Figure 1 shows the overall scheme of a standard MS imaging (MSI) experiment. It comprises tissue preparation, matrix application [for matrix-assisted laser desorption ionization (MALDI) applications], MSI data acquisition, followed by data analysis and image construction. Acquisition of MS data is divided into two processes. The first process involves desorbing analyte molecules from the tissue followed by ionization to create positively or negatively charged ions. Desorption and ionization occurs in what is often referred to as the ionization source of the mass spectrometer. Depending on the probing beam that is used to interrogate tissue samples, a variety of ionization sources are available. Matrix-assisted laser desorption ionization (Caprioli et al., 1997; Chaurand et al., 1999; Stoeckli et al., 2001), desorption electrospray ionization (DESI) (Takats et al., 2004; Wiseman et al., 2008; Manicke et al., 2009; Mueller et al., 2011), and secondary ion mass spectrometry (SIMS) (Colliver et al., 1997; Vickerman, 2011) are the most common ionization sources, and they utilize a laser beam, a solvent stream, and an ion beam, respectively, as the probe. Laser ablation electrospray ionization (LAESI) (Nemes and Vertes, 2007; Nemes et al., 2008, 2009) uses laser irradiation to desorb the analyte molecules and a solvent stream to ionize the molecules. Table 1 summarizes the major characteristics of these commonly used ionization sources.
Once analyte molecules have been desorbed from the tissue surface and ionized, they are introduced into a mass analyzer and sorted on the basis of their mass to charge ratios (m/z). Table 2 lists a few commercially available mass analyzers and some of their important characteristics for MALDI MSI experiments. Time-of-flight (TOF)-MS was initially the only analyzer that was available for MALDI MSI, but now a wider variety of mass analyzers are available for MSI from most manufacturers (Garrett et al., 2007; Cornett et al., 2008; Strupat et al., 2009). Atmospheric pressure (AP) MALDI (Spengler and Hubert, 2002; Koestler et al., 2008; Römpp et al., 2010), DESI, and LAESI can be used with any mass spectrometer with an ESI source. Secondary ion mass spectrometry is commonly used with a TOF analyzer, and recently its adaptation to a quadrupole (Q)-TOF has been demonstrated (Carado et al., 2008). After a mass spectrum is acquired from an individual spot on the tissue, the sample plate is moved to a new position for the next spectral acquisition, and thousands or more spectra are obtained by rastering hundreds of x and y positions. The collected spectra are collated and chemical images of each ion of interest can be generated. More details about ionization, mass analysis, and image production are discussed in other review articles (Chughtai and Heeren, 2010), but basic information is described below along with some recent advances.
The first mass spectrometer to be used for imaging applications was TOF-SIMS, initially developed for elemental species (Castaing and Slodzian, 1962; Liebl, 1967). Secondary ion mass spectrometry uses a primary ion beam to desorb and ionize analyte molecules, which are then referred to as secondary ions. The single biggest advantage of SIMS sources compared with other ionization methods is their spatial resolution. Spatial resolution values of less than 100 nm are regularly reported with SIMS methods (Ostrowski et al., 2004; Piehowski et al., 2008), which compares to a typical high resolution of 10–20 μm with MALDI. In SIMS analysis, the primary ions bombard the sample surface with very high energy (typically 5–40 keV) and in high-ion-current mode, which can sputter individual atoms from the sample surface for elemental analysis. At lower ion currents (<1013 ions cm−2) the surface damage is minimized, and the desorption/ionization of intact molecules on the surface becomes possible (Vickerman, 2011). This process, called ‘static SIMS’, still creates a large number of molecular fragmented ions, and limits the size of the biological molecule that can be analyzed. Because of such high fragmentation and inherent low ionization efficiency, SIMS imaging has not been as widely used as other ionization sources for biological applications, in spite of its long history (Heeren et al., 2009).
The technique that has received the most widespread use as the ionization source for MSI experiments is MALDI. The exact mechanism of MALDI is not well understood (Karas and Kruger, 2003), but it is generally accepted that the process involves photon absorption by the matrix, local surface heating, and explosion/desorption of the surface molecules. Ionization can occur during the desorption process or in the high-density gas plasma that is initially formed, called the laser plume (Jaskolla and Karas, 2011). Co-crystallization of the matrix with the analyte molecules is considered to be essential, particularly for macromolecules such as polypeptides and proteins. The choice of matrix depends on many factors including, but not limited to, the class of analyte molecules being investigated, the molecular weight of the analytes, and the sample composition. A comprehensive description of matrix choices and the factors that need to be considered in selecting the matrix has been reviewed (Hillenkamp and Peter-Katalini, 2007). In some instances, the analyte molecules or other native species have adequate absorption at the wavelength of the laser (Li et al., 2006; Hoelscher et al., 2009), and data acquisition can be performed without a matrix, simplifying the sample preparation process.
