and buffered media. However, if possible, it is advisable to run your experiment in an enclosed environment where both temperature and gas are controlled, providing as normal an environment as possible for the cells. Therefore live-cell imaging systems have an incubation chamber built around the microscope.

Adherent cell lines are the easiest to work with but non-adherent ones can also be studied in these systems. The cells can be observed using fluorescence, differential interference contrast (DIC) or phase contrast, or a combination of the above, depending on the nature of the experiment. Cells can be fluorescently labeled through transfection of constructs carrying a marker such as green fluorescent protein (GFP) or by introducing vital dyes that are compatible with living cells and bind to specific cellular components such as calcium or DNA.

Some live-cell imaging systems have a multi-positioning stage allowing the observation of multiple cells over multiple wells. There is also considerable variation in the level of sophistication of the chambers in which the cells are mounted. These range from homemade chambers to commercial flow-through chambers. These set-ups allow compounds to be added to the cells and subsequent changes in cell behaviour to be studied, (e.g. calcium, pH, cellular movement etc.).

When capturing images of living cells, you need to be aware of the fact that all light is toxic to cells if they are exposed to it at high intensities. When using visible light, a green filter must be placed in the light path. Cells should only be exposed to the light for as long as it takes to gain a good image. The rule is always to expose your cells for as short a time as possible with as low light levels as possible to obtain a good image. This means that low-light-level cameras are essential.

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LV-SEM

Low vacuum scanning electron microscopy
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Scanning electron microscopes can be operated in a variety of vacuum states (or modes): high vacuum for conventional SEM and low vacuum (reduced vacuum or variable pressure) and environmental SEM (ESEM) for viewing samples in their natural state without the need for desiccation.

For viewing samples using a high vacuum mode,... Show more »

Scanning electron microscopes can be operated in a variety of vacuum states (or modes): high vacuum for conventional SEM and low vacuum (reduced vacuum or variable pressure) and environmental SEM (ESEM) for viewing samples in their natural state without the need for desiccation.

For viewing samples using a high vacuum mode, samples must be completely dry and conductive. Nonconductive samples are usually coated with a thin layer of gold, platinum or carbon. However this can limit further use of the sample. In the low vacuum mode and in ESEM, samples can be viewed without being conductive. They do not need to be coated or to have extensive sample preparation such as fixation and dehydration. The absence of coating is ideal when compositional analysis using spectroscopic techniques is required as it can be undertaken on the native sample without being compromised by the coating material.

An advantage of using the low vacuum (LV) mode in SEM is that the pressure can be adjusted in the sample chamber until the artefact of “electron charging” is removed from images. This charging artefact occurs when electrons from the electron beam build up in a nonconductive sample. The extra electrons then discharge from the sample unpredictably, causing lines and streaks on the generating image, or they repel the beam, causing jumps in the image or the appearance of black patches.

In LV mode samples are imaged using backscattered electron imaging (BSE) and therefore, nonconductive, uncoated samples viewed in this way can provide information about composition via the contrast of the image: whiter regions have a higher average atomic number than darker regions.

The LV mode can also be used to freeze-dry samples. The sample is placed on a mount, plunged into liquid nitrogen and then placed on the SEM machine stage. The chamber is then pumped free from air (evacuated). It takes about 10 minutes to remove the water from the frozen sample, as it warms up. It is then ready for viewing. This technique works best on samples that have some basic structural integrity, such as plant tissue.

A wide variety of samples can be viewed and analysed using the LV mode; for example insect tissues, plant material, biological and non-biological polymers such as hydrogels, particulate samples, and geological materials.

It should be noted that while LV mode allows adjustment of the pressure within the sample chamberthis is not to the degree achieved in an ESEM.ESEM, like LV SEM, has the advantage over conventional SEM of being able to image samples in their natural state. It can be used in what is termed the wet mode where samples containing water can be viewed without the need for desiccation. Relative humidity (RH) can be controlled within the chamber by adjusting the temperature of the conventional stage (±20º C) along with the pressure. For example a relative humidity of 100% can be achieved by combination of low temperature (e.g. 4º C) and high water vapour pressure (e.g. 6.1 Torr). The advantage of using 100% RH is that the sample is not being dehydrated as it is being imaged. Water can also be condensed on the samples by going above 100% RH.

Dynamic experiments can also be carried out on wet samples in real time, involving heating on a specialised hot-stage, anywhere up to 1500º C, cooling, wetting and drying. The samples can be imaged while these dynamic processes are occurring. Some examples of experiments that can be undertaken in the ESEM include the determination and imaging of melting dynamics for physical science materials; determination of crystallisation dynamics; and imaging of biological processes, for example pollen tube growth in real time through wetting of pollen.

