The Carlsberg Foundation Research Infrastructure Grant has contributed to establishing mass spectrometry imaging (MSI) as a research field in Denmark. The MSI instrumentation has been – and continues to be – used in research projects within pharmacology, drug delivery and plant biology. MSI contributes with highly detailed information about the spatial distribution of compounds, which cannot be obtained by conventional techniques such as liquid chromatography-mass spectrometry (LC-MS). By Associate Professor Christian Janfelt, Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen The Carlsberg Foundation Research Infrastructure Grant has contributed to establishing mass spectrometry imaging (MSI) as a research field in Denmark. MSI relies on the recording of mass spectra in every point on the surface of a sample, from which images can be generated of every detected compound. The images are extremely specific and information rich thanks to the use of mass spectrometry (MS). In particular lipids are often imaged by MSI, since the lipid profile is characteristic to the different tissue types and can thus be used for tissue classification. Knowing the distribution of drugs and metabolites relative to such features in biological tissue is of great importance in understanding biological phenomena and processes. The MSI instrumentation has been – and continues to be – used in research projects within pharmacology, drug delivery and plant biology, some of which are mentioned below. MSI contributes with highly detailed information about the spatial distribution of compounds, which cannot be obtained by conventional techniques such as liquid chromatography-mass spectrometry (LC-MS). MSI contributes to a variety of research fields beneficial to society such as development of new treatments for skin cancer, improved food crops and new drug delivery technologies for better pharmaceutical products. “These new techniques are absolutely amazing because we can now accurately identify and locate all smaller organic molecules in a tissue section. It is reminiscent of the discovery of the microscope about 300 years ago as the new micro world was suddenly visible. These techniques will be very important for our understanding of disease processes, thereby helping in the development of new drugs and see how they work in a tissue.” - Harald S. Hansen, Professor in Pharmacology, University of Copenhagen. Which Compounds Are Where? MSI Provides the Answers As one of the most widely used analytical techniques, LC-MS is known for its high sensitivity and specificity – even in highly complex samples, drugs and metabolites can be detected and identified. However, since LC-MS and related techniques work on liquid samples, analyse of solid samples such as plant or animal tissue must be performed on liquid extracts of the samples, and information about the distribution of the detected compounds in the sample prior to extraction is therefore not available. As an alternative, mass spectrometry techniques like Desorption electrospray ionisation (DESI) and matrix-assisted laser desorption/ionisation (MALDI) (cf. the adjacent fact boxes) use a spray of charged droplets or a laser beam to ionise compounds from the samples surface. Therefore, such ionization techniques can be used for imaging by scanning the sample below the ionisation spot, while mass spectra are recorded from every point on the sample. Thus, after some hours of analysis, several thousand mass spectra have been recorded, and from this collection of mass spectra, specific images may be generated, one for each peak in the mass spectra; these images can then be overlaid in colours to show how different analytes are localised relatively to each other (Figure 1). This approach is referred to as mass spectrometry imaging (MSI), and it provides information about distributions which cannot be obtained by any other techniques. Figure 1: The principle of MSI, illustrated on a leaf of Hypericum performatum (St John’s wort). A spectrum is recorded from every point on a sample, and from this collection of mass spectra, individual images can be generated from each of the detected compounds. From . Imaging of Tissue Biomarkers – Histology by Mass Spectrometry Most MSI studies today are performed on cryo-sections of animal tissue, e.g. for tissue diagnostics or drug distribution analysis. Many endogenous compounds are imaged in such studies, and the lipids are particular interesting, since they constitute the major part of all cellular membranes. It turns out that the lipid profile observed in a mass spectrum is characteristic to the cell type being analysed, and thereby imaging of lipids can be used for example for very accurate localization of cancer tissue relative to healthy tissue. We have used MSI in the study of brain ischemia in mice, studying the inflammation, which occurs e.g. in the event of a stroke, and the subsequent resolution and repair phase. We found biomarkers for the dead neurons as well as for the macrophages, which on behalf of the immune system resolve the infarct. Figure 2: MALDI images reveal the development of brain ischemia in mice over the course from 2 hours to 20 days. Top row