Advantages of multimodal imaging by elemental and molecular mass spectrometry
by Dr Ann-Christin Niehoff
Multimodal imaging by mass spectrometry offers a spatially resolved analysis of tissue sections as an additional tool in clinical research. Here, matrix-assisted laser desorption/ionization mass spectrometry for molecular imaging and laser ablation inductively coupled plasma mass spectrometry for elemental imaging are used to tackle two drug applications.
Mass spectrometry imaging
In recent years, mass spectrometry imaging (MSI) has gained more and more interest in the field of clinical, biological and pharmaceutical research. In contrast to hyphenated chromatographic techniques (e.g. LC-MS or GC-MS), MSI provides spatially resolved information while maintaining high sensitivity. With today’s techniques, high spatial resolution down to the low micrometre range can be achieved and is therefore a good combination with existing clinical imaging approaches from pathology.
Multimodal imaging describes the combination of different imaging techniques, as none by itself is a gold standard to answer all questions. Since MSI works with tissue sections, it can be combined easily with various microscopy applications, providing an additional input to clinical histology. Although protocols for different kinds of tissue sections exist, the preference here is to work on cryosections rather than formalin-fixed paraffin-embedded sections; this helps to avoid wash out of analytes from the tissue during the fixation and embedding steps.
Different MSI techniques can be used to focus on molecular or elemental imaging. In this article, the focus will be on matrixassisted laser desorption ionization mass spectrometry (MALDIMS) for molecular and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for elemental imaging.
MALDI-MSI
MALDI-MS is the most frequently used imaging technique in molecular MS. The analysis requires coating of the tissue section with a matrix, typically a small organic compound, and performing soft ionization of desorbed molecules by a pulsed laser. Ionization efficiency is highly dependent on molecular structure, matrices and laser wavelength. By scanning over the sample, a full mass spectrum is generated for each pixel.
Using the tandem MS (MS/MS) mode, fragmentation studies can provide information on molecular structures. Matrix preparation is one of the critical aspects and a potential disadvantage of MALDI-MS, Microscopy and Imaging in the Clinical Lab June 2020 21 | Figure1. Multimodal imaging of myocardial infarction Microscopic images of the two parallel sections (a & b) with the area of myocardial infarct (marked with a black line), quantified distribution of gadolinium determined with LA-ICP-MS (c) at 15|μm spot size, distribution of the ligand from Gadofluorine P (d) at 40|μm spot size, as well as the structure of the ligand and the theoretical spectrum (cyan bars) and the measured spectra (black line) with MALDI-MS (e). as it may influence limits of detection and spatial resolution due to analyte extraction of the sample by the matrix. Different instruments for matrix preparation are therefore commercially available to improve homogenous distribution and reproducibility. Owing to matrix effects in molecular MS, quantification is challenging, but is possible to achieve for single analytes via internal standards or standard addition with matrix matched standards.
Here, matrix was sublimated using the iMLayer (Shimadzu). MALDI-MS experiments were performed with the iMScope TRIO (Shimadzu), equipped with a fluorescence microscope, atmospheric pressure MALDI-source and an ion trap/time-of-flight (IT-TOF) mass analyser. IMAGEREVEAL MS (Shimadzu) was used for data analysis.
LA-ICP-MSI
In the field of elemental MSI, LA-ICP-MS provides major, minor and trace elemental information on surfaces and tissue sections. A laser is scanned over the sample and the ablated material is transported by a carrier gas into the ICP source, where the particles are atomized and ionized. To obtain spatially resolved images, transient signals of the respective analyte are required.
As mass analyser, quadrupoles are most frequently used. Although less matrix dependent than MALDI-MS, a fundamental aspect of recent research is method development for reliable quantification strategies, mainly via matrix matched standards. The major disadvantages of LA-ICP-MS are its destructive nature with loss of molecular information.
In this study, experiments were performed with the LSX-G2+ laser ablation system (Teledyne Cetac Technologies) coupled to the quadrupole based ICPMS-2030 (Shimadzu).
Complementary bioimaging of Gadofluorine P in myocardial infarction in mice
Magnetic resonance imaging is a widely used imaging technique in daily clinical practice. To enhance contrast during this examination, several different contrast agents are available. While most gadoliniumbased contrast agents (GBCAs) distribute systemically, some targetspecific GBCAs are under investigation as well. Gadofluorine P is one of these target-specific contrast agents and shows high affinity towards the collagen-rich extracellular matrix which is secreted in the event of myocardial infarction (MI) [1].