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Metastable Induced Electron Spectroscopy

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Metastable induced electron spectroscopy allows surface analysis with excellent sensitivity. It measures the electron density on the very surface of the sample facilitating the analysis of surface composition and electronic structure. It can be applied to a large range of different samples including liquid surfaces generated as... Show more »

Metastable induced electron spectroscopy allows surface analysis with excellent sensitivity. It measures the electron density on the very surface of the sample facilitating the analysis of surface composition and electronic structure. It can be applied to a large range of different samples including liquid surfaces generated as surfaces of thin liquid films, and solid surfaces such as polymers and insulators mounted on a manipulator. Examples described in the literature include the analysis of catalysis, corrosion, surface coatings with monolayers, surfactant solutions, self-assembled monolayers and nanoparticles. MIES averages the surface composition over an area of 1 mm2 up to a few mm2.
In a MIES experiment the valence electrons, those that facilitate all chemical bonds, are excited with metastable helium atoms and their binding energy is determined by measuring the kinetic energy of emitted electrons, in a similar way to UV-photoelectron spectroscopy. The spectra show, however, only features that are related to those electron orbitals in the outermost layer. The same molecule placed in two different orientations on a surface will result in two readily distinguishable spectra. For instance, if alkane molecules are adsorbed onto graphite they lie lengthwise along the surface, exposing both the CH2 backbone as well as the CH3 terminal groups. If alkanethiolate molecules are adsorbed onto gold through the thiol group at one end only the terminal CH3 groups at the other ends of the molecules will be exposed to the environment. MIES spectra generated from these two samples would distinguish the different orientation of the molecules on the surface by detecting either the CH2 or CH3 groups constituting the outermost layer.
As an example the MIE spectrum of the surface of a silicon waver modified with 3-aminopropyltriethoxysilane (APTES) is shown in the figure below. The spectrum shows contributions of all valence electron orbitals of the molecule. However, predominantly the orbitals forming the C-C backbone and the CH2 groups are detected which shows that the majority of the APTES molecules are lying flat on the surface. A surface with upright standing APTES molecules would predominantly show the C-N bonding and the non-bonding N orbitals.

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Mineral Liberation Analysis

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Mineral liberation analysis (MLA) is an automated mineral analysis system based on a scanning electron microscope. Four EDS detectors are used simultaneously to rapidly determine the mineral phases in a polished section of rock. Sophisticated software differentiates the mineral from the mounting resin so that analysis time is... Show more »

Mineral liberation analysis (MLA) is an automated mineral analysis system based on a scanning electron microscope. Four EDS detectors are used simultaneously to rapidly determine the mineral phases in a polished section of rock. Sophisticated software differentiates the mineral from the mounting resin so that analysis time is devoted only to the rock itself. The results are visually presented in colour-coded mineral maps, which are readily interpreted by mineralogists and process metallurgists.

With four EDS detectors, instruments can analyse up to 200 points per second, making them a serious tool for improving productivity in the analysis of rocks in geoscience research and the mining industry. Theycan resolve down to around 3 µm within polished samples. Associated software can analyse the results in numerous ways, listing grain size, minerals, and so forth.

The system needs to be set up for an individual type of sample and the correct type of files for comparison must be available in the software system.

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Multiphoton Fluorescence Microscopy

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Multiphoton microscopy is essentially an extension of confocal imaging. Very short, intense pulses of light in the near infrared are used for illumination and fluorescence is excited when two photons hit the fluorochrome molecule simultaneously, and behave as one photon of half the wavelength. The intensity of a single pulse is... Show more »

Multiphoton microscopy is essentially an extension of confocal imaging. Very short, intense pulses of light in the near infrared are used for illumination and fluorescence is excited when two photons hit the fluorochrome molecule simultaneously, and behave as one photon of half the wavelength. The intensity of a single pulse is very high, but the spaces are much longer than the pulses, so that averaged over time the irradiation is comparable to confocal microscopy. Since two-photon events will only take place at the focus of the excitation spot, optical sectioning is automatic without any confocal pinhole. This means that detection can be made more efficient, particularly in a scattering sample, and it also means that that there will be no bleaching above and below the focal plane.

The longer wavelengths used in multiphoton imaging penetrate better into thick samples, and are also less damaging to living cells, than the wavelengths used for conventional fluorescence. Other key benefits are the ability to excite fluorochromes such as DAPI and calcium ratio dyes which would normally require near UV, and a wide wavelength selection since the lasers used can be tuned through the range 700–1000 nm.

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NMR

Nuclear magnetic resonance spectroscopy
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Nuclear magnetic resonance (NMR) spectroscopy is most frequently used by chemists and biochemists to study organic molecules and has application in a wide range of disciplines including physics, engineering, plant biology, soil science, medicine, pharmacy, sports science and marine archaeology. It is based on the fact that certain... Show more »

Nuclear magnetic resonance (NMR) spectroscopy is most frequently used by chemists and biochemists to study organic molecules and has application in a wide range of disciplines including physics, engineering, plant biology, soil science, medicine, pharmacy, sports science and marine archaeology. It is based on the fact that certain nuclei (e.g. 1H, 13C, 31P) can absorb energy from the radio frequency range of the electromagnetic spectrum, when placed in a magnetic field. The utility of NMR arises because the frequency of the absorbed energy is characteristic of the chemical environment of each nucleus in the sample. Further interactions via the chemical bonds or through space can provide information showing the interconnection, proximity and angular relationship of nearby nuclei. This information can be used to determine the structure of a molecule or in the case of a mixture, the nature and quantities of the different components.

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NanoSIMS

Nano Secondary Ion Mass Spectrometry
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NanoSIMS is a new generation of ion-microprobe analysis that combines high spatial resolution with high sensitivity, offering highly versatile imaging and analysis opportunities across the fields of biology, earth science and materials science. The technique employs secondary ion mass spectrometry (SIMS), where a high-energy ion... Show more »

NanoSIMS is a new generation of ion-microprobe analysis that combines high spatial resolution with high sensitivity, offering highly versatile imaging and analysis opportunities across the fields of biology, earth science and materials science. The technique employs secondary ion mass spectrometry (SIMS), where a high-energy ion beam ablates material from a sample surface and this material is then analysed in a mass spectrometer. The advantage of nanoSIMS is that a newcoaxial lens design allows the primary beam to be focused down to 50 nm, compared to the tens-of-micrometres typical in conventional SIMS.

