GCIB-SEM: 3D electron microscopy with < 10nm isotropic resolution

GCIB-SEM is a new technique that combines high resolution electron microscopy with the damage free sputtering of gas cluster ions to produce incredible 3D tomography with less than 10 nm isotropic resolution.

Over the last two decades, gas cluster ion beams (GCIB) have become increasingly popular as add-on components for ultra-high vacuum techniques such as XPS, SPM, and SIMS. Due to their excellent combination of fast yet low-damage sputtering, GCIBs have been widely adopted as depth profiling ion beams, or as a means of cleaning samples in situ.

Very low impact energies, as little as 1 eV per atom, means cluster ions sputter material without modifying the surface chemistry, i.e. without breaking bonds. This makes GCIBs particularly effective for high-resolution depth profiling of soft materials such as polymers and organic matter.

GCIB 10S cluster schematic, and PET C 1s XPS spectrum comparing Ar1 and Ar2000.
The GCIB 10S is a powerful tool for damage-free depth profiling of polymers, organics, and other soft materials, delivering consistently superior results over monatomic beams.

Traditional sputter beams such as Ar1 typically have impact energies in the kilovolt range, resulting in not only large amounts of fragmentation to surface molecules, but also penetration of the ions beneath the surface causing further damage. This damage shows up in XPS and SIMS spectra, and limits the depth resolution of the technique.

Cluster beams also sputter soft, organic material much faster than hard, inorganic materials, making them extremely useful for removing adventitious carbon and other surface contamination without damaging the substrate — ideal for cleaning surfaces prior to analysis.

It is no surprise then that GCIBs have become so popular as add-on components for surface analysis instrumentation.

The GCIB 10S

  • 10 kV argon cluster ion source
  • Selectable clusters from Ar1 to > Ar3000
  • Real-time cluster measurement & adjustment
  • Sample current imaging
  • Gate valve for quick & easy servicing
  • Large spot size and wide scan field for even removal of material

The versatile nature of the GCIB makes it a useful tool in a variety of other techniques as well, beyond strictly surface science. In particular, the GCIB has recently been shown to be powerful tool in electron microscopy. A new technique pioneered by researchers at HHMI Janelia Research Campus combines high resolution electron microscopy with the damage free sputtering of gas cluster ions to produce incredible 3D tomography with less than 10 nm isotropic resolution.

Published in Nature Methods in 2019 the GCIB-SEM system developed by Hayworth et al. consists of a GCIB 10S from Ionoptika mounted on a Zeiss Ultra SEM. Using 1 µm thick serial sections of brain tissue, high-resolution electron imaging was interleaved with wide-area ion milling until the entire section was consumed. Full experimental details can be found in the paper linked above.

Figure detailing results achieved using GCIB SEM, by Hayworth et al
GCIB-SEM is a powerful technique for acquiring extremely detailed 3D maps on an unprecedented scale. Images from a GCIB-SEM run performed on three sequential 500-nm-thick sections of mouse cortex. bioRxiv: http://dx.doi.org/10.1101/563239.

The result is a 3D data set hundreds of microns in area by tens of microns deep, with less than 10 nm isotropic resolution throughout. Such a high resolution data set then allows researchers to map the brain structure in incredible detail. The figure above shows a 15 x 15 x 10 µm section of mouse brain, the detail of which is truly remarkable. Panel e shows a single spiney dentritic process with axons synapsing on it, while panel f shows various high-resolution 2D and 3D views of a single spiney synapse.

Other technologies used to perform similar experiments include FIB-SEM and diamond knife based sectioning, however both have their drawbacks. FIB provides the necessary resolution, but is thus far incompatible with the high-throughput needed for larger volumes, while diamond knife techniques are highly compatible with larger volumes, but lack the consistency needed at such thin cuts.

In contrast, the GCIB 10S mills away just the top few nanometres of the surface resulting in an improvement in depth resolution of a factor of 3 or more over other techniques, whilst simultaneously improving sectioning reliability. The rapid, wide area milling afforded by the GCIB 10S is also compatible with the new multi-beam SEM systems now on the market, which will enable even larger volumes to be analysed with no loss of resolution.

GCIB-SEM

  • Large-area and fast (up to 450 µm3 s-1).
  • Can be automated and is highly scalable.
  • Consistent performance over large volumes.
  • Simple, easy to maintain, and reliable.
  • Improves z resolution by a factor of 3 or more.

GCIB-SEM is a powerful technique for exploring complex materials and structures in three dimensions with extraordinary detail. For this application, control of the cluster size and current is critical to the result. Unlike other gas cluster beams, the GCIB 10S lets the user take complete control of the experiment. With real-time cluster measurement, cluster size can be tuned to the users’ needs and the settings saved for later use.

Real-time cluster measurement on the GCIB 10S

The GCIB 10S is easily installed on a range of instrumentation, from XPS and SIMS, to electron microscopes, Auger, and more. To speak with us and find out how the GCIB 10S might be right for your application, or to request a brochure, please get in touch via our Contact Page.

Why you shouldn’t overlook C60 beams just yet

C60 cluster ion beams are a fantastic tool for analyzing both hard and soft materials. Composed of sixty carbon atoms arranged into a football shape, C60 ions combine several different features making it a great all-rounder ion beam. This is why we always recommend customers to consider including a C60 beam when specifying their J105.

As the C60 molecule is larger (approx. 7 Å) than the lattice constant for most materials, it does not experience channeling the way smaller ions such as bismuth do. As such C60 beams exhibit incredibly uniform sputter rates across a wide range of materials, and even on challenging poly-crystalline materials where there is a range of crystal orientations.

As a cluster ion, C60 also produces very shallow craters with very little, if any, subsurface damage, so etch cycles are not needed to remove damaged layers when performing depth profiles or 3D imaging. As the J105 samples 100% of the analysis volume, high sensitivity is guaranteed, and combined with spot sizes as low as 300 nm, C60 is a powerful beam for delivering maximum resolution in 2D and 3D.