In this application, mice underwent injection of Gadofluorine P solution as contrast agents 6|weeks after an induced MI. Afterwards the mice were sacrificed and the hearts were removed for cryosections preparation. By multimodal imaging, LA-ICP-MS was used to generate quantified elemental imaging of gadolinium, while MALDI-MS validated the findings (Fig. 1) and could further provide information for phospholipids and heme b distribution (data not shown).
Figure 1 shows the microscopic images (a & b) of the two thin sections analysed. With LA-ICP-MS (c), a homogeneous distribution of the gadolinium in the healthy myocardium with an average concentration of about 50|μg/g was detected. The infarct region contained two times higher gadolinium concentrations of about 110|μg/g with maximum values up to 370|μg/g.
Higher gadolinium concentrations could also be found in the ventricle due to the intravenous administration of the contrast agent. These distributions could be verified with MALDI-MS imaging (d).
In this experiment, only the protonated ligand of Gadofluorine P rather than the intact complex could be detected (e). The main peak (m/z 1168.39) was used to create the image, which showed good correlation to the gadolinium distribution determined with LA-ICP-MS. The highest intensities of the molecular probe were found in MI and ventricle regions, whereas healthy myocardium showed low and homogenous intensities.
Multimodal imaging of photosensitizers in 3D tumour cell models
Photodynamic therapy offers an alternative cancer treatment. A photosensitive compound (photosensitizer; PS) is administered and the tumour is subsequently irradiated. The activation of the PS leads to the formation of a reactive oxygen species and subsequently to cell apoptosis. One main challenge in the development of PS is the hydrophobic character of the compounds, which hinders tissue penetration. Additionally, the orally administered compound needs to pass through the mucus layer in the gastrointestinal tract. Thus, the determination of the penetration depth of these compounds is of great interest.
The use of 3D tumour spheroids enables in vitro drug screening, while simulating the tumour environment better than 2D cell cultures. The photosensitizer 5,10,15,20-tetrakis (3-hydroxy-phenyl)-porphyrin (mTHPP) and its palladiumtagged analogue mTHPP-Pd were analysed in this study. Here, multimodal imaging is used to visualize the penetration depth of mTHPP and the lipid distribution in 3D tumour spheroid by MALDI-MS (5|μm spot size) as well as to quantify the drug by LA-ICP-MS (7|μm spot size) [2,3].
The MALDI-MS and LA-ICP-MS images of a tumour spheroid treated with mTHPP or mTHPP-Pd are shown in Figure 2. In the microscopic image, an almost spherical tissue section with a diameter of approx. 550|μm can be seen. The distribution map of mTHPP shows a ring-shaped distribution, which can be precisely correlated with the outer cell layer of the tumour spheroid. The PS is distributed homogeneously inside the outer layer and not around the spheroid, although it does not penetrate deeper into the tissue.
Nevertheless, the MALDI-MS experiments revealed that the PS can be detected as an intact molecule without substantial decomposition during the sample preparation. The LA-ICP-MS results for a spheroid incubated with mTHPP-Pd show the same distribution as the mTHPP detected by MALDI-MS. Since the metal-tagged PS is needed for ICP-MS analysis, only spheroids treated with this compound could be investigated. Conversely, this modification of the molecule could no longer be detected using MALDI-MS. Owing to the loading with palladium, the preferred protonation sites of the molecule are unavailable, impairing the detection.
However, before LA-ICP-MS experiments, MALDI-MS can be used to identify phospholipids as shown in Figure 3. Palladium concentrations up to 10|μg/g with an average of 1.9|μg/g were detected (Fig. 3b). This represents an enrichment of PS by a factor of 3 (average) up to 18 (highest concentration) compared to the incubation concentration. The phospholipids PC(34:1), PC(34:0) and PC(30:0) could be detected and show different distributions coherent with the different metabolic zones in a tumour spheroid.
Conclusion
In conclusion, the two applications shown provide an example of how to add MSI to clinical research. Multimodal imaging has successfully been performed to address drug penetration and enrichment in different kinds of tissue based on the combination of elemental imaging and molecular imaging by LA-ICP-MS and MALDI-MS.
The author
Ann-Christin Niehoff PhD
European Innovation Center, Shimadzu Europa GmbH,
47259 Duisburg, Germany
E-mail: acn@shimadzu.eu