NanoSIMS uses a rastered ion beam to scan across a sample surface in a manner similar to an SEM. The secondary ions that are sputtered from the sample are sorted by their atomic mass in a magnetic-sector mass spectrometer. The ions are collected and counted by electron multipliers at the exit of the mass spectrometer. Multiple detectors allow parallel mapping of up to five ion species, simultaneously. The recorded data set simply consists of the number of counts recorded for each pixel of the scan, for each ion species. These data are then reconstructed to produce an image showing the surface composition of the sample.

As the mass spectrometer distinguishes between ions by mass, it is possible to distinguish different isotopes of the same element. Furthermore, the high mass resolution allows the separation of overlapping peaks on the same mass – for example, 13C and 12C1H at 13 atomic mass units. This allows the precise measurement of isotope ratios by tuning the detectors to measure, for example, 12C and 13C simultaneously.

The high-resolution ion imaging capability is a powerful and unique feature of nanoSIMS, and is highly suitable for elemental or isotopic mapping at the sub-micrometre scale. This is a particularly powerful technique for mapping the distribution of isotopic tracers (e.g. 15N or 13C in biological experiments). Additionally, high-resolution linescans can also be obtained, which can be used to show diffusion profiles across phase boundaries. As the technique is destructive, it can also be used for depth profiling, in particular where layers are too thin to be resolved though other techniques. Combining depth profiling with imaging allows 3-D images to be obtained. Due to the fine-scale beam, depth profiling is generally limited to less than a micrometre in depth.

The main advantage of nanoSIMS is also its chief limitation: as the size of the primary beam gets smaller, the sample interaction volume gets smaller and the number of secondary ions sputtered from the surface decreases. It can therefore take a long time to acquire an image, as each pixel requires a longer dwell time. Another major limitation is in quantification. Different elements have different secondary ion yields, and these ion yields change between different matrices. This makes it very difficult to calibrate the instrument for absolute concentrations, especially in fine-scale, multi-component systems. Where quantification is required, it is necessary to analyse a well-characterised standard with an identical matrix, concurrently.

The benefits of SIMS in general are that it is very sensitive, with a large dynamic range where major and trace elements can be measured simultaneously. It can also detect most of the elements in the periodic table, although some elements require a high degree of fine-tuning. Secondary ion yields are dependent on the ionisation potential or electron affinity of the desired element. In nanoSIMS, negative secondary ions (C–, O–, Si–, CN–, S–) can only be generated with a Cs+ primary beam, where as positive secondary ions (Na+, Ca+, Si+, Fe+, etc) can only be generated with a O- primary beam. The Cs+ primary can be focussed down to less than 50 nm, while the O- primary beam can only achieve around 200–150 nm, leading to a small discrepancy between positive and negative ions images.

Examples of analyses performed using nanoSIMS:

Biological

Mapping metals in cultured cells after treatment with anti-cancer compounds.
Mapping and measurement of isotopic labels (15N) in bacterial communities at the scale of individual microbes.
Measuring nutrient uptake by plant roots, fungi and bacteria in soil.
Sub-cellular distribution of metals in the leaves of hyper-accumulating plants.
Earth and planetary science

Distribution of ‘invisible’ Au in ore-bearing pyrite.
C and N mapping in ancient sedimentary structures to determine biogenicity.
Element distributions in primitive meteorites.
Variations in elemental ratios as climate proxies in biomineralised marine microorganisms.
Materials science

Composition changes with depth through thin-films and doped semiconductors.
Composition changes around sulphide inclusions in stainless steel.
Composition of phases within stress-corrosion cracks in steel.
Samples must be vacuum-compatible (10-9 torr), solid, flat and polished. They must also be electrically conductive. Insulating samples can be coated with Au, Pt or C to provide conductivity, and an electron flood gun can be used to compensate charge build up when using the Cs+ primary beam. Sample charging is less of an issue when using the O– primary.

Sample holders can accommodate 25 mm, 12.5 mm and 10 mm discs, and it is usually possible to embed irregular shaped samples in resin or Wood's metal, and then grind and polish to the desired grade. Geological glass slides do not fit unless they are cut down to fit inside the 25 mm mount. Biological materials must be fixed and sectioned (500 nm to 1 µm sections work best), mounted flat on either 2.5 mm diameter Si wafer or Au-coated Al stubs. TEM samples mounted on standard 3 mm formvar-backed Cu grids can also be accommodated, although it is difficult to get a strong signal from very thin TEM sections (less than 100 nm).

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Optical Waveguide Lightmode Spectroscopy

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Techniques that enable researchers to monitor the dynamic binding of macromolecules (such as proteins) to a surface, for example onto surface-immobilised antibodies, have a large range of applications in the life sciences, chemistry and biomedical device engineering.

Optical waveguide lightmode spectroscopy (OWLS) is such a... Show more »

Techniques that enable researchers to monitor the dynamic binding of macromolecules (such as proteins) to a surface, for example onto surface-immobilised antibodies, have a large range of applications in the life sciences, chemistry and biomedical device engineering.