shows microscope images of histologically stained tissue, while the mid row shows MALDI images of a biomarkers for dead neurons, while the bottom row shows MALDI images of a biomarker for the macrophages. From . By imaging brains from mice, which were euthanised at different time points after the onset of the ischemia, we could monitor the stepwise resolution of the infarct (Figure 2). Our recently acquired instrumentation for MALDI imaging provides an exceptional spatial resolution of 5 µm, delivering images of microscopic details as seen in the images of rat testis in Figure 3. Here, MS images based on molecular information make it relatively simple to localise the different cell types in testis. These initial results have led to collaboration with Dept. of Growth and Reproduction at Copenhagen University Hospital, focusing on localisation of compounds of importance for the human spermatogenesis. Imaging of Drugs and Metabolites – Does the Drug Make its Way to the Target and Where Is it Metabolised? In development of new drugs, it is important to study the fate of a drug in the organism – how is the drug absorbed and where in the organism it is transformed? Here, MSI can be used to trace the drug in the organism, and the selectivity of MS allows for specific imaging of the different metabolites of the drug. Figure 4 shows how a drug can be followed in a mouse. The antidepressive drug amitriptyline was dosed in the body cavity of a mouse and already 15 minutes later, the major metabolite nortriptyline was detected in the liver and kidneys. It is also observed that the original drug was detected not only in the abdominal cavity but also in the fatty tissue near the skin, which may be due to the highly lipophilic structure of the drug molecule. Figure 4: Whole-body DESI imaging of a mouse dosed with the antidepressive drug amitriptyline. The drug is shown in red, the metabolite nortriptyline is shown in blue, and an endogenous lipid (in green) is used to display the profile of the animal. From . In order for a drug to be absorbed and exert its effect in the organism, the drug must penetrate barriers such as the skin or the intestinal wall. In a study on drug delivery in skin, we were able to show that the hair follicles play an important role in the absorption of the commonly used local analgesic lidocaine. Also, by detection of the metabolite 3-OH lidocaine, we were able to demonstrate that metabolism is not just a feature of the liver but also takes place in the subcutaneous fatty tissue in skin (Figure 5a). “The MSI technology allows to visualise drug biodistribution in skin and for the first time, we’re able to confirm improved and homogeneous drug uptake in a highly specific manner. The MSI technique continues to be a vital feature of our work, pursuing to develop new, targeted, non-invasive treatments to patients with skin cancer.” - Merete Hædersdal, MD, Professor in Dermatology, Bispebjerg Hospital. Indeed, the penetration of drugs through the skin is a major concern, also in potential treatment of skin cancer by chemotherapy directly on the skin. Work is in progress at Bispebjerg Hospital on such chemotherapy treatment, where a laser is used to burn channels in the skin for efficient delivery of drugs to the tumour. We can show by conventional LC-MS analysis of skin sections, that the drugs can be delivered to 2 mm depth in the skin, but MSI was necessary to show that the drug not only remains by the laser channels but also distributes homogenously to the target tissue (Figure 5b). We are planning future studies on real tumour samples where images of the chemotherapy drug are combined with images of tumour biomarkers in order to ensure sufficient delivery of the drug to the tumour. Figure 5: a) DESI-MSI study of delivery of lidocaine through skin. Lidocaine (in red) is seen to at 1.6 mm depth to be highly abundant near the hair follicles, suggesting that they play a role in delivery of the drug. The metabolite 3-OH-lidocaine (in green) is also detected in the adipose (fat) tissue. From . b) MALDI-MSI study of laser mediated delivery of 5FU through skin for treatment of skin cancer. The control tissue (left) shows very limited and random delivery of drug even to a low depth of 100 µm. In contrast the laser treated tissue (right) shows significance abundance of the drug; the laser channels are observed in a regular pattern, but the drug is observed to be relatively homogenously distributed throughout the tissue. From . Imaging of Plant Metabolites – Understanding Biosynthesis of Natural Products The new MSI techniques have been widely embraced by the plant science community since localisation of the different plant metabolites is important for understanding their biosynthesis and function. Since plants unlike most animals are very immobile, they have developed a vast number of metabolites over millions of years having functions in communication, attraction of insects for pollination and for determent of possible herbivores. The MSI-example in Figure 1 shows a leaf of Hypericum perforatum, which is the first plant we studied by MSI  and since then, we have contributed to a number of MSI studies of different plant