Both MALDI and SIMS create singly charged ions, usually protonated or deprotonated ions ([M+H]+ or [M-H]−), but sometimes other adduct ions can be formed (e.g. [M+K]+ or [M+Na]+). Because the majority of ions created by these techniques are singly charged, the mass range of the mass analyzer is an important consideration if the experiment calls for imaging of large biological molecules such as proteins. Recently, Trimpin and co-workers showed that MALDI can also produce multiply charged ions for peptides and proteins with specific matrices and instrument conditions (Trimpin et al., 2009).
Desorption electrospray ionization desorbs and ionizes molecules from the surface of the sample by utilizing a plume of highly charged solvent molecules typically produced from a modified traditional ESI source (Dill et al., 2009). This method is energetically very soft, and is able to desorb and ionize a large range of molecules in a plant tissue sample without the use of a matrix (Mueller et al., 2011). In typical DESI experiments, the size of the ESI plume limits the spatial resolution of the technique. More recently, a nano-DESI source has been developed that could improve the spatial resolution of this technique (Roach et al., 2010). Additional molecules can be added to the solvent which can enhance the desorption/ionization processes for certain classes of molecules (Badu-Tawiah and Cooks, 2010). The DESI ionization source is capable of producing multiply charged ions, [M+nH]n+, which effectively lowers the (m/z) values of the macromolecular ions. Desorption electrospray ionization sources are especially useful for ionizing polar compounds, but more hydrophobic compounds can also be detected using appropriate solvents.
Another ionization method that is capable of producing multiply charged ions is LAESI. This technique separates the desorption and ionization process into two distinct steps (Nemes and Vertes, 2007). First, infrared (IR) laser photons focused on the sample surface are absorbed by native water molecules and create an ablation event. The ablated molecules, mostly cytoplasmic metabolites inside the tissue, are post-ionized by electrospraying with appropriate solvents. As in DESI, LAESI has the advantage of producing multiply charged ions, and the experiment can be performed in the ambient atmospheric environment. Laser ablation electrospray ionization has a unique advantage in that it can easily achieve depth profiling; each ablation event removes 30–40 μm of material from the tissue surface. Multiple acquisition of two-dimensional MSI data provides a means of stacking three-dimensional views of the distribution of analyte molecules (Nemes et al., 2009).
Secondary ion mass spectrometry and most MALDI ion sources operate in a vacuum chamber, which leads to the loss of volatile compounds and complicates issues associated with sample handling due to the removal of water from the sample. The DESI and LAESI techniques operate exclusively at atmospheric pressure (AP) and can minimize sample handling and associated issues. Recently, an AP MALDI technique has been developed that allows sampling in an ambient environment; however, this approach suffers from significant ion losses during the transfer of the ions from the source to the vacuum of the mass analyzer. Detailed mechanistic studies are being conducted on the formation of the laser plume in AP, and the transportation of the resulting ions into the mass analyzer to improve the ion transfer efficiency (Schmitz et al., 2010, 2011). Various ambient ionization methods have been developed or adapted for MSI (e.g. femtosecond laser ablation followed by electrospray ionization, Judge et al., 2011; low temperature plasma ionization, Liu et al., 2010; IR laser ablation followed by metastable chemical ionization, Galhena et al., 2010b). Continuing developments and applications are expected in MSI with ambient ionization conditions.
The second main component in MS data acquisition is the mass analyzer. Many different commercial mass analyzers are available for MALDI MSI (Table 2). There are several important characteristics that need to be considered in terms of selecting the appropriate analyzer for MSI: (i) mass resolution and tandem MS (MS/MS) capability, which is important for confident identification of analytes; (ii) spatial resolution, which is important for obtaining high-quality images; and (iii) MS scan speed, which enables acquisition of the image in a reasonable time frame. Tandem MS experiments fragment a selected ‘precursor’ ion and subsequently collect a mass spectrum of the fragment ions. Information garnered from MS/MS or multiple MS/MS experiments (i.e. MSn) can be used to provide structural information about the precursor ion. There is no single instrument that satisfactorily meets all these criteria, and therefore it is inevitable that an experimenter will have to sacrifice one or more of these characteristics. For example, high-resolution mass spectrometers [i.e. Fourier transform ion cyclotron resonance (FTICR) or Orbitrap] typically have slower scan speeds (see Table 2).
