Ambient Mass Spectrometry Imaging: A Comparison of Desorption Ionization by Sonic Spray and Electrospray
- 1.5k Downloads
Easy ambient sonic spray ionization (EASI) and desorption electrospray ionization (DESI) were used for imaging of a number of samples, including sections of rat brain and imprints of plant material on porous Teflon. A novel approach termed Displaced Dual-mode Imaging was utilized for the direct comparison of the two methods: Images were recorded with the individual rows alternating between EASI and DESI, yielding a separate image for each technique recorded under perfectly similar conditions on the same sample. EASI works reliably for imaging of all samples, but the choice of spray solvent and flow rate is more critical in tissue imaging with EASI than with DESI. The overall sensitivity of EASI is, in general, slightly lower than that of DESI, and the representation of the dynamic range is different in images of the two techniques for some samples. However, for abundant compounds, EASI works well, resulting in images of similar quality as DESI. EASI can thus be used in imaging experiments where the application of high voltage is impractical or undesirable. The present study is in its nature also a comparison of the characteristics of the two techniques, showing results also applicable for non-imaging work, with regards to sensitivity and experimental conditions.
Key wordsEasy ambient sonic spray ionization Desorption electrospray ionization Imaging mass spectrometry Ambient mass spectrometry Displaced dual-mode imaging
With the introduction of desorption electrospray ionization (DESI) in 2004 and direct analysis in real time (DART) in 2005, it became possible to perform mass-spectrometric analysis directly from surfaces and under ambient conditions [1, 2]. The possible applications of the techniques range from direct analysis of tablets [3, 4] and profiling of tissues  to detection of explosives [6, 7] and analysis of liquid samples or extracts deposited on surfaces [8, 9], benefiting from the rapid detection offered by these direct techniques.
Since 2004, a variety of related techniques have been invented and applied for many different purposes [10, 11, 12]. As for DESI, several of the techniques are desorption analogues of already established spray techniques (e.g., desorption atmospheric pressure chemical ionization (DAPCI) , desorption atmospheric pressure photo ionization (DAPPI) , and nanospray DESI [14, 15], inspired by APCI, APPI, and nanospray, respectively.
Sonic spray ionization (SSI)  is an alternative to electrospray ionization, using no high voltage and thus relying solely on the pressure of the nebulizer gas to induce ionization. Charged droplets and, consequently, gas-phase ions are formed because of an unbalanced charge distribution during droplet formation in the supersonic spray. Compared to ESI, SSI results in a softer ionization of (e.g., amino acids) since the ions have lower internal energies than those produced in ESI [17, 18]. The desorption analogue of SSI was presented in 2006 by Haddad and co-workers, first under the name desorption sonic spray ionization (DeSSI)  and later renamed to easy ambient sonic-spray ionization (EASI) . EASI is one of the simplest of the ambient ionization techniques and can be used with any mass spectrometer with an atmospheric pressure inlet. It does not require high voltage or radiation sources and uses only compressed nitrogen and a spray solvent for ionization.
EASI has proven to be efficient in desorbing and ionizing compounds from different types of surfaces, such as drug tablets , thin layer chromatographic (TLC) plates [21, 22, 23], and paper [24, 25, 26, 27]. EASI has been utilized to analyze drugs trapped inside the pores of a molecularly imprinted polymer  and for real-time studies of the polymerization of organo-functionalized silanes  and degradation of drugs . A combination of DESI-imaging and non-imaging EASI has been suggested for very specific recognition of counterfeit banknotes . Recently, EASI was used for lipid profiling of liver tissue, however, only as liver extracts deposited on a paper surface  rather than direct analysis of the liver tissue as previously presented with DESI .
DESI imaging has since its introduction in 2006  been used mainly for tissue analysis, (e.g., in imaging of drugs in tissue  and examinations of diseased tissue [34, 35, 36], but also for imaging of plant material [37, 38], studies of metabolite exchange between bacteria , defence mechanisms of seaweed , and analysis of inks  and chemical fingerprints .