Optical waveguide lightmode spectroscopy (OWLS) is such a technique. It is a very sensitive method that measures alterations in the refractive index in a medium, such as water, near a solid sensor surface when molecules adsorb to that sensor surface. As such it does not need the molecules to be labelled with fluorescent or radioactive tags as is often required in other analytical methods. The technique has a time resolution of seconds, which allows real-time adsorption kinetics to be measured.

The set-up comprises a flow-through cell over the test surface, which sits immediately above a wave-guiding film through which light from a He-Ne laser is diffracted by an optical grating to initiate its propagation along the waveguide by total internal reflection. The test surface is positioned within the resulting evanescent field and it is this field that probes the optical properties of the solution. A solution containing the molecules of interest can be pumped into the cell so that adsorption (and desorption if the solution is replaced by saline) can occur to the active surface. Binding can be detected at levels down to ngs/cm2.

Some of the numerous applications of OWLS include protein–DNA interactions, ligand–receptor binding, investigations of lipid bilayers, behaviour of biocompatible materials, monitoring of environmental pollution, and the interaction of surfaces with blood plasma and serum. It can also be used to measure the attachment of living cells to biomaterials surfaces, making it a valuable tool for the development of medical devices and antibacterial coatings.

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Phase Contrast Microscopy

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Transmission optical microscopy illuminates the sample from one side and images it from the other. Optimal illumination is vital for a good quality image. Some samples are naturally dark, or coloured, but most biological specimens will require staining. There is a huge range of stains available, many of them highly specific,... Show more »

Transmission optical microscopy illuminates the sample from one side and images it from the other. Optimal illumination is vital for a good quality image. Some samples are naturally dark, or coloured, but most biological specimens will require staining. There is a huge range of stains available, many of them highly specific, though certain formulations such as haematoxylin and eosin, or Masson’s trichrome, are quite routine in histology. Typically samples will be fixed, embedded in paraffin wax or a water-miscible plastic resin, and sectioned. Paraffin sections are then washed with a solvent such as xylene to remove the wax before staining. Resin sections are stained without removal of the resin. A coverslip is affixed with a suitable mounting medium, which should match the refractive index of glass.

Optical contrast techniques provide a method of observing detail in material, such as living cells, which cannot be stained. Phase contrast introduces contrast based on refractive index, while differential interference contrast (DIC) gives contrast based on the local rate of change in refractive index, which gives an artificial relief appearance to the image. Typically phase contrast is better for thin samples such as cell monolayers or bacteria, while DIC is better for thicker samples such as embryos or protozoa. Living cells will need to be in an aqueous medium and for high resolution it is therefore necessary to use water immersion lenses, which are designed for the refractive index of water.

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Polarized Light Microscopy

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Transmission optical microscopy illuminates the sample from one side and images it from the other. Optimal illumination is vital for a good quality image. Some samples are naturally dark, or coloured, but most biological specimens will require staining. There is a huge range of stains available, many of them highly specific,... Show more »

Transmission optical microscopy illuminates the sample from one side and images it from the other. Optimal illumination is vital for a good quality image. Some samples are naturally dark, or coloured, but most biological specimens will require staining. There is a huge range of stains available, many of them highly specific, though certain formulations such as haematoxylin and eosin, or Masson’s trichrome, are quite routine in histology. Typically samples will be fixed, embedded in paraffin wax or a water-miscible plastic resin, and sectioned. Paraffin sections are then washed with a solvent such as xylene to remove the wax before staining. Resin sections are stained without removal of the resin. A coverslip is affixed with a suitable mounting medium, which should match the refractive index of glass.
Optical contrast techniques provide a method of observing detail in material, such as living cells, which cannot be stained. Phase contrast introduces contrast based on refractive index, while differential interference contrast (DIC) gives contrast based on the local rate of change in refractive index, which gives an artificial relief appearance to the image. Typically phase contrast is better for thin samples such as cell monolayers or bacteria, while DIC is better for thicker samples such as embryos or protozoa. Living cells will need to be in an aqueous medium and for high resolution it is therefore necessary to use water immersion lenses, which are designed for the refractive index of water.

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Raman Spectroscopy

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Raster Image Correlation Spectroscopy

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Raster image correlation spectroscopy (RICS) exploits the time-related information that exists in sequential laser-scanned images to correlate fluorescence intensity fluctuations over time on a pixel-by-pixel basis. Data from fluctuations in fluorescence intensity can be analysed as a function of time and space by autocorrelation... Show more »

Raster image correlation spectroscopy (RICS) exploits the time-related information that exists in sequential laser-scanned images to correlate fluorescence intensity fluctuations over time on a pixel-by-pixel basis. Data from fluctuations in fluorescence intensity can be analysed as a function of time and space by autocorrelation mathematics to reveal information on diffusion, binding, flow and the state of molecular aggregation. RICS is able to provide highly detailed positional and time-resolved data on the movement of the labeled molecules in the microsecond to second range with single molecule sensitivity. Importantly, RICS can reveal fast molecular movement by filtering out the slower bulk cell movement, which is invaluable as many cellular components are constantly in motion. RICS software has been designed to be suitable for use on data from most commercially available confocal laser scanning microscopes.