NiCr Standard Depth Profile C60
C60 depth profile through the NIST NiCr standard showing <5 nm depth resolution. As there is no need to perform etch cycles to remove damaged layers, depth resolution on the J105 is limited only by the crater depth of the ion beam.

The figure below shows a 3D image of a semiconductor stack alongside a depth profile through the same, performed with a 40kV C60 beam in positive ion mode. The sample consists of layers of InSb, Al, and GaAs respectively, covered in a protective photoresist layer.

3D SIMS image of InSbAlGaAs Stack with depth profil
The J105 has one mode of operation, so amazing 3D images, high-resolution 2D images, as well as detailed depth profiles can all be obtained from a single data set.

The resulting 3D SIMS image shows the layers in amazing clarity, with very sharp interfaces. As the J105 always samples 100% of the analysis volume, high sensitivity is guaranteed. The detailed depth profile through the sample also shows the presence of dissolved Al within the InSb layer, as well as the presence of Sb in the pure Al layer.

The 40kV C60 beam is ideal for this type of sample or application due to the combination of soft organic, inorganic, and hard metallic layers within the same sample. Combined with spot sizes as low as 300 nm, C60 is a powerful beam for delivering maximum resolution in 2D and 3D, no matter what type of sample you have.

How the J105 SIMS works: An introductory guide

The J105 SIMS is a state-of-the-art 3D imaging ToF SIMS combining innovative design with cutting-edge science that has redefined ToF SIMS. Designed to exploit the benefits of cluster ion beams, the J105 delivers exceptional sensitivity to molecular ions, 3D MS imaging, and consistent performance across all samples.

In this article, we aim to give you an overview of how the J105 SIMS works, as it is quite different to other ToF SIMS. We will guide you through the various features of the instrument and explain their purpose, how they work, and what the benefits are.

How the J105 SIMS works: an introductory guide

1.      The Ion Beam

The J105 was designed to get around many of the limitations faced by traditional ToF SIMS instruments, particularly for biological samples. One of the ways this is achieved is by not pulsing the primary ion beam, but instead running it in DC, or continuous mode. This is a major advantage and is what makes the J105 a very different instrument to most other ToF SIMS.

One of the biggest advantages of having a continuous beam is that any ion beam, no matter what size, can be used as the primary source. This gives the user a lot of choice when designing their experiment. We’ll cover the intricacies of different ion beams in a different article, but for the purposes of this discussion we’ll focus on gas cluster ion beams (GCIB).

A GCIB typically consists of thousands of constituent atoms, giving it a collective molecular weight anywhere from 100,000 g/mol upwards. Under typical acceleration voltages (kV), such a large ion moves very slowly, requiring longer pulses and on a conventional ToF would result in poor mass resolution. By running in continuous (or long pulsing) mode, the J105 is able to get around this issue and take full advantage of the benefits of using GCIBs.

The other major advantage of running in DC mode is that focusing the ion beam to a fine spot can be prioritized without affecting the performance of the mass spectrometer. With our most powerful GCIB, the GCIB SM, the optics have been designed to enable spot sizes of just 1.5 µm, combining greater spatial resolution with high-sensitivity mass spectrometry. The benefits of this are clear, and have been highlighted recently by the pioneering work published in Science.

Benefits: Simultaneous high-sensitivity mass spectrometry with high-spatial resolution.

View of a sample through a window

2.      The Extraction Optics

As the primary beam is not pulsed, in order to determine a time-of-flight the secondary ion beam is pulsed instead. This is done by the Buncher, but the extraction optics play a key role is controlling the secondary ion beam prior to that step.

Secondary ions extracted from the surface contain a lot of energy making them difficult to control. In order to form the secondary ions into a controlled beam, they enter an RF quadrupole filled with N2, which slows the ions down through the process of collisional cooling.

This is a crucial step, as it decouples the effects of the primary beam and the sample from the secondary ions. By effectively wiping the memory of any interaction on the surface, this step enables the J105 to analyse samples with complex topography without any loss of mass spec performance.

Benefits: Consistent performance that is independent of ion beam or sample topography.

The analysis chamber of the J105 SIMS

3.      The Buncher

In many ways the heart of the instrument, the Buncher is what takes a continuous stream of secondary ions coming from the quad and forms them into a very short pulse. In order to measure the time-of-flight without pulsing the primary beam or the extraction, the Buncher creates an asymmetric pulse that focuses all ions of the same mass to a single time focus, T0. This is an essential step, and is what ultimately determines the mass resolution.

Benefits: High mass accuracy, high mass resolution.

J105 SIMS reflectron & mass analyser

4.      Tandem MS & Time-of-Flight

As with any form of mass spectrometry, definitively assigning peaks requires a secondary validation step. One way to do this is through tandem MS, whereby a parent ion is selected to undergo fragmentation and the resulting spectrum is used to determine the exact form of the parent. The J105 SIMS was the first SIMS instrument to introduce tandem MS, and is included as standard on all our instruments.

When a user selects an ion of interest, it is directed into a high-energy collision cell filled with N2, producing characteristic fragment ions. Whether running an MS1 or MS2 experiment, ions then enter the 1500 mm long reflectron before being detected.

Benefits: Tandem MS for accurate peak identification, high mass resolution.

The J105 SIMS contains several innovative design features that combine to produce an instrument like no other, optimized to enable both maximum sensitivity and maximum spatial resolution simultaneously from any ion beam. Consistent performance is guaranteed, as the mass spectrometer delivers high mass resolution (> 10,000) and mass accuracy (< 5 ppm) that are completely independent of the ion beam and the sample environment.