EASI has not been applied for either direct tissue analysis or imaging. In addition, EASI and DESI have never been compared directly on the same samples. Thus, the scope of this study is to explore and compare these two related techniques in a number of imaging experiments of various samples. Imaging experiments are well suited for the comparison of ionization techniques, since the generation of an image inherently provides a pixel-to-pixel comparison, making it easy to distinguish between signal and noise. It is striking how little signal from a peak is actually needed to provide a good image. Even small peaks that would normally be dismissed as noise may in the comparison with regions in the image, where the compound is not present, indeed turn out to originate from an actual analyte. Therefore, in comparisons of sensitivity, which is all about signal-to-noise ratios, imaging experiments are well suited, although they of course do not provide any quantitation or limits of detection.
LC-MS grade acetonitrile was purchased from VWR International (Herlev, Denmark) and water was prepared with a Millipore Direct-Q3 UV system (Billerica, MA, USA). All standard compounds were purchased from Sigma-Aldrich Denmark A/S (Copenhagen, Denmark). Porous Teflon (1.5 mm thick, medium pore size of 7 μm, pore volume of 36 %) was purchased from Berghof (Eningen, Germany).
2.2 Tissue Sections
The brain was removed from a 9-week old male Sprague-Dawley rat (Taconic, Ry, Denmark), immediately put on dry ice for rapid freezing, and subsequently placed in a –80 °C freezer. The frozen brain was mounted on a cryotome sample holder under –25 °C with water as the only adhesive. The brain was cut in slices of 14 μm thickness using a Leica CM3050S cryotome (Leica Microsystems, Wetzlar, Germany). Subsequently, the slices were thaw-mounted on glass slides (four sections per glass slide) and stored at –80 °C until time of analysis. On the day of analysis, the sample slide was taken directly from the freezer to a vacuum dessiccator for 10 min prior to DESI analysis.
2.3 Imprints of Plant Material
Plants of St. John’s Wort (Hypericum perforatum) were harvested in Denmark in September 2011. Imprints were made by pressing the leaves in a sandwich of porous Teflon, leaf, tissue paper, and silicone rubber (1 mm thick) between two aluminum plates in a vice, as described in further details elsewhere .
Mass spectrometric data were recorded using a Thermo Fisher Scientific LTQ XL Linear Ion Trap Mass Spectrometer (San Jose, CA, USA) equipped with a custom-built DESI imaging ion source. The DESI imaging ion source, described in detail elsewhere , was based on a motorized microscope stage from Märzhäuser Wetzlar (Wetzlar, Germany) and controlled by an in-house written software. The mass spectra were collected with Xcalibur 2.0 software (Thermo Fisher Scientific) and converted to image files (imzML files) using a conversion tool (www.maldi-msi.org). The images were generated with Data Cube Explorer from AMOLF, Amsterdam, The Netherlands.
The MS parameters were as follows for negative ion mode experiments: 300 °C heated capillary temperature, –5 kV spray voltage for DESI experiments (0 kV for EASI experiments), –35 V capillary voltage, –110 V tube lens voltage, and automatic gain control (AGC) off. In positive ion mode the capillary voltage was 30 V, the tube lens voltage was 120 V, and the spray voltage was +5 kV for DESI and 0 V for EASI.
For the imaging of rat brain (negative ion mode: scan range m/z 500–1,000, positive ion mode: scan range m/z 600–1,000), the geometric parameters were optimized for EASI, using a nebulizer gas pressure of 10 bar and a 10 μL/min flow of methanol. The ion injection time was 100 ms, and four microscans were averaged for each pixel in the image. A spatial resolution of 150 μm was used. The DESI images were recorded under the same conditions, only with ±5 kV high voltage applied to the ion source. Imaging of the plant imprint (negative ion mode, scan range m/z 300–700) was performed with a nebulizer gas pressure of 10 bar and a 2 μL/min flow of acetonitrile and water (50:50). The injection time was 50 ms with six microscans, and the spatial resolution was 100 μm. All images were recorded in a displaced dual-mode imaging approach, as discussed below, such that EASI and DESI images were recorded simultaneously of the same sample. The geometry optimization was always performed for EASI and used for both ionization modes.
