In secondary ion mass spectrometry, a highly energetic ion beam is used to “sputter” material from a sample surface. This primary ion beam impacts the sample surface with energies ranging from a few hundred eV to more than 10 keV, causing a cascade-collision within the top few atomic layers that releases material from the surface. The sputtered material consists of atoms, atom clusters, molecular fragments, and backscattered primary ions. The small proportion of material that is ionized (secondary ions) can be extracted into the mass spectrometer using electrostatic fields. The amount of ionized material is dependent on the ionization efficiency of the element within a specific matrix, and on the primary ion used to sputter the sample, and can vary over many orders of magnitude.

SIMS works in both “static” and “dynamic” regimes depending on the current density of the primary ion beam. In static SIMS, the primary ion dose is typically <10¹² ions/cm², which results in a situation where each impinging primary ion interacts with an essentially pristine area of the sample surface. This leads to the liberation of low-energy secondary ions from within the topmost monolayers only—the surface is not actively eroded by the primary beam—hence, the term “static.” The ejected secondary ions have different velocities depending on their mass, and by measuring the time it takes for the ions to arrive at the detector, it is possible to differentiate between different elements, molecules, and molecular fragments [1]. The pulsed primary ion beam, typically consisting of cluster projectiles such as C₆₀ or Bi₃₀₀, can be rastered over the sample surface, producing images with lateral resolution up to 100 nm, which record the entire mass spectrum (up to tens of thousands of amu) on each pixel. The technique, however, must trade off lateral resolution for mass resolution and sensitivity and, even under optimum conditions, cannot achieve the mass separation necessary to provide high-precision isotopic analyses. The term “static SIMS” has generally been replaced by “ToF-SIMS” (time-of-flight-SIMS).

Dynamic SIMS, by contrast, uses a high-energy beam that actively erodes the sample surface, producing a higher secondary ion flux but destroying the molecular bonds between the atoms in the sample. Dynamic SIMS typically employs magnetic-sector mass analyzers, where secondary ions are deflected in a magnetic field depending on their mass. Double-focusing mass spectrometers also use an electrostatic sector to filter the secondary ions by kinetic energy either before or after the magnetic-sector. The so-called “sector-field” mass analyzers allow high mass resolution to be achieved with high transmission, providing optimal conditions for high-precision elemental and isotopic analyses. The development of dynamic SIMS over recent years has pushed towards higher sensitivity and better lateral resolution.

While both ToF-SIMS and dynamic SIMS have clear advantages and disadvantages over one another, this paper is primarily concerned with the development and application of high lateral resolution dynamic SIMS for use in biological and biomedical research.

SIMS is a well-established technique in the semiconductor industry, where it has been traditionally used to measure the concentration of dopants and in device failure analysis. The geological community has employed SIMS to quantify trace elements in minerals at concentrations too low for detection by more typical methods such as electron microprobe. It has also proved to be effective in the analysis of stable isotopes in minerals. The lateral resolution, however, has typically been limited to tens of microns because of the geometry of the probe-forming ion optics and as such has attracted little interest from the biological community. As originally proposed by Burns [2], however, there are clear advantages in applying SIMS to biological samples, not least because of the high sensitivity to biologically relevant elements such as C, N, O, S, Na, and K, but also for the potential to detect stable minor isotopes, such as ¹³C and ¹⁵N. Nevertheless, the overriding limitation was the restricted lateral resolution that struggled to differentiate cellular components with any detail.

The NanoSIMS 50, conceived by Georges Slodzian [3] and developed by CAMECA (Gennevilliers, France), was designed for imaging with submicron lateral resolution. This is achieved by a novel coaxial lens configuration, which positions the primary probe-forming lens parallel and very close to the sample, allowing the beam to be focused to a very small diameter. The primary ion beam impacts the sample surface at 90°, with the secondary ions extracted back through the same lens assembly. As the coaxial lens uses common focusing optics for both the primary and secondary ion beams, the polarity of the secondary ions must be opposite to that of the primary ions. Currently, two primary ion sources are commercially available for the NanoSIMS: a Cs⁺ source for the generation of negative secondary ions, which can be focused to a sub-50 nm probe diameter, and a duoplasmatron source that can produce O⁻ and O₂ ⁺ primary ions, with a smallest beam diameter of about 150 nm. Using the Cs⁺ primary beam also allows secondary electrons, generated during the sputtering process, to be extracted to a photo multiplier and used for imaging.