The J105 SIMS is the ideal tool for a wide range of applications and sample types, including biological research, pharmaceuticals, thin films, polymers, energy applications and many more. To find out if the J105 might be the right instrument for you, or to arrange a demonstration, please get in touch via our Contact Page.

Drug detection with high-sensitivity using ToF SIMS

The high attrition rate of pharmaceutical drug compounds adds enormously to the cost of those that make it to market, so there is an urgent and growing need to identify failure at earlier stages of drug development.

In order to do so, researchers require as much information as possible. Specifically, there is a need to measure the concentration of a drug at the target in order to accurately predict its pharmacological effect. This then requires a means of generating label-free sub-cellular imaging, as fluorescent labels may affect drug chemistry, altering results.

Time of flight secondary ion mass spectrometry (ToF SIMS) is a powerful tool for label-free chemical imaging, having typically very high lateral resolution capable of resolving sub-cellular features with 3D analysis capabilities.

ToF SIMS is thus a potentially powerful analysis tool for the screening of new drug compounds. However, the use of high energy projectiles for ToF SIMS analysis can cause molecules to fragment, preventing the molecular ion from being detected. This can lead to a lot of ambiguity, for example distinguishing between a drug compound and its metabolites.

Another possible stumbling block is the issue of sensitivity, particularly for those compounds of most interest. In a recent study by the National Physical Laboratory (NPL), Vorng et al. demonstrate that the sensitivity in ToF SIMS is proportional to the Log P of that compound, such that compounds with low or negative Log P values are extremely difficult to detect.  

Log P, or partition coefficient, is a measure of hydrophobicity, and is a major factor used in pre-clinical assessment of a compound’s druglikeness.  It is advisable that a drug candidate be as hydrophilic as possible while still retaining adequate binding affinity to the therapeutic protein target, i.e. that the Log P be as low (or negative) as practicable. This presents an obvious problem for the use of ToF SIMS as an analytical tool in this context.

Cluster beam colliding with a surface.

We have recently led the development of a new type of ion source for ToF SIMS that provides unparalleled sensitivity particularly for organic species. Available exclusively on the J105 SIMS, the Water Cluster Source simultaneously reduces fragmentation while increasing ionization, for truly unparalleled sensitivity of drugs, metabolites, biomarkers, lipids, peptides and more.

Combining this new ion source with the already impressive sensitivity of the J105 SIMS, even low Log P compounds can be detected in tissue and cells, with direct, label-free imaging of the molecular compounds at sub-cellular resolutions.

To demonstrate this, we doped tissue homogenate with 4 different pharmaceutical compounds that span the range of Log P from -0.8 to 7.6. The relationship between sensitivity and Log P reported by NPL is observed in this data, however the slope of the line is greatly reduced, with only a factor of 40 between the highest and lowest values.

ToF SIMS sensitivity to drugs as a function of Log P
ToF SIMS sensitivity of four different drugs using the Water Source. Sensitivity shows a linear relationship to the partition coefficient, Log P, though the slope is not steep.

As a comparison, we performed the same experiments with a state-of-the-art Ar gas cluster ion beam and plotted the yield against that of the new Water Source. The Water Source increased sensitivity by an order of magnitude in most cases, with the largest increase being for those compounds with the lowest Log P values. This indicates that the improvement in sensitivity is greatest for those compounds that need it the most.

Comparing sensitivity of argon and water cluster beams for four different drugs
Comparing sensitivity of a state-of-the-art Ar cluster source with the Water Source. Sensitivity improves by roughly an order of magnitude when using water, with the largest increase for those compounds with lower Log P values.

As a final demonstration of the capabilities of the J105 with the Water Source, we performed tandem MS analysis on the homogenate samples. Tandem MS is an important step for confirming any assignment in mass spectrometry, however the inefficiency of the process often means it can only be performed on high intensity peaks. With the boost in sensitivity provided by the Water Source, tandem MS analysis is possible even on compounds with relatively low Log P values, such as ciprofloxacin.

Tandem MS analysis of the drug ciprofloxacin
Tandem MS performed on the J105 SIMS with a Water Source. Greater sensitivity allows definitive confirmation of many more peaks.

ToF SIMS is a potentially powerful analysis tool for the screening of new drug compounds, however research is hampered by the inherently low sensitivity to many drug candidates. The J105 SIMS in combination with the Water Cluster Source provides unparalleled sensitivity to drug compounds, particularly in complex matrices such as tissue and cells, even for low Log P compounds. This unprecedented sensitivity combined with sub-cellular imaging and high-resolution 3D imaging mean the J105 SIMS is a powerful tool for drug analysis.

To learn more about how the J105 SIMS can benefit your research or to set up a demonstration, get in touch via our Contact Page.

Cocaine metabolite imaging in fingerprints with Water Cluster SIMS

Detection of drug compounds and their metabolites in natural environments is a critical topic for both forensic and pharmaceutical applications, and requires overcoming some of the limitations in existing microscopic and analytical techniques.

Time of Flight Secondary Ion Mass Spectrometry (ToF SIMS) is a powerful analytical technique capable of providing detailed chemical and spatial information about a surface, and as such has recently been employed in a number of forensic studies for drug and metabolite detection. However, ToF SIMS can suffer from low sensitivity due to insufficient ionisation efficiency, and this is particularly true for complex biomaterials, i.e. those of most interest to forensic and medical analysts.

Recently, we have led the development of a powerful unique gas cluster ion beam (GCIB) using water clusters. The Water Cluster Source is capable of enhancing ion yields by many orders of magnitude compared to other conventional ion beams (C60+, Bi3+ etc.), and is particularly effective for biomolecular imaging and 3D analysis of organics such as tissue, cells, fingerprints, etc.

Plot displaying increase in signal intensity using water clusters
Water clusters enhance sensitivity to intact biomolecules such as lipids, even compared to current state-of-the-art GCIB technology.