The nebulizer gas pressure was tested in the range of 5–15 bar. A major improvement in the EASI signal was observed up to 10 bar, above which no further improvement was observed. For convenience and to avoid unnecessary smearing of the sample, the nebulizer gas pressure was kept at 10 bar in all experiments reported in this paper.
3 Results and Discussion
3.1 Considerations on Experimental Conditions
While DESI imaging is typically performed at solvent flow rates of 1–2 μL/min in order to avoid smearing of the sample leading to degraded spatial resolution, EASI analysis is usually performed a higher flow rates (up to 20 μL/min). DESI is typically performed using mixtures of acetonitrile (or methanol) and water as spray solvent, while mixtures of methanol and water or often pure methanol has been used for EASI.
EASI imaging was possible using a low solvent flow rate, but for tissue samples the geometry optimization was difficult and we were not able to perform any imaging in positive ion mode. However, we found that the optimum way of optimizing the spray geometry for tissue imaging with DESI was to turn off the high voltage and thus optimize in EASI mode. Since the spray geometry is very critical in low flow EASI, the optimal geometry settings could be recognized because of sudden and distinct changes in signal. When the optimal EASI geometry was found, the spray voltage was turned back on, resulting in optimal settings for DESI. By increasing the flow rate of the spray solvent (≥5 μL/min) more intense and reliable signals were obtained for EASI. Aqueous solvent mixtures at these high flow rates smear the tissue samples, making imaging impossible. However, by using pure methanol as spray solvent, solvent flows up to 20 μL/min were possible without damaging the sample. A flow of 10 μL/min of pure methanol was found to be ideal for imaging of tissue sections in EASI as well as in DESI. With this, in an imaging context unusually high flow rate, the geometry optimization became very simple for both EASI and DESI, providing straightforward and reproducible EASI images of brain tissue in negative as well as in positive ion mode.
The sensitivities of EASI and DESI were tested by analyzing standard solutions deposited on a porous Teflon surface, using a 2 μL/min flow of acetonitrile and water (50:50) and a 10 μL/min flow of methanol, respectively. Although 10 μL/min of pure methanol is optimal for EASI analysis of tissue samples, a 2 μL/min flow of the acetonitrile/water mixture was equally good or better for analysis of the standards on Teflon. DESI and EASI showed almost similar sensitivities for compounds, which are easily ionized such as methadone (tens of pg for both techniques). However, for compounds that are more difficult to ionize (e.g., paracetamol) the difference between the two techniques is greater in favor of DESI. The details and results of these experiments are found in the Supplementary Material.
3.2 Displaced Dual-Mode Imaging
In order to make a direct comparison of the two ionization techniques, we designed an experiment where the imaging stage was scanning the sample with for example 100 μm spatial resolution in the x-direction but in lanes separated by a 50 μm distance. Every second lane was scanned in EASI and DESI, respectively, thus generating one EASI image and one DESI image, both with 100 μm resolution in both directions. In this way, the same sample was imaged with both techniques, at the same time, and under the same conditions, thus enabling the best possible comparison of the two techniques. In experiments with aqueous (highly conductive) solvents, an equilibration time of 10 s between each row was sufficient, whereas 50 s were needed between each row for equilibration of the spray when pure methanol (weakly conductive) was used as spray solvent. We term this approach displaced dual-mode imaging (DDI) and foresee a number of other potential applications of it, as discussed below.
3.3 Imaging of Rat Brain Tissue
It is worth noticing that the spatial resolution of the two techniques is identical, also when compared with images obtained with a low flow of acetonitrile/water (Figure S2). The high flow of methanol does not cause any smearing of the sample.
3.4 Imaging of Plant Imprints
In another comparison of the two techniques, we performed imaging of Teflon imprints of leaves of Hypericum perforatum, a sample known from a previous study . This sample is ideal for this experiment because the porous Teflon surface is well defined and easy to optimize on, and the plant—studied in hundreds of papers—contains a vast number of different compounds.