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Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy services
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Scanning electron microscopy (SEM) – imaging
Scanning electron microscopy (SEM) – in-situ techniques
Scanning electron microscopy (SEM) – spectroscopy

Scanning electron microscope (SEM) imaging is one of the most commonly used techniques to image the surface of samples in the nanometre to centimetre scale. Inside an SEM, the... Show more »

Scanning electron microscopy (SEM) – imaging
Scanning electron microscopy (SEM) – in-situ techniques
Scanning electron microscopy (SEM) – spectroscopy

Scanning electron microscope (SEM) imaging is one of the most commonly used techniques to image the surface of samples in the nanometre to centimetre scale. Inside an SEM, the electron beam is scanned across the surface of the sample and the resulting signal is detected and used to build up an image. The interaction between the electron beam and the sample results in a number of different signals, and these can be detected using a variety of specialised detectors. The two most commonly detected signals are secondary electrons (SE) and backscattered electrons (BSE).

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Surface Plasmon Resonance (SPR)

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Scanning Tunneling Microscopy

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Scanning tunelling microscopy (STM) can only be applied conducting samples and is generally used for very-high-resolution imaging – less than a nanometre. The instrument can provide information on atomic or molecular order in materials in addition to map the location of electronic states. It creates images in a very similar manner... Show more »

Scanning tunelling microscopy (STM) can only be applied conducting samples and is generally used for very-high-resolution imaging – less than a nanometre. The instrument can provide information on atomic or molecular order in materials in addition to map the location of electronic states. It creates images in a very similar manner to atomic force microscopy (AFM). However, the one major difference is that STM uses current conducted between an atomically sharp probe and the substrate to build the image.

One of the capabilities that distinguishes STM from most techniques is that atomic resolution is possible for some systems. Such resolution does require careful sample preparation and generally can only be used on systems that are quite stable over long periods. As with AFM, there are two modes of operation, generally know as constant height and constant current. Constant height experiments can be done more quickly but the sample properties have to be well known and the samples flat. Otherwise there is a risk of crashing the tip into the surface, which will generally render the probe unusable. The constant current mode is slower but follows the contours of the electronic states of the surface and hence tip crashes are less likely.

Sample size is restricted to a maximum area of 1 x 1 cm and with a maximum sample thickness of approximately 3 mm.

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Scanning Near Field Microscopy

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NSOM reveals the topography and chemical composition of the sample surface down to a lateral resolution of 20 nm and vertical resolution of 2–5 nm, giving an image and/or spectra.

NSOM reveals the topography and chemical composition of the sample surface down to a lateral resolution of 20 nm and vertical resolution of 2–5 nm, giving an image and/or spectra.

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SIMS

Secondary Ion Mass Spectometry
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Secondary ion mass spectrometry (SIMS) is one of the most sensitive surface analytical techniques available for high analytical precision of in-situ quantitative measurements in solid materials. Its combines high transmission, high mass resolving power, and unique features that make it one of the most versatile and powerful mass... Show more »

Secondary ion mass spectrometry (SIMS) is one of the most sensitive surface analytical techniques available for high analytical precision of in-situ quantitative measurements in solid materials. Its combines high transmission, high mass resolving power, and unique features that make it one of the most versatile and powerful mass spectrometry techniques available.

In SIMS, a high-energy ion beam ablates material from a sample surface and secondary ions are then separated according to mass/charge (m/z) ratio in a mass spectrometer. Although capable of imaging at sub-mm spatial resolution, it is typically operated using a 10–20 mm beam in order to optimise sensitivity. Typical analyses that may be performed are quantification of trace elements in semiconductor or geological materials and spatially resolved, in-situ, stable isotope ratio measurements. The IMS 1280 at the University of Western Australia can detect up to five ion species simultaneously or can detect masses is sequence by cycling the magnetic field of the mass spectrometer.

Although SIMS often requires less sample preparation than other analytical techniques, for in-situ isotope ratio analyses, high-quality sample preparation is mandatory. Sample geometries of less than 7 mm thick and up to 25 mm in diameter can be accommodated. Geological samples are most often cast in vacuum-compatible, 25 mm epoxy plugs, ground, and polished flat. Samples and standards should be cast in the centre 15 mm of the mount and minimal relief should exist between samples and surrounding epoxy. Insulating samples can be accommodated, but should be sputter coated with (typically) 30 nm of gold.

Although SIMS can detect every element in the periodic table, detection limits span more than five orders of magnitude. For species with low ionisation potential or high electron affinity, detection limits are typically sub-ppb. Although quantification of trace elements is one of the most widely utilised strengths of SIMS, secondary ion yields are highly matrix dependent, thus matrix matched standards are mandatory for optimum accuracy. Homogeneously doped standards are desirable but are often unavailable for a given matrix. Thus, ion implants are often used as standards where absolute quantification is required.

Stable isotope ratio analyses are the most common use of SIMS. In order to optimise precision for a given analysis, multicolection is desirable. Multicollection is possible for single elements from Li to U. D/H analyses are feasible, but magnet switching is required.

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Single Particle Analysis

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SPA provides a critical resolution bridge between high-end structural techniques such as X-ray crystallography and nuclear magnetic resonance (NMR), and cellular tomography. SPA allows single molecules as well as macromolecular assemblies to be imaged then reconstructed at relatively high resolution for detailed analysis of their... Show more »

SPA provides a critical resolution bridge between high-end structural techniques such as X-ray crystallography and nuclear magnetic resonance (NMR), and cellular tomography. SPA allows single molecules as well as macromolecular assemblies to be imaged then reconstructed at relatively high resolution for detailed analysis of their 3-D structure. Although SPA can also be used in combination with the acquisition of a limited tilt series of 2-D images taken as the specimen is tilted at different angles in the TEM, SPA fundamentally relies on using the large number of identical particles, oriented randomly and captured in each field of view to compute a 3-D model of the particle structure.