High mass resolution is achieved through the use of a double-focusing sector-field spectrometer, consisting of an electrostatic filter (to filter secondary ions according to their kinetic energy) and a magnetic-sector (to separate ions by their mass). The geometry of the mass spectrometer has been optimized to give high transmission and high mass resolution at high lateral resolution, such that a M/ΔM mass resolution of >5,000 with a 100 nm beam diameter is achievable with only an ~25 % loss in transmission. Most significantly, the mass resolution is high enough to separate, for example, ¹³C from ¹²C¹H on mass 13 and ¹²C¹⁵N from ¹³C¹⁴N on mass 27 (see Fig. 1).

The secondary ions are focused along the plane of the magnetic-sector where up to seven detectors can be positioned, allowing a large number of mass combinations to be measured. This multicollection capability allows the same microvolume to be sampled on up to seven detectors simultaneously, minimizing the loss of material and increasing throughput. The latest generation of NanoSIMS instruments comes equipped with both electron multipliers (EM) and faraday cup (FC) detectors and electronics that are housed under vacuum and thermally regulated. Imaging is achieved by scanning the focused primary ion beam across the sample, recording the number of incoming ions at the electron multipliers for each pixel of the scan (raster).

Early dynamic SIMS instruments provided insight into the realm of possibilities available for the biosciences [4]. The advent of high-resolution NanoSIMS resulted in a rapid increase in dynamic SIMS applications throughout the biological and biomedical sciences [5, 6]. For biology, NanoSIMS provides the opportunity to both spatially and temporally study dynamic physiological events—via the detection of either intrinsic or labeled isotopes—at a scale previously unattainable via other SIMS or X-ray detection methods. This subcellular imaging capability, combined with a high sensitivity for biological elements, has resulted in a powerful tool for investigating biological processes at a scale relevant to tissues, cells, and even single microorganisms (see Fig. 2). Areas of research that have utilized NanoSIMS are broad, ranging from medical to environmental applications.

In medicine, NanoSIMS has been successful at locating and measuring heavy metals (e.g., Au and Pt) in cells, introduced via anticancer therapeutics [7, 8], demonstrating the potential in drug delivery studies [9]. Natural iron deposition has also been identified in neural tissues associated with degenerative brain diseases [10, 11]. Changes in Ca distribution have also been identified with secondary degeneration of nerve cells following neurotrauma [12]. The use of labeled isotopes has been used to demonstrate stem cell division and metabolism [13] and the rate of protein turnover in hair cell stereocilia [14].

In environmental studies, metal uptake by plants has been investigated [15–17]. In soil, nutrient cycling has been demonstrated between microbes and plants, using ¹⁵N isotopic labels [18]. In fact, NanoSIMS has proved highly adept at deconvoluting the complex relationships between microbes and other organisms. Mapping isotopic labels has shown how bacteria fix nitrogen in bivalves [19] and how marine symbionts transfer nitrogen from seawater to corals [20].

In the last few years, the NanoSIMS has proved to be a revolutionary new tool for microbiologists [21]. The potential to simultaneously determine metabolic activity and identity of naturally occurring microorganisms had been demonstrated using conventional SIMS to analyze indigenous microbes in anoxic marine sediments [22]. Combining fluorescence in situ hybridization (FISH) with SIMS measurements of carbon isotopes found that cell aggregates binding a specific archeal probe were strongly depleted in ¹³C, indicating a methane-based metabolism. This FISH-SIMS approach is even better suited to NanoSIMS, as it can provide this information at the length-scale of an individual bacterium (~1 μm). Several variations on this approach, termed element-labeled fluorescent in situ hybridization (EL-FISH) or halogen-labeled in situ hybridization (HISH), have been reported [23–26]. But perhaps the most promising approach is catalyzed reporter deposition FISH (CARD-FISH), which deposits high concentrations of fluorine-containing fluorophores in target cells [27].