Time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis depends on a high energy focused ion beam that functions as a primary ion source ejecting analytes only from the top atomic monolayers of a sample surface, referred to as secondary ions. The secondary ions are separated in a time-of-flight mass analyzer based on their mass-to-charge ratio (m/z) before they hit the detector [1–3].

Traditionally, ToF-SIMS has been applied in the domain of surface physics and semiconductor industry, but over the last decade it has redirected toward biomedical applications as well. Especially in mass spectrometry imaging -based biological tissue analysis, one has to contend with a high level of complexity concerning the molecules that are found on the sample surface. In many cases, it is predictable and generally accepted which compounds are detected in specific samples . Although the mass resolution and mass accuracy in ToF analyzers have improved over the years, particularly for higher masses this is not sufficient for unambiguous peak identification.

Tandem mass spectrometry is considered the benchmark technique to identify and elucidate structures of unknown molecular species and distinguish isomers of which there are multiplicities in biological samples. Controlled fragmentation of ions after they are formed in the source of a mass spectrometer results in a unique fragment pattern for every compound and is therefore an extremely useful tool for identification and elucidation of molecules.

Matrix-assisted laser desorption/ionization (MALDI) is often the ionization technique of choice for tandem MS imaging in biological tissues, but has several inherent limitations compared to ToF-SIMS imaging. First, in contrast to ToF-SIMS, MALDI needs a homogeneously applied chemical matrix to absorb the laser energy, to extract and ionize compounds from the sample. This makes the sample preparation time consuming and results in the perturbation of the intrinsic molecular chemistry and loss of lateral resolution . Furthermore, photon ionization of the analytes in a MALDI ion source is very destructive and usually consumes the sample, whereas in ToF-SIMS only sub nanometer thick layers are removed from the sample surface. In addition, the typical spatial resolution of MALDI imaging is above 30 μm, ToF-SIMS imaging can reach submicron spatial resolution. Lastly, current instrument designs do not allow simultaneous collection of MS¹ and MS² imaging data sets.

Recently, a novel and patented PHI NanoTOF II TRIFT mass spectrometer was developed, equipped with a second time-of-flight analyzer designed for tandem MS imaging [4–6]. The ion optics of the first mass analyser allow selecting a precursor from the stream of generated ions to be analyzed in the tandem time-of-flight mass analyzer simultaneously. This makes the combination of surface screening with general MS imaging with targeted identification with tandem MS imaging possible (Fig. 1).

In this chapter, we show an example of a tandem TOF-SIMS imaging experiment performed on a whole-body section prepared from a diseased zebrafish. The fish was part of a study on tuberculosis and therefore infected with Mycobacterium marinum as a model for human tuberculosis infection. Without further preparation, the samples were analyzed in negative ion mode using a PHI nanoTOF II TOF-TOF mass spectrometer equipped with a liquid metal ion gun (Bi₃ ⁺), a gas collision cell, and a second ToF detector. ToF-SIMS tandem imaging MS was used to localize and identify lipid species and fatty acids as well as the host membrane phospholipids that are believed to play an important role in the bacterial inflammation cascade.

Zebrafish (Danio rerio) are considered a suitable model to study infectious diseases such as tuberculosis (TB) and to aid in the development of treatments [7]. Mycobacterium marinum bacteria cause tuberculosis in fish, and has a very similar disease process compared to Mycobacterium tuberculosis, which is responsible for tuberculosis infections in humans.