The ideal scenario for structural studies by SPA is to first immobilse the purified preparation of molecules or protein complexes onto TEM specimen supports (grids) using a fast-freezing approach like 'plunge-freezing', which permits the ultra-rapid rates of cooling required to achieve sample vitrification without the formation of crystalline ice. The composite 3-D image is produced by first sorting the arbitrarily oriented particles into different classes based on the different orientations of the particles on the grid. The examples obtained for each different class are then averaged (class-averaging) to generate a composite image for each class that now displays a relatively high signal-to-noise ratio. All of these composite images representing different views of the particle in various orientations are finally combined to compute an overall 3-D model. SPA is particularly well suited to structures that display a high degree of symmetry, as is the case for a large number of viruses.

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Small Angle X-ray Scattering

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Small-angle X-ray scattering (SAXS) is used to measures the elastic scattering from inhomogeneities within a material over an angular range of approximately 0.05–5°. These scattering profiles can be used to determine information about the sizes and shapes of, and the distance and nature of the interactions between, these... Show more »

Small-angle X-ray scattering (SAXS) is used to measures the elastic scattering from inhomogeneities within a material over an angular range of approximately 0.05–5°. These scattering profiles can be used to determine information about the sizes and shapes of, and the distance and nature of the interactions between, these inhomogeneities. The ranges of sizes or distances that can be determined are typically on the order of a few to tens of nanometres and the samples can be liquid or solid in nature. It must be noted however, that the data generated by SAXS are averaged across the sample. This technique does not give an image showing specific pores or particles as do some other techniques. Nevertheless it can be very valuable in providing information about non-crystalline materials.

Typical examples of the types of information that can be determined include measurements of: the size and shape of nanoparticles, polymers, proteins or micelles in solution; measurements of pore size and interpore spacing in mesoporous materials; characteristic length scales in partially ordered systems, e.g. block copolymers, composites and gels; and interparticle interactions in colloidal dispersions. Hence SAXS has many applications in the fields of structural biology, chemistry, physics and engineering and sample types: e.g. polymers, pharmaceuticals, cosmetics, foods, catalysts, coal, membranes, and proteins.

The SAXS instrument is equipped with both CCD and image plate detection. The system uses a sealed X-ray tube and a Kratky camera (i.e. line collimation) which allows for the detection of dilute or weakly scattering systems. However the systems must be isotropic in nature (i.e. non-oriented). Measurements can be made of a variety of sample types including liquid dispersions, films, gels and finely ground powders over a range of temperature; approximately -20–100°C, depending on the nature of the sample.

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Surface Characterization

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Transmission Electron Microscopy (TEM)

Transmission Electron Microscopy services
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Transmission electron microscopy (TEM) – diffraction
Transmission electron microscopy (TEM) – imaging
Transmission electron microscopy (TEM) – in-situ techniques
Transmission electron microscopy (TEM) – spectroscopy

Transmission electron microscopy (TEM) – diffraction
Transmission electron microscopy (TEM) – imaging
Transmission electron microscopy (TEM) – in-situ techniques
Transmission electron microscopy (TEM) – spectroscopy

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TOF-SIMS

Time of flight secondary ion mass spectrometry
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Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is an analytical technique used to image and record organic and inorganic mass spectral data of solid materials. It is a highly sensitive technique that provides chemical information regarding elemental, isotopic and molecular structure. It involves the analysis of ionised... Show more »

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is an analytical technique used to image and record organic and inorganic mass spectral data of solid materials. It is a highly sensitive technique that provides chemical information regarding elemental, isotopic and molecular structure. It involves the analysis of ionised particles that are emitted when the surface is bombarded with an energetic primary ion beam. Emitted particles are accelerated to constant kinetic energy into the time-of-flight chamber, where mass separation is achieved according to mass-to-charge ratio. It is a highly surface sensitive technique, as only the secondary ions generated from the outer 1–2 nm region have enough energy to escape the surface for detection and analysis.

Although not a quantitative technique, ToF-SIMS can provide a qualitative surface chemistry comparison between samples. Using ‘bunched’ instrument settings, high mass resolution spectra can be collected from the surface. By using ‘unbunched’ settings, spatial resolution is optimised (sub-micrometre for Au1), so that images and chemical maps of the surface can be produced by the rastered beam. If data is saved to RAW format during an acquisition, these images may be produced in retrospect, as a full mass spectrum is saved for every pixel of the rastered area.

A range of samples can be analysed by ToF-SIMS, with the requirement that the sample must be ultra-high vacuum (10-7Pa) compatible. Films or layers on solid substrates are easily loaded, while powders and mineral slurries can be pressed into indium foil and mounted on silicon for analysis. Small (<20 mm diameter) samples are back-mounted in the 9-sample holder, while larger samples can be front-mounted to the stage plate. A vice holder is also available for mounting samples that require cross-sectional analysis.

Temperature control of samples is available by using the hot/cold stage module. This can range from cooling down to liquid nitrogen temperature to heating up to approximately 200ºC. A unique feature of this particular instrument is the custom-built preparation chamber, where a sample may be subjected to any number of experiments or heating/cooling under vacuum, before introduction into the analysis chamber.