The higher sensitivity of DESI observed in the spectra in Figure 5 is also expressed in the images of various compounds from the H. perforatum plant from the EASI and DESI experiments. As seen in Figure 6, DESI was capable of imaging several low abundance compounds. From the DESI data we could generate images of quercetin (m/z 301.0, Figure 6d) (known to accumulate in the black glands and therefore have the same distribution as hypericin ), an oxidation product of hyperforin (m/z 583.4, Figure 6e)  (which therefore has the same distribution as hyperforin), and rutin (m/z 609.2, Figure 6f), which is homogeneously distributed throughout the leaf surface and also previously shown to be found in the H. perforatum leaves . None of these compounds showed up in images generated by EASI.
3.5 Imaging of Dyes on Paper and PMMA in Positive Ion Mode
EASI has previously been used for analysis of inks and dyes, and was therefore expected to be suitable for imaging of such samples. Thus, letters written using red and blue permanent markers on paper and PMMA were used for testing of the imaging performance of EASI in the positive ion mode. The red pen provides a signal in positive ion mode at m/z 443, corresponding to Rhodamine 6G, and the blue pen provides a signal in positive ion mode at m/z 478, corresponding to Basic Blue 7, as well as a minor signal at m/z 443 due to Rhodamine B. The two isomeric rhodamines can be distinguished by MS/MS: Rhodamine 6G yields a major fragment at m/z 415, while the major fragment of Rhodamine B is m/z 399. It should be noted that all three dyes are cationic in their natural state, typically occurring as chloride salts and thus easily ionized. The critical point is therefore extraction and desorption of these compounds from the surface.
The imaging performances of EASI and DESI of the three dyes on paper and PMMA were equally good and with comparable absolute signal intensities. EASI appears to be quite tolerant with respect to contaminations (e.g., in the solvent line); the ion of m/z 443 is always the base peak, even on contaminated systems. In comparison, DESI is more vulnerable to contamination and shows several peaks in the background on a contaminated system. In EASI with its lower number of charges available, there seems to be a more pronounced preference for donating the limited amount of charges to the compound that is the most easily ionized, in this case the cationic dyes. In case of the blue dye, the m/z 443 peak (Rhodamine B) is slightly less prevalent, relative to Basic Blue 7 at m/z 478, in the EASI spectra than in the DESI spectra. The spatial resolution of the two techniques is similar, providing very comparable images down to a resolution of about 100 μm. The experimental details and results of these experiments are found in the Supplementary Material.
We have shown that EASI can be used for imaging as well as direct analysis of tissue sections. For analytes such as dyes, the performance is similar to DESI, even presenting a larger tolerance of contaminants in the analytical system. Imaging of tissue samples in negative and positive ion modes was possible although optimization of the solvent composition and flow rate is more crucial in EASI than with DESI. A 10 μL/min flow of methanol seems to be optimal, providing images with very high success ratios. These solvent settings also work well for DESI imaging of tissue, demanding very little in terms of geometry optimization in both positive and negative ion mode. The need for a higher solvent flow rate in EASI is probably due to the lower number of charges on the droplets, which is then compensated for by generation a larger number of droplets via an increased flow rate. The EASI images turned out a little differently than the corresponding DESI images, indicating differences in the dynamic range or some degree of ion suppression. However, EASI is well-suited for imaging of a variety of samples and analytes, in particular when the analyte is of relatively high abundance or easily ionized. EASI imaging can thus prove useful in imaging applications where high voltage (DESI) is impractical or undesirable (e.g., in potential imaging of living organisms).
In conclusion, we have introduced displaced dual-mode imaging, a new method to record two images with different ionization settings of the very same sample simultaneously, by using half the pixel size in the y-direction as in the x-directly and change the ionization settings for every second row in the image. We used it here to make a reliable estimation of the impact of the high voltage on the sensitivity in desorption imaging experiment by generating simultaneous images with and without high voltage. This enabled a direct comparison of the sensitivity and performance of EASI and DESI, which is otherwise difficult to obtain because of difficulties in reproducing geometries, samples, and spray conditions. A more general application, however, would be use it as a method to record simultaneous imaging in positive and negative ion modes of the same sample. This would be a good extension of the recent work by Cooks and co-workers, where one sample was used for consecutive DESI imaging, MALDI imaging, and H&E staining .