In this application note, an experimental fingerprint detection approach using the Water Cluster Source identifies traces of ingested cocaine on human skin. The use of the J105 SIMS equipped with the Water Cluster Source (Water Cluster SIMS) provides both visualisation of the latent fingerprint as well as discrimination between contact-only and ingested cocaine by looking for metabolites of the drug excreted through the skin.

Detectable levels of metabolite in a fingerprint are extremely low, for instance 25 mg of ingested cocaine excretes less than 2.5 ng/mL in sweat,1 and previous attempts using other mass spectrometry imaging (MSI) techniques such as MALDI and DESI were unsuccessful. Using Water Cluster SIMS, it was possible not only to detect the metabolite, but also to generate a high-contrast chemical map of the entire fingerprint.

The fingerprint specimen, provided by University of Surrey, was collected on a piece of silicon wafer from a donor who had previously ingested cocaine,2 then a ToF-SIMS analysis was acquired on an 18×6 mm2 area with a 70 kV (H2O)29k+ primary ion beam in the J105 SIMS.

Figure 1(a) shows the chemical image of the 290.14 m/z signal, demonstrating the characteristic fingerprint features with ridges, valleys, as well as sweat pores. Due to the high mass accuracy of the J105 SIMS, this signal is confidently annotated as the cocaine metabolite benzoylecgonine (BZE, C16H20NO4+). Figure 1(b) shows a colour overlay of BZE (magenta) and the cocaine molecular ion (C17H22NO4+, 304.15 m/z – yellow). As expected, cocaine was observed in particulate form (see arrow) due to direct contact of the donor with the powder, and is not co-localised with BZE.

ToF SIMS image of cocaine metabolite BZE in a fingerprint.
Figure 1(a) Positive ion image of BZE (C16H20NO4+, 290.14 m/z) in a fingerprint. (b) Overlay positive ion image with BZE (magenta) and cocaine (C17H22NO4+, 304.15 m/z – yellow). (c) BZE peak, with high mass accuracy and high mass resolution.

These images, with the small amounts of BZE and cocaine present, demonstrate the benefits of Water Cluster SIMS for enhancing sensitivity, particularly for trace detection of organic compounds in complex sample matrixes.

The J105 SIMS is a powerful tool for 2D and 3D molecular imaging, providing high sensitivity analysis with a range of powerful features. Now featuring the new Water Cluster Source, the J105 takes another leap forward to offer even greater sensitivity and to intact molecular ions. This exciting new technology has been shown to dramatically improve the imaging of drug metabolites ingested by the body, and is a powerful tool for visualising molecular information in a wide range of applications.

To find out more about how the J105 SIMS can benefit your research, get in touch via our Contact Page.


References

  1. Kacinko, S. L., Barnes, A.J. et al. , Disposition of Cocaine and Its Metabolites in Human Sweat after Controlled Cocaine Administration, Clinical Chemistry, 51, 2085 (2005). https://doi.org/10.1373/clinchem.2005.054338
  2. Jang, M., Costa, C., Bunch, J. et al. On the relevance of cocaine detection in a fingerprint. Sci Rep 10, 1974 (2020). https://doi.org/10.1038/s41598-020-58856-0

High-resolution molecular imaging ToF SIMS

Historically ToF SIMS has not been sensitive to intact molecules due to the excessive fragmentation caused by the primary ion beam. Now however, thanks to the progress in gas cluster ion beam (GCIB) technology over the last decade, sensitivity to intact molecular species in ToF SIMS has increased by several orders of magnitude, making it possible to achieve molecular imaging with the high-spatial resolution traditionally associated with SIMS.

The development of high-energy gas cluster beams with small spot sizes has dramatically altered the sensitivity to intact molecular species. This is enabled by the unique design of the J105 SIMS, which allows any ion beam to be used without impacting performance. So large gas cluster beams may be used while still maintaining high mass resolution, and thereby greatly improving molecular sensitivity.

It is now possible to map the distribution of lipids in biological tissue with higher resolution than ever before. This is illustrated in Figure 1, where two sphingolipid species and a glycerophospholipid species are imaged within rodent cerebellum tissue. The inset line scan demonstrates the sharp drop off in C24-OH signal, on the order of a few microns, giving researchers unparalleled clarity into the structure of their sample.

Figure 1. 2 μm per pixel lipid mapping in rodent brain tissue, analysed using a 40kV Ar4000 beam. Boundaries between sphingolipid species C24 (m/z 890.6 – blue), and C24-OH (m/z 906.6 – green), and the glycerophospholipid species PI(38:4) (m/z 885.6 – red) are clearly resolved. Inset: line scan drawn across the C24-OH signal. Data courtesy of the University of Gothenburg.

As damage to the sample molecules is minimised, a volume of material can be analysed, not just a static dose limit, resulting in higher signals and the ability to depth profile without wasting material. Significantly, for a given dose, larger cluster beams have been shown to produce higher signals from most large molecules, as illustrated in Figure 2.

Figure 2. High signal intensity with low fragmentation. (a) Normalised signal intensity for molecular and significant fragment signals from Irganox 1010 with an Ar4000 beam, showing much higher ion yields for higher beam energy. (b) Normalised signal ratios comparing levels of fragmentation for four different beam energies. Data courtesy of the University of Gothenburg.

Figure 3 shows the mass spectrum and corresponding image of the DG region of a rodent hippocampus. Using a 30 kV [CO2]3k+ beam, the distribution of GM1(36:1), GM1(38:1), and ST(18:0) were mapped at a pixel density of 2 μm per pixel. A wealth of information is contained within the spectrum, with detailed phospholipids, cardiolipin species, and high-mass ganglioside species all clearly present and identified.