The instrument contains four ion sources. Two of these can be used for both analysis and sputtering (Au and C60), while the caesium and gas guns are purely for sputtering. The Au liquid metal ion gun (LMIG) can be operated using either monatomic primary ions (Au1) or cluster ions (Au2 or Au3). Due to their larger size, use of cluster ions can help to increase the yield of higher mass fragments. Similarly, use of the C60 ion gun can help to increase the yield of much higher mass fragments, potentially into the 1000’s amu range. Hence the C60 is very important to the analysis of polymers, and also when depth profiling organic materials, as the organic information is retained during sputtering.

Operation of the Au or C60 analysis sources in conjunction with any of the sputtering sources can be used to perform depth profiles of materials. Analysis and sputtering phases are alternated to remove and then analyse the underlying layer of the material. If depth profiles are performed with the analysis phase optimised for spatial resolution, the 3-D images of the depth profile may be produced.

Data analysis is an important part of the technique, usually taking longer than the actual acquisition of the data itself. In terms of mass spectra, inorganic components are easily identified based upon their expected mass in the spectra. Organic components are usually not as easily identified, due to the fragmentation of their structures down to CxHy, CxHyOz, CxHyNz, etc. fragments. Unlike other forms of mass spectroscopy, it is uncommon to measure a molecular ion (M+ or M-) in ToF-SIMS. While other indicative peaks may be present, organic analysis often comes down to the careful comparison of the ratios of the fragment ions. Hence, ToF-SIMS is often coupled with multivariate data analysis as the amount and complexity of the data can be high. For example, principal component analysis can be used to help characterise differences between absorbed proteins, as well as the relative amount and even the conformation of those proteins.

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Pyrolysis/Thermal Analysis

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Thermal analysis measures dimensional or phase changes as a function of time or temperature under a controlled atmosphere and static or dynamic conditions.

Thermal analysis measures dimensional or phase changes as a function of time or temperature under a controlled atmosphere and static or dynamic conditions.

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TIMS

Thermal Ionization Mass Spectrometry
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TIRF

Total Internal Reflection Microscopy
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Total internal reflection fluorescence (TIRF) microscopy is an imaging technique that overcomes the limited axial (or depth) resolution of conventional fluorescence microscopy. It is most commonly used for fluorescence microscopy of the cell membranes of live cells. It exploits the formation of an electromagnetic field, or... Show more »

Total internal reflection fluorescence (TIRF) microscopy is an imaging technique that overcomes the limited axial (or depth) resolution of conventional fluorescence microscopy. It is most commonly used for fluorescence microscopy of the cell membranes of live cells. It exploits the formation of an electromagnetic field, or evanescent wave, that is generated when laser light undergoes TIR at the interface between a glass cover slip (or similar) and an aqueous or cellular sample. The evanescent wave excites fluorescent molecules in the sample, but only to a depth of approximately 100 nm, thereby giving outstanding axial resolution.

Traditional wide-field fluorescent signals from membrane molecules tend to be dwarfed by cytoplasmic fluorescence, TIRF microscopy is widely used to distinguish biological processes that occur at cell surfaces from those that occur within the cytoplasm. Such processes include adhesion of cells to surfaces, cellular secretion processes, and binding of proteins to receptors at cell surfaces. TIRF is particularly valuable for examining dynamic and single molecule events (like endocytosis and exocytosis, and protein trafficking) occurring on or near the cell membrane of living cells.

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X-Ray Diffraction (XRD)

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X-ray diffraction (XRD) uses X-rays to investigate and quantify the crystalline nature of materials by measuring the diffraction of X-rays from the planes of atoms within the material. It is sensitive to both the type of and relative position of atoms in the material as well as the length scale over which the crystalline order... Show more »

X-ray diffraction (XRD) uses X-rays to investigate and quantify the crystalline nature of materials by measuring the diffraction of X-rays from the planes of atoms within the material. It is sensitive to both the type of and relative position of atoms in the material as well as the length scale over which the crystalline order persists. It can, therefore, be used to measure the crystalline content of materials; identify the crystalline phases present (including the quantification of mixtures in favourable cases); determine the spacing between lattice planes and the length scales over which they persist; and to study preferential ordering and epitaxial growth of crystallites. In essence it probes length scales from approximately sub angstroms to a few nm and is sensitive to ordering over tens of nanometres.

The samples for analysis are typically in the form of finely divided powders, but diffraction can also be obtained from surfaces, provided they are relatively flat and not too rough. Moreover the materials can be of a vast array of types, including inorganic, organic, polymers, metals, composites and thin films. The potential applications cover almost all research fields, e.g. metallurgy, pharmaceuticals, earth sciences, polymers and composites, microelectronics and nanotechnology. Powder XRD can also be applied to study the pseudo-crystalline structure of mesoporous materials and colloidal crystals provided that the length scales are in the correct range.

In addition to XRD it is also possible to carry X-ray reflectometry experiments of thin (< 200 nm) films on atomically smooth surfaces (e.g. silicon wafers).

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XRF

X-Ray Fluorescence
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X-Ray Reflectivity

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X-ray Photoelectron Spectrometry

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X-ray photoelectron spectroscopy (XPS), also known as electron spectroscopy for chemical analysis (ESCA), provides elemental (down to approximately 0.1 atom %) and chemical information (oxidation state) of the outermost approximately 10 nm of any solid. It is non-destructive and can be used with both amorphous and crystalline... Show more »

X-ray photoelectron spectroscopy (XPS), also known as electron spectroscopy for chemical analysis (ESCA), provides elemental (down to approximately 0.1 atom %) and chemical information (oxidation state) of the outermost approximately 10 nm of any solid. It is non-destructive and can be used with both amorphous and crystalline materials.