materials [7-12]. Results from MSI can be combined with genetics to obtain more detailed information about where and how the plants synthesize different natural products. “Our research aims to understand plant’s specialised metabolism and discover metabolic pathways for production of compounds of high value in medicine, in the flavour and fragrance industry, for optimisation of crops for climate change, etc. Distribution analysis of metabolites has been lacking behind compared to techniques for studying localisation of gene expression, but this has changed with the advent of robust MSI techniques with much improved spatial resolution, and such analyses will undoubtedly become a gold standard in future pathway discovery in plants.” - Birger Lindberg Møller, Professor in Plant Biochemistry, and Nanna Bjarnholt, Associate Professor in Plant Biochemistry, University of Copenhagen. Support from the Carlsberg Foundation for MSI and Considerations on SSR Support from the Carlsberg Foundation has been crucial for establishing our research in MSI and for all the results presented above. In 2009, the Carlsberg Foundation contributed to the purchase of a mass spectrometer which was used for DESI imaging, and in 2015 another Carlsberg Foundation grant contributed to an Orbitrap mass spectrometer for MALDI imaging. Both grants have in interplay with concurrent grants from the Danish Council for Independent Research (for the imaging ion sources used with the mass spectrometers) been essential in building up the expertise in MSI in Denmark and in the development of my career since my start as Assistant Professor in 2008. At the moment, approx. 25 scientific papers on MSI have resulted from the two mass spectrometers purchased with support from the Carlsberg Foundation. Some of these papers deal with important issues in society such as cancer treatment and development of future food crops. Other papers present new methods which may help in the development of new and better drug products for treatment of a variety of diseases. References  N. Bjarnholt, B. Li, J. D’Alvise, C. Janfelt, Mass spectrometry imaging of plant metabolites – principles and possibilities. Nat. Prod. Rep. 2014, 31, 818–837.  M. M. B. Nielsen, K. L. Lambertsen, B. H. Clausen, M. Meyer, D. Bhandari, S. T. Larsen, S. S. Poulsen, B. Spengler, C. Janfelt, H. S. Hansen, Mass spectrometry imaging of biomarker lipids for phagocytosis and signalling during focal cerebral ischaemia. Scientific Reports 2016, 6, 39571.  S. Okutan, H. S. Hansen, C. Janfelt, A simplified approach to cryo-sectioning of mice for whole-body imaging of drugs and metabolites with Desorption Electrospray Ionization Mass Spectrometry Imaging. Proteomics 2016, 16, 1634–1641.  J. D’Alvise, R. Mortensen, S. H. Hansen, C. Janfelt, Detection of follicular transport of lidocaine and metabolism in adipose tissue in pig ear skin by DESI Mass Spectrometry Imaging. Anal. Bioanal. Chem. 2014, 406, 3735–3742.  E. Wenande, U. H. Olesen, M. M. Nielsen, C. Janfelt, S. H. Hansen, R. Anderson, M. Haedersdal, Fractional laser-assisted topical delivery leads to enhanced, accelerated and deeper cutaneous 5-fluorouracil uptake. Expert Opinion on Drug Delivery 2016, 14, 307-317.  J. Thunig, S. H. Hansen, C. Janfelt, Analysis of Secondary Plant Metabolites by Indirect Desorption Electrospray Ionization Imaging Mass Spectrometry. Anal. Chem. 2011, 83, 3256-3259.  L. Kato, A. P. Moraes, C. M. A. de Oliveira, B. G. Vaz, L. de Almeida Gonçalves, E. C. e Silva, C. Janfelt, The Spatial Distribution of Alkaloids in Psychotria prunifolia (Kunth) Steyerm and Palicourea coriacea (Cham.) K. Schum Leaves Analysed by Desorption Electrospray Ionisation Mass Spectrometry Imaging. Phytochem. Anal. 2017, in press.  B. Li, D. R. Bhandari, C. Janfelt, A. Römpp, B. Spengler, Natural products in Glycyrrhiza glabra (licorice) rhizome imaged at the cellular level by atmospheric pressure matrix‐assisted laser desorption/ionization tandem mass spectrometry imaging. The Plant Journal 2014, 80, 161-171.  B. Li, N. Bjarnholt, S. H. Hansen, C. Janfelt, Characterization of barley leaf tissue using direct and indirect desorption electrospray ionization imaging mass spectrometry. J. Mass Spectrom. 2011, 46, 1241-1246.  B. Li, S. H. Hansen, C. Janfelt, Direct imaging of plant metabolites in leaves and petals by desorption electrospray ionization mass spectrometry. Int. J. Mass Spectrom. 2013, 348, 15-22.  B. Li, C. Knudsen, N. K. Hansen, K. Jørgensen, R. Kannangara, S. Bak, A. Takos, F. Rook, S. H. Hansen, B. L. Møller, C. Janfelt, N. Bjarnholt, Visualizing metabolite distribution and enzymatic conversion in plant tissues by desorption electrospray ionization mass spectrometry imaging. The Plant Journal 2013, 74, 1059-1071.  A. Kucharíková, K. Kimáková, C. Janfelt, E. Čellárová, Interspecific variation in localization of hypericins and phloroglucinols in the genus Hypericum as revealed by desorption electrospray ionization mass spectrometry imaging. Physiologia plantarum 2016, 157, 2-12.  M. Kompauer, S. Heiles, B. Spengler, Atmospheric pressure MALDI mass spectrometry imaging of tissues and cells at 1.4-um lateral resolution. Nature methods 2017, 14, 90-96.