Figure 3. Negative SIMS spectrum and corresponding image from DG region of rodent hippocampus, showing the range of phospholipid, ganglioside, and cardiolipin species detected. Analysis performed using a 30 kV [CO2]3k+ beam at 2 μm per pixel. Data courtesy of Pennsylvania State University.

Large molecular species such as lipids play an important role in basic cellular processes. As such it is crucial to have the correct tools with which to study these systems. The J105 SIMS, alongside the development of new gas cluster ion sources, is pushing the capabilities of ToF-SIMS, both in terms of the mass detection limits, and the limits of spatial resolving power, enabling researchers to probe further and discover more.

For further information about our instruments or to arrange a demonstration, please get in touch via our Contact page.

Cherry blossoms mount fuji

Detecting pollutants in a Cherry Blossom leaf

Plant samples such as leaves are a challenging sample for ToF SIMS. Composed of insulating materials such as cellulose (cell walls) and lipophilic coatings (cuticular layer), charge build up can affect measurement quality. Using an electron gun during analysis can alleviate the charging effect and enables 3D analysis of the surface of a leaf.

Cherry blossom leaves (Prunus serrulata) collected in a busy city were analysed on the J105 SIMS using a 40 kV C60 ion beam. Pieces of blossom leaves were mounted onto double sided tape, attached to a sample stub, and gently pressed down in the corners to ensure best possible contact without deforming the leaf surface. Overview images were acquired on both sides of the leaf surface, with a spatial resolution of 1 µm per pixel and a primary ion dose of 2.2×1013 ions/cm2.

Experimental Conditions

Ion Beam:40kV C60+
Dose:2.2×1013 ions/cm2
Spatial Resolution:1 μm
Charge Compensation:60V Electron Gun, 25V Stage Bias

Without charge compensation, no secondary ions could be detected. Applying an ever-increasing stage bias would produce secondary ions temporarily. Only a combination of charge compensation methods via a 25 V pulsed stage bias and electrons emitted at 60 V beam energy enable us to generate an image of the leaf surface as well as steady signal during depth profiling.

Figure 1. Analysing the surface of a cherry blossom leaf.

Surface analysis reveals the outline of single plant cells. The outlines of the cells contain CaOH (m/z 56.97), while inorganic compounds such as K2O+ (m/z 93.92), Na2Cl+ (m/z 80.95), and Fe+ (m/z 55.93) are unevenly dispersed on the surface of the leaf. All compounds identified across the uneven leaf surface have a mass accuracy < 5 ppm (Table 1).

Analysis also revealed the surface to be coated with an even layer of organic compounds represented by molecules containing aromatic structures, e.g. tropylium ion, C7H7+ (m/z 91.05). Wax coatings on plants take the form of long aliphatic carbon chains, so aromatic structures such as these are unexpected and may indicate the presence of gasoline pollutants such as BTX (benzene, toluene, xylene).

Analysis of complex, insulating, and uneven samples such as these is made routine on the J105 SIMS.

Depth profile analysis reveals that as the cells are etched away, the layer of aromatic compounds reappears on the underside of the sample. Additionally, potassium containing substances are detected that are not present on the surface and only occur within certain cell walls (Figure 1 inset, green).

Imaging depth profile through a leaf showing CaOH (red), K2O (green), and C7H7 (blue).

Repeating the analysis on the lower epidermis reveals a high concentration of aromatic signals surrounding the stomata (Figure 2, green). It is known that plants can absorb pollutants such as BTX, mainly through the stomata, giving further evidence to the origin of these compounds.

Analysis of lower epidermis. Concentration of aromatic signals such as C7H7+ around the stomata may indicate uptake of pollutants such as BTX.

Analysis of complex, insulating, and uneven samples such as these is made routine on the J105 SIMS. Performing high-resolution 3D analysis with high sensitivity creates a more complete picture, enabling a greater understanding of the sample and its environment.

We gratefully acknowledge NESAC/BIO and the University of Washington for the use of their data in this work.

For further information about our instruments or to arrange a demonstration, please get in touch via our Contact page.

ion beams and their applications

Ion Beams and their Applications

Ion beams come in many shapes and sizes; different source technologies produce a wide range of different beam types, some have flexible sources that can accommodate a range of elements, while some are broad beams, and some analytical. It can be a mine field if you don’t know what you’re looking for. In this application note, we shed light on Ionoptika’s range of ion beams.

1. Sputter vs. Analytical Ion Beams

Ionoptika’s range of ion beams can be split into two groups based on their applications or purpose; sputter beams, and analytical beams.

Sputter Beams

While all ion beams will sputter a surface, we make this distinction based on the area and speed with which this occurs. Sputter beams have three characteristic features: high current, large spot size, and wide field of view. They are designed to deliver a large dose of ions over a wide area as quickly as possible, in order to optimize etch rates.

Sputter beams are often used to remove material prior to analysis using a separate analytical technique, e.g. SIMS, XPS, SEM, TEM, Auger etc. This can be for cleaning purposes, or used as a means of depth profiling through the sample.

Analytical Beams

Rather than being used to facilitate analysis using a separate technique, analytical beams are designed to perform the analysis themselves. They also have three characteristic features; wide energy range, small spot size, and variable current control. This gives the user incredibly fine control over the beam characteristics, which enables them to optimize their experiment.

Analytical beams are primarily used for mass spectrometry, for example SIMS (secondary ion mass spectrometry), and have also been installed on MALDI (matrix assisted laser desorption ionization) instruments.

2. C60 Beams

Carbon-60, or just C60, is a type of fullerene molecule, consisting of sixty carbon atoms formed into a hollow sphere, not unlike the shape of a soccer ball. The first C60 ion beam was produced by Ionoptika, in collaboration with the University of Manchester in 2002, and since then more than 140 units have been sold worldwide.