The most widely used instrument for MSI is MALDI-TOF. The operating principle behind a TOF mass analyzer is relatively straightforward. Ions are pulse-injected into a flight tube, which is field-free. The velocity of the ions inside the tube (v) depends on the (m/z) values of the ions [v = (2 zV/m)1/2, where V is the accelerating voltage], and thus the corresponding arrival time of the ions at the mass detector is given by t = L/v, where L is the length of the flight tube. Mass spectra can be obtained by measuring the arrival time of each ion at the detector; ions with low (m/z) values arrive earlier, and ions with high (m/z) values arrive later. The MALDI technique interfaces very well with TOF MS because both are pulse-based (i.e. the laser pulse can be used for both ion production and initiation of time measurement). Another advantage of TOF for MSI is the scan speed. TOF MS is able to record a mass spectrum at unparalleled speeds (as fast as 100 Hz), reducing data acquisition time in MSI. Commercial TOF mass analyzers provide mass resolution up to 40 000 (m Δm−1) in reflective mode, while maintaining fast scan speeds. Tandem MS experiments cannot be performed with a single TOF mass analyzer. To perform MS/MS experiments, a TOF/TOF or a Q-TOF hybrid mass spectrometer is required. In both cases an additional component, either another flight tube (in the case of TOF/TOF) or a quadrupole mass analyzer (in the case of Q-TOF), allows the system to select precursor ions for the subsequent fragmentation and fragment analysis in TOF-MS. Mass resolution for precursor selection is limited in TOF/TOF, but it offers the advantage of high-energy fragmentation, which is usually not available with other MS/MS instruments.
Ion-storage based mass analyzers have also been coupled with a MALDI source. These include an ion trap (IT) (Garrett and Yost, 2006), FTICR (Taban et al., 2007), or Orbitrap (Landgraf et al., 2009). In these types of analyzers, ions are stored inside the cell and their m/z values are determined by extracting individual ions (in IT) or through image current measurement (in FTICR and Orbitrap). Alternatively, the stored ions can be subjected to MS/MS analysis. Unlike other typical tandem mass analyzers in which selection of the precursor ion, fragmentation, and measurement of the (m/z) values of the fragments occur at different physical locations (MS/MS in space), MS/MS in ITs occur within the same space by sequential processes of precursor ion selection, fragmentation, and fragment spectrum acquisition (MS/MS in time). Excellent sensitivity and the ability to conduct multiple fragmentation MS cycles (i.e. MSn) are unique advantages of IT mass analyzers. Another very practical advantage of the IT technique, which has particular application in MSI, is the fact that there is almost no surface charging effect because low voltage is needed for ion extraction, so non-conducting surfaces (e.g. a glass slide) or thick tissue samples can be used in a MALDI IT.
The operation principles of Fourier transform (FT) type mass analyzers (i.e. FTICR and Orbitrap) are similar. Both analyzers trap ions in a cyclotron or orbiting motion inside a cell, either with magnetic or electric fields. Cyclotron or orbiting frequency is a function of (m/z), and Fourier transformation of ion current measured as a function of time provides the mass spectrum. Orbitrap and FTICR mass spectrometers provide ultrahigh mass resolving power, 100 000 or greater. However, this is at the expense of scan speed, which typically is 1 sec or longer. With a higher magnetic field, FTICR can perform mass analysis at a resolving power exceeding 1 000 000, but such analyzers store and measure ions with an even longer time. The slow scan speed makes imaging with FTICR challenging for large tissue samples, but it may be well suited for specialized applications. For example, the high mass resolving power of FTICR is able to distinguish ions with an m/z difference of only 0.001, even with extremely complex spectra (Kaiser et al., 2011).
Most MSI data are obtained in ‘microprobe’ mode, sampling one small area of the tissue at a time, then moving the MALDI plate to the next spot. An alternative approach is ‘microscope’ mode (Luxembourg et al., 2006; Harada et al., 2009), where a large area is sampled simultaneously and the spatial information of the ions is preserved during the ion flight, and recorded simultaneously with a position-sensitive detector. This approach has several advantages. First, the spatial resolution is not limited by the probe beam size, but rather by the detector pixel size, ion optical magnification, and the perfection of the ion optics. These parameters can be easily controlled to provide spatial resolution down to a few microns. Second, data acquisition is much faster, because data can be simultaneously acquired from hundreds or thousands of pixels. Currently, several technical barriers limit the wide application of this data acquisition mode. First, only TOF or magnetic sector type mass separation allows the preservation of the original spatial information of the ions; hence, high-resolution MS or MS/MS is not possible. Second, it has a limited field of view (i.e. 80 × 80 pixels), which limits sampling to small areas only. In addition, the current detectors for microscope mode have a limited response time and dynamic range. After the removal of these limitations, this technique will find its niche in imaging applications where fast data acquisition and ultrahigh spatial resolution are needed.
Software for data analysis
There are currently two major issues regarding the software for analyzing MSI data. One issue is that each commercial instrument typically uses a unique software platform and there is no consensus on how to integrate image data obtained with different instruments. This can be especially problematic when a research group has multiple MSI instruments or when image data have to be exchanged with other research groups. A common format for MSI data has been developed for this purpose, imzML (http://www.maldi-msi.org) (Römpp et al., 2011), but as yet it is not commonly used by researchers.
Another extremely useful goal of several software ventures is to extract common or previously unknown image features through automatic analysis of large data sets (Amstalden van Hove et al., 2010; McDonnell et al., 2010). Currently, most analysis of imaging data is performed manually, which is not only a time-consuming process but may also result in missing important image features. The automated analysis of large imaging data sets requires a huge computational resource, and their full utilization needs not only software developments but probably also developments in hardware.