The authors thank Niels Wellner for providing the rat brain and Janina Thunig for useful discussions about compounds in Hypericum perforatum. Support from the Carlsberg Foundation, The Danish Council for Independent Research | Natural Sciences and the Working Environment Research Fund, Denmark, is gratefully acknowledged.
- 4.Nyadong, L., Late, S., Green, M.D., Banga, A., Fernández, F.M.: Direct quantitation of active ingredients in solid artesunate antimalarials by noncovalent complex forming reactive desorption electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 19, 380–388 (2008)CrossRefGoogle Scholar
- 6.Takats, Z., Cotte-Rodriguez, I., Talaty, N., Chen, H., Cooks, R.G.: Direct, trace level detection of explosives on ambient surfaces by desorption electrospray ionization mass spectrometry. Chem. Commun. 1950–1952 (2005)Google Scholar
- 11.Alberici, R., Simas, R., Sanvido, G., Romão, W., Lalli, P., Benassi, M., Cunha, I., Eberlin, M.: Ambient mass spectrometry: bringing MS into the “real world.” Anal. Bioanal. Chem. 398 (2010)Google Scholar
- 22.Eberlin, L.S., Abdelnur, P.V., Passero, A., de Sa, G.F., Daroda, R.J., de Souza, V., Eberlin, M.N.: Analysis of biodiesel and biodiesel-petrodiesel blends by high performance thin layer chromatography combined with easy ambient sonic-spray ionization mass spectrometry. Analyst 134, 1652–1657 (2009)CrossRefGoogle Scholar
- 31.Eberlin, L.S., Haddad, R., Sarabia Neto, R.C., Cosso, R.G., Maia, D.R.J., Maldaner, A.O., Zacca, J.J., Sanvido, G.B., Romao, W., Vaz, B.G., Ifa, D.R., Dill, A., Cooks, R.G., Eberlin, M.N.: Instantaneous chemical profiles of banknotes by ambient mass spectrometry. Analyst 135, 2533–2539 (2010)CrossRefGoogle Scholar
- 33.Kertesz, V., Van Berkel, G.J., Vavrek, M., Koeplinger, K.A., Schneider, B.B., Covey, T.R.: Comparison of drug distribution images from whole-body thin tissue sections obtained using desorption electrospray ionization tandem mass spectrometry and autoradiography. Anal. Chem. 80, 5168–5177 (2008)CrossRefGoogle Scholar
- 35.Masterson, T., Dill, A., Eberlin, L., Mattarozzi, M., Cheng, L., Beck, S., Bianchi, F., Cooks, R.: Distinctive glycerophospholipid profiles of human seminoma and adjacent normal tissues by desorption electrospray ionization imaging mass spectrometry. J. Am. Soc. Mass Spectrom. 22, 1326–1333 (2011)CrossRefGoogle Scholar
- 36.Janfelt, C., Wellner, N., Leger, P.-L., Kokesch-Himmelreich, J., Hansen, S.H., Charriaut-Marlangue, C., Hansen, H.S.: Visualization by mass spectrometry of 2-dimensional changes in rat brain lipids, including N-acylphosphatidylethanolamines, during neonatal brain ischemia. FASEB J. 26, 2667–2673 (2012)CrossRefGoogle Scholar
- 40.Lane, A.L., Nyadong, L., Galhena, A.S., Shearer, T.L., Stout, E.P., Parry, R.M., Kwasnik, M., Wang, M.D., Hay, M.E., Fernandez, F.M., Kubanek, J.: Desorption electrospray ionization mass spectrometry reveals surface-mediated antifungal chemical defense of a tropical seaweed. Proc. Natl. Acad. Sci. U. S. A. 106, 7314–7319 (2009)CrossRefGoogle Scholar
- 43.Holscher, D., Shroff, R., Knop, K., Gottschaldt, M., Crecelius, A., Schneider, B., Heckel, D.G., Schubert, U.S., Svatos, A.: Matrix-free UV-laser desorption/ionization (LDI) mass spectrometric imaging at the single-cell level: distribution of secondary metabolites of Arabidopsis thaliana and Hypericum species. Plant J. 60, 907–918 (2009)CrossRefGoogle Scholar