Compared to monatomic ion beams, C60 beams result in a much “gentler” sputtering action, greatly reducing the damage caused to sub-surface layers. Preferential sputtering – normally an issue for monatomic beams – is largely non-existent for C60, while the etch rate of is also relatively consistent across different material types. This makes C60 a very powerful, very consistent sputter source.

As an analytical beam, the gentle sputtering action of C60 also reduces the fragmentation of larger molecules, resulting in an enhanced molecular signal intensity. For techniques such as SIMS, this can be incredibly important, as it significantly increases the usable mass range of the technique.

3. Gas Cluster Ion Beams

Gas cluster ion beams (GCIB) are high-energy beams of cluster ions, ideal for the sputtering and analysis of organic matter. They are an incredibly versatile ion source, as both the beam-type and the properties of the beam can be varied as needed. This allows the user to tune the beam to the needs of their experiment.

The source operates through the adiabatic expansion of gas in a vacuum, causing rapid cooling and resulting in cluster formation. The clusters are then ionized through electron bombardment and accelerated through the column. The size of cluster is a vital parameter, and may be tuned over a wide range by adjusting the source conditions.

GCIB for Organic Analysis

GCIBs are the ideal choice for sputtering organic matter. Etch rates of organic materials are several orders of magnitude higher than metallic or semiconductor materials. This makes cluster beams such as the GCIB 10S an excellent tool for cleaning surfaces prior to analysis. The large cluster species also produce very little fragmentation or sub-surface damage – performing better than C60 on both fronts.

In order to maximize the benefits of GCIBs for SIMS, the beam must be operated as high energy. This is because the secondary ion yield shows a non-linear increase as a function of beam energy. We therefore currently offer two variants of analytical beam: the 40kV GCIB 40, and the 70kV GCIB SM.

The J105 SIMS utilizes the benefits of gas cluster beams for organic analysis. The combination of the gentle sputter action of large cluster ions with increased secondary ion yield has extended the usable mass-range to > m/z 2500.

Choice of gas

The versatility of GCIBs comes from having a choice of input gas. Argon has historically been a favorite, as it is an inert gas that forms clusters easily. Ar/CO2 mixtures are also common – the CO2 acting as a cluster formation agent. However pure CO2 gas is now the favored option for many of our customers using GCIBs for SIMS.

The stronger van der Waals forces between CO2 molecules results in much larger clusters than would be available for Ar – up to 20,000 in some cases. This gives users much greater control over the all important E/n value (energy per nucleon). Researchers have shown that optimizing E/n results in an enhancement of the secondary ion signal. It is also thought that the presence of O ions at the surface improves the probability of ionization – further enhancing ion yield.

We have recently developed a water vapor (H2O) source for GCIBs. This is currently available as an optional add-on for our GCIB-40 and GCIB-SM products.

4. Liquid Metal Ion Beams

Liquid metal ion beams, also known as LMIS, or LMIG, are a well established source technology. The source operates by a liquid metal reservoir feeding a blunt tungsten tip, from which ions are extracted by a strong electric field. The source design is elegant and reliable, and hence has been used in focused ion beam (FIB) systems for decades. Ionoptika offer a 25 kV LMIG system in two variants; the IOG 25AU gold-cluster system, and IOG 25GA gallium system.

Liquid metal beams produce monatomic or small-cluster ion beams, such as Au+, Ga+, and Au3+. As such, they are characterized by very small spot sizes (< 100 nm). This makes them ideal for high-resolution analysis applications.

The high-energy smaller ions can penetrate, or channel, far beneath the surface before dissipating their energy. This causes significant sub-surface damage, and as such depth profiling can be unreliable. It also results in significant fragmentation, making LMIG sources much more suited to the analysis of hard materials.

5. Duoplasmatron Ion Beams

Duoplasmatrons are an incredibly high-brightness ion source. The ions are generated as gas is slowly fed into an arc chamber, where it ignites as plasma. The plasma is then extracted and focused through the column. The nature of the source allows for many different source gases to be used. This means that duoplasmatron sources are a highly flexible source of ions.

Duoplasmatron ion beams are monatomic, and do not form clusters. This however results in much lower energy distributions, enabling smaller spot sizes. Combined with a high-brightness source, this leads to a very high current density beam, ideal for hard materials or where fragmentation is not a concern.

Ionoptika produce two variants of the duoplasmatron ion beam: the FLIG® 5 – a low energy sputter source, and the IOG 30D – a 30 kV high-current analytical beam.

FLIG – Floating Low Energy Ion Beam

The FLIG® 5 is a unique ion beam system as it is based on floating column design. The design enables very low energy operation (down to 200 eV), while still delivering high current. Operating at such low impact energies greatly reduces the penetration depth of the beam, which in turn improves the depth resolution. Both O2 and Cs variants of the FLIG are available.

6. Conclusion

The table below compares Ionoptika’s various ion beam products under a number of categories discussed in this article.

Analytical Ion Beams:

Ionoptika ion beam product comparison table

Sputter Ion Beams:

Ionoptika Product Comparison Table Sputter beams

SIMS showing Cr signal of M1.6 screw thread

ToF SIMS of Rough or Uneven Samples

A common problem faced by TOF SIMS analysis is loss of peak resolution and mass accuracy on samples with rough or uneven topography. The J105 SIMS does not suffer from these issues, experiencing no loss in mass accuracy across even the roughest of samples

The ability to obtain consistent, accurate results is key to all analytical techniques, and none more so than ToF SIMS. Providing detailed chemical information about a surface, ToF SIMS is an extremely versatile surface analysis technique. The results, however, can be very sensitive to sample environment; insulating samples, or changes in sample height can affect the measurement and result in inconsistent, or inaccurate results.

In this application note we explain the reasons why sample roughness can be a problem, and show how the unique design of the J105 SIMS has solved this issue.