Soft X-rays (1486.6 eV) are used to bombard the sample under an ultra-high vacuum resulting in the ejection of the core-level electrons (the photoelectrons) from the surface atoms. The measured binding energy provides the elemental and chemical information.

Sub-surface layers can be analysed after removal of the outer layers by in-situ ion beam milling. Bulk analysis can be performed after appropriate sample preparation such as crushing (to provide a fresh new surface), fracturing and scraping.

Typically, data is taken from an area of approximately 800 x 300 µm. The smallest area that can be analysed is approximately 15 nm. Elemental imaging, typically over 200 x 200 µm enables the area of analysis to be identified. Data acquisition time is from 5–60 minutes depending on the extent of chemical information required.

There is no specific sample preparation but it is critical that sample is NOT handled as surface contamination is always an issue considering that the detected electrons are originating from only the top 10 nm.

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microCT

X-ray microtomography
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X-ray microtomography (micro-CT) has emerged over the last few years as a leading frontier in characterisation across almost the entire range of medical, science and engineering disciplines. This form of microscopy is often referred to as non-destructive, since its aim is to image internal structure without the need to section or... Show more »

X-ray microtomography (micro-CT) has emerged over the last few years as a leading frontier in characterisation across almost the entire range of medical, science and engineering disciplines. This form of microscopy is often referred to as non-destructive, since its aim is to image internal structure without the need to section or otherwise damage the specimen. Since this ability to see into the specimen is not limited to one particular view X-ray microtomography is naturally three-dimensional in its context.

This technique is based on a similar principle to medical CT-scanning, except with a much higher resolution. The specimen is incrementally rotated over 180° or 360° in a beam of X-rays in steps of between 0.1° and 1°. A series of projection images are digitally acquired at each rotational position that map the absorption of the X-rays in the specimen at that angle. This series of projection images are subsequently processed via a technique called filtered-back-projection to generate a stack of individual slices that include both the surface detail, as well as internal structural detail. These slices are referred to as the axial slices since they are perpendicular to the axis of specimen rotation in the original scan. The axial slices can be viewed individually or, since they are spatially aligned, a specialised visualisation software can be used to produce an interactive 3-D rendering that effectively reconstructs the original sample in a computational space that supports whole object viewing, cutting in any direction, animation, and analysis of individual components within the specimen. The technique allows internal structural details to be revealed in a broad range of materials but is particularly well suited to porous specimens or materials where there is a large density difference between adjacent structural components.

Differential staining of components within the samples can enhance density differences and therefore extend the range of samples that can be usefully imaged. The available resolution is a function of sample size and density but in general ranges from about 15 µm down to 1.5 µm. Sample size is usually in the order of 1 mm up to 10 mm in diameter, but larger samples can be used if the average density is not too large.

As well as modeling the structure of the material it is also possible to simulate deformation and failure of structures using measured or standard data. In the material sciences there is a constant need to validate simulation with physical measurement, and vice versa. However, there are a limited range of tools for the specific materials problem of calculating the transport and mechanical properties directly from tomograms. These are typically computationally intensive. Tool boxes include multi-phase segmentation, finite element modelling, fluid flow, conduction, morphological measures/filtering as well as the ability to perform direct image-based registration in which the tomograms of successively disturbed (dissolved, fractured, cleaned, etc) specimens can be correlated back to an original undisturbed state in 3-D.

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X-ray Nanotomography

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X-ray nanotomography (namo-CT) takes X-ray tomography into new realms at the sub-micrometre scale. It is a non-destructive technique, because its aim is to image internal structure without the need to section or otherwise damage the specimen. This ability to see into the specimen is not limited to one particular view – X-ray... Show more »

X-ray nanotomography (namo-CT) takes X-ray tomography into new realms at the sub-micrometre scale. It is a non-destructive technique, because its aim is to image internal structure without the need to section or otherwise damage the specimen. This ability to see into the specimen is not limited to one particular view – X-ray nanotomography is naturally three-dimensional in its context – so the resulting data may be viewed and/or re-sliced at any angle to see the features of most interest.

The technique allows internal structural detail to be imaged either as 2-D slices or as 3-D rendered volumes. With specimen thickness in the order of 200 μm, 3-D sub-volumes of 64 μm cubed or 16 μm cubed can be imaged at resolutions of 150 nm or 50 nm respectively.

Although functionally similar to microtomography, the higher resolution achieved in nanotomography demands that particular attention be paid to the alignment of the projection images that are acquired as the specimen is incrementally rotated over 180° in steps of between 0.1° and 1°. To facilitate this alignment a 3 μm gold particle is placed on the specimen and subsequently used as a fiducial marker for correction of spatial misalignments. Following alignment of the projection image series, the projection data are subsequently converted into a stack of axial slices and viewed in 3-D.

Specimen preparation is a critical part of this process, with samples of somewhat less than 0.5 mm being mounted on steel pins for scanning. Typically a low-resolution micro-CT scan will be done as a preliminary assessment to establish the orientation of the sample and to find regions of interest for the higher resolution nano-CT scan. Scan durations are long, ranging from 12 hours for a simple preliminary scan to up to 168 hours (one week) for a very high-quality scan. In addition to the standard X-ray absorption mode of operation, the scanner can also be used in a phase-contrast mode that provides further enhancement of structural details, particularly at material boundaries.

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