Why Roughness is a Problem for ToF SIMS

Time of flight mass spectrometers determine the mass-to-charge ratio of ions by measuring the time taken to travel a known distance under an electric field. The accuracy of this measurement – known as the mass-resolution – is determined by how precisely this flight time can be measured.

In conventional ToF SIMS, good measurement accuracy is achieved by generating very short pulses (in nanoseconds) of the primary ion beam. If the sample is completely uniform, the variation in flight time of ions with the same mass will be minimal. Thus the experiment will be quite accurate.

Say that our sample is not uniform however, but has a height variation caused by a slight tilt when mounting. In this case, ions of equal mass coming from different parts of the sample will have to travel slightly different paths, and thus will have slightly different flight times. Our experiment will not be as accurate this time, and will show broader peaks as ions of the same mass arrive at slightly different times.

What about a sample with a lot of surface roughness or height variation, such as the surface of a meteorite, or a coronary stent? In these cases it should be clear that accurate results will be difficult to obtain.

There is also a secondary issue caused by samples with rich topography, particularly those that are naturally insulating. Topographic features may result in the build up of localized surface charge. This localized charge distorts the local electric field, and can thus affect the flight path of ions at these locations.

Solving the Problem: Decoupling The Mass Spectrometer

Animated GIF of J105 SIMS Operation

Schematic of the J105 SIMS operation, illustrating the path of primary (green) and secondary (red and blue) ions through the instrument.

In order to solve this problem, the sample and its environment must be decoupled from the mass spectrometer. However, in order to do this, a new method of producing the first time-focus is required.

The J105 SIMS has been designed such that the primary beam is decoupled from the time-of-flight measurement. Instead, a shaped-field buncher and timed ion gate sit after the electrostatic analyzer, generating the first time-focus for the time-of-flight measurement. By cooling the ions prior to passing them into the buncher, the energy spread is reduced to within 1 eV, removing any effects of the sample.

As a result of this design, the distance traveled by the ions prior to reaching the spectrometer has no effect on the measured flight time. The mass spectrometer is therefore independent of the local environment on the sample.  This thus enables the J105 to analyze otherwise challenging samples, producing consistent, accurate results with no loss of resolution.

The following examples demonstrate the capabilities of the J105 to analyse rough, uneven, topographic samples without compromising mass resolution or mass accuracy.

Example #1: M1.6 Screw Thread

The figure below shows the total ion image as obtained from an M1.6 screw thread. This sample was chosen for its difficult topography, having a thread depth of approximately 300 μm. Despite the challenging nature of the sample, highly consistent results are obtained no matter what point on the surface is chosen.

The spectrum surrounding the Cr+ peak (nominally 51.94051 amu) is shown to the right of the image for a range of selected areas across the sample. The areas chosen span the full 300 μm depth of the screw thread.

Despite this large variation in height across the sample, the variation in peak center across the chosen points is only 0.0002 amu, which corresponds to a mass error of less than 5 ppm across the complete depth range.

Total Ion Image of M1.6 screw thread

Left Total ion image of an M1.6 screw thread, obtained on the J105 SIMS. Right Comparison of the Cr+ peak (nominal mass 51.94051 amu) from spectra obtained at various locations across the screw thread sample (height variation approximately 300 μm). Variation in mass accuracy across the 300 μm depth range of the sample shows less than 5 ppm deviation from the nominal value.

Example #2: Frog Embryo

The figure below shows the total ion image of a frog embryo during multiple stages of development over a 5.5 hr period. The embryo is a sphere approximately 1.1 mm in diameter – a very challenging samples for ToF SIMS.

Despite the challenging nature of the sample, uniform signal is obtained from almost all areas of the hemisphere without affecting the mass calibration.

Total ion image of developing Xenopus laevis embryo

Total ion image of a Xenopus laevis embryo at various stages of development, from 2 cells to many. Embryo is approximately 1.1 mm in diameter, in a field of view of 1200 x 1200 μm. Strong signal is obtained from all parts of the hemisphere, and the mass calibration did not change over the image. This research was originally published in the Journal of Lipid Research. Tian, H. J. Lipid Res. 2014. 55: 1970-1980. Published with permission from original author.

Conclusions

Rough, uneven, or highly topographic samples can cause problems in ToF SIMS analysis, resulting in loss of mass accuracy and mass resolution. Unfortunately, most real world samples tend not to be perfectly uniform.

The J105 SIMS solves this problem by decoupling the mass spectrometer from the primary beam, thereby removing any effects of the sample from the time of flight measurement. The result is a ToF SIMS capable of analyzing rough, topographic samples without compromising mass resolution or mass accuracy.

The J105 SIMS combines flexibility with high performance, delivering unprecedented performance on biological and inorganic samples alike. For more information, visit the J105 product page, read our Application Notes, or Contact Us to discuss your interest further.

3D SIMS overlay of a single Tetrahymena cell in water ice on a silicon substrate.

Depth Profiling in ToF-SIMS with the J105 SIMS

Depth profiling is a powerful technique in surface analysis for examining interfaces and exploring the 3-dimensional structures of a material, to which SIMS is uniquely suited.

Many modern instruments are equipped with a sputter ion beam in addition to their primary analysis beam for depth profiling. This enables users to perform “etch-cycles” in-between analysis cycles, thereby building up a stack of 2D images and generating a 3-dimensional view of the sample.

There are limitations to the use of SIMS for depth-profiling however. Unlike non-destructive techniques such as XPS, SIMS alters the surface during analysis. In fact, fragmentation and migration of species can occur up to 20 nm below the surface. Etch-cycles therefore perform several duties. 1. They enable 3D image capture in a realistic time-frame, and 2. they remove the damaged layers, leaving behind a relatively clean surface for the subsequent analysis cycle. This process has some obvious drawbacks, most notably that a significant amount of material is lost during each cycle. This can severely limit the depth resolution.

As the J105 SIMS uses a dual-stage ToF analyzer, any beam may be employed as the primary analysis beam, including C60 and gas cluster beams. In this case, additional etch-cycles are not required – analysis and low-damage etching are continuous and concurrent. This means that no data is lost and all the material is sampled, making the J105 SIMS an extremely accurate tool for depth profile analysis.

No etch-only cycles on the J105 SIMS

As the J105 uses a DC beam, there is no need to interlace an etching beam with the analysis beam as analysis and low-damage etching are continuous and concurrent.

Organic Samples

In 2015, NPL led a VAMAS study comparing compositional analysis of an organic layered structure using both SIMS and XPS. The stack consisted of layers of Irganox 1010 and either Irganox 1098 (denoted MMK) or Fmoc-pentafluoro-Lphenylalanine (denoted MMF) in well defined ratios.

Chemical structure of Irganox 1010 and Irganox 1098

Chemical structure of (a) Irganox 1010, and (b) Irganox 1098.

Compared with a number of instruments using a Bin+ analysis beam, the J105 SIMS, using a 40 kV Ar4000+ beam, achieved the highest depth resolution of any instrument, demonstrating 12.8 nm. The figure below shows an example depth profile of such an organic stack structure.

VAMAS depth profile using J105 SIMS

Depth profile of a stacked Irganox 1010/Irganox 1098 sample. The J105 SIMS demonstrated a depth resolution of 12.8 nm – the highest of any instrument in the study.

HeLa cells

Depth profiling may also be used to produce 3D chemical maps of complex structures. The example below shows a 3D reconstruction of HeLa cells – an immortal human cervical cell line. The lipid phosphocholine head group (m/z 184.1), shown in green, represents the cell membrane, while the nucleic acid adenine (m/z 135.1), shown in red, represents the nucleus.

3D chemical map of HeLa cells

3D chemical mapping of HeLa cells using a 40 kV C60 beam. Phosphocholine (m/z 184.1 – green) and adenine (m/z 135.1 – red) represent membrane and nucleus respectively. Data courtesy of John Fletcher.

Such 3D reconstructions, detailed by Fletcher, Rabbani, and Henderson et al. here, require extensive post-processing of the data to convert the 2D layers – which have no height information – into a 3D image that is representative of the actual structure. The result, however, is a stunning reconstruction of the cell structure, that is made possible by the ability of the J105 SIMS to capture a complete spectrum from each voxel without loss of data.

The animation below reveals how the cell membrane is gradually removed to reveal the internal contents – in this case, depicted by the void created in cells.

Animated GIF of depth profile through HeLa cells with J105 SIMS

Depth profiling through HeLa cells using a 50 kV (CO2)5000+ beam at 1 μm per pixel. The lipid layer (phosphocholine) @ m/z 184.1 – representing the cell membrane, is gradually etched away, revealing the contents of the nucleus (adenine) @ m/z 135.1. Data courtesy of Hua Tian.

Inorganic Samples

The properties of poly-atomic ion beams such as gas clusters and C60+ are particularly useful for the analysis of organic samples, and have allowed molecular depth profiling to become a reality. However, that does not mean that such ion beams do not also provide benefits on inorganic samples. In fact, C60 ion beams are uniquely useful for use on both organic and inorganic samples.

Sputtering under C60+ has been shown to be less sensitive to variation in material, incidence angle, and crystallinity of the sample and has been show to offer improvements in quantitation and reproducibility on glass/mineral standards, and therefore provide an excellent solution for depth profiling on these types of samples.

NIST Ni:Cr Standard

The performance of 40 keV C60+ on a metal multilayer sample is demonstrated on the Ni:Cr standard reference material from NIST. The sample comprises alternating layers of Ni and Cr on a silicon wafer substrate. The plot on the left shows the signal from the m/z 52, and 58 mass channels corresponding to the Cr and Ni respectively. The Ni layers are 66 nm thick and the Cr layers 53 nm. A depth resolution of ca. 5 nm is calculated (16-84% rising edge of 1st Ni layer). This is close to the expected crater depth of a single C60+ impact.

Depth profile through NIST Ni:Cr standard with J105 SIMS

40 keV C60+ depth profile through the NIST Ni:Cr standard showing < 5 nm depth resolution.

Mixed Metal Oxide Sample

The example below is a depth profile through a mixed metal oxide material of relevance in hydrogen fuel cell fabrication. The experiment was performed using a C60+ 40 keV primary ion beam rastered over a 150 × 150 µm2 field of view. The J105 provides excellent signal to noise as all the material is collected during the depth profile similar to a dynamic-SIMS instrument but with the parallel mass detection and mass range of a time-of-flight analyzer. Decoupling the mass spectrometry from the ion generation process also means that the mass resolution is not compromised, and mass accuracy and calibration does not drift as the sample is eroded. Depth profiles and 3D images are generated retrospectively from the “image stack” with the outermost pixels discarded to remove any crater-wall artifacts.

Depth profile through fuel cell using J105 SIMS

Variation in secondary ion signal as a function of primary ion beam fluence for Cr+, Fe+, CeO+, and TiO+ displayed in the profile and 3D reconstruction in green, grey, red, and blue respectively. 150 x 150 μm2 field of view, z depth approximately 600 nm.

The composition of this sample is in fact much more complex than shown in the above plot and 3D rendering, and contains Al, Mn, Cr, Ce, Fe, Ti and oxides of these metals all of which are observed clearly in the mass spectra through the profile, in addition there is a thin layer of Na and K on the very surface of the sample (not shown).

Conclusions

The J105 SIMS provides an excellent solution for depth profiling of samples. The ability to use a variety of cluster beams for continuous and concurrent analysis and low-damage etching, without the need for “etch-only” cycles, enables high depth resolution on any sample type. If you would like more information, or would like to speak to a member of our team, please get in touch via our Contact page.