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Elucidating the distribution of plant metabolites from native tissues with LD-LTP MSI Abigail Moreno-Pedraza, Ignacio Rosas-Román, Nancy Shyrley García-Rojas, Héctor Guillén-Alonso, Cesaré Ovando-Vázquez, David Díaz-Ramírez, Jessica Cuevas-Contreras, Fredd Vergara, Nayelli Marsch-Martínez, Jorge Molina-Torres, and Robert Winkler Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04406 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

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Analytical Chemistry

Elucidating the distribution of plant metabolites from native tissues with LD-LTP MSI Abigail Moreno-Pedraza,† Ignacio Rosas-Román,† Nancy Shyrley García-Rojas,† Héctor Guillén-Alonso,† Cesaré Ovando-Vázquez,†,‡ David Díaz-Ramírez,†

Jessica Cuevas-Contreras,† Fredd Vergara,¶ Nayelli Marsch-Martínez,† Jorge Molina-Torres,† and Robert Winkler∗,†,§

†Center for Research and Advanced Studies (CINVESTAV) Irapuato, Department of Biochemistry and Biotechnology, Km. 9.6 Libramiento Norte Carr. Irapuato-León, 36824 Irapuato Gto., Mexico

‡CONACYT Potosino Insitute of Scientific and Technological Research (IPYCIT), National Supercomputing Center, Camino a la Presa San José 2055, Col. Lomas 4ta Sección, 78216 San Luis Potosí S.L.P., Mexico

¶German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany

§Max Planck Institute for Chemical Ecology, Mass Spectrometry Group, Beutenberg Campus, Hans-Knoell-Strasse 8, 07745 Jena, Germany E-mail: [email protected] Phone: +52 (462) 623 9635

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Abstract Secondary metabolites of plants have important biological functions, which often depend on their localization in tissues. Ideally, a fresh untreated material should be directly analyzed to obtain a realistic view of the true sample chemistry. Therefore, there is a large interest for ambient mass-spectrometry-based imaging (MSI) methods. Our aim was to simplify this technology and to find an optimal combination of desorption/ionization principles for a fast ambient MSI of macroscopic plant samples. We coupled a 405-nm continuous-wave (CW) ultraviolet (UV) diode laser to a three-dimensionally (3D) printed low-temperature-plasma (LTP) probe. By moving the sample with a RepRap-based sampling stage, we could perform imaging of

samples up to 16 × 16 cm2 . We demonstrate the system performance by mapping mescaline in a San Pedro cactus (Echinopsis pachanoi) cross-section, tropane alkaloids in jimsonweed (Datura stramonium) fruits and seeds, and nicotine in tobacco (Nicotiana tabacum) seedlings. In all cases, the anatomical regions of enriched compound concentrations were correctly depicted. The modular design of the LD-LTP MSI platform which is mainly assembled from commercial and 3D-printed components facilitates its adoption by other research groups. The use of the CW-UV laser for desorption enables fast imaging measurements. A complete tobacco seedling with an image size of 9.2 × 15.0 mm2 was analyzed at a pixel size of 100 ×

100 μm2 (14,043 mass scans), in less than 2 h. Natural products can be measured directly from native tissues, which inspires a broad use of LD-LTP MSI in plant chemistry studies.

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Introduction Plants produce secondary metabolites, which are ecologically important for their defense against herbivores or microbial pathogens. Therefore, the localization of compounds is indicative of their biological function. 1 For example, the feeding pattern of the cotton bollworm (Helicoverpa armigera) on Arabidopsis thaliana leaves reassembles the areas with a reduced content of glucosinolates. 2 In these studies, matrix assisted laser desorption/ionization (MALDI) time of flight (ToF) mass spectrometry (MS) has been used, which is an established platform for biological mass spectrometry imaging (MSI). However, conventional desorption/ionization methods require physical conditions, which are not compatible with life, such as vacuum, high temperature, and solvent and matrix addition. The first “ambient” ionization technique was desorption electrospray ionization (DESI). 3 The sample is still exposed to charged solvents, but the object can remain at ambient temperature and pressure, and no matrix application is needed. DESI was successfully employed for MSI, providing a lateral resolution of 150 µm for analyses of illicit substances on latent fingerprints. 4 The resolution was further improved by nano-DESI, a solvent extraction method, which enabled, for example, the mapping of nicotine in rat brain sections with a pixel size of 27 × 150 μm2 . 5

Another important direction of the ambient MSI method development was based on the use of lasers. MALDI was employed to operate under ambient conditions. Lateral resolution of up to 1.4 μm have been reported for atmospheric pressure (AP)-MALDI, which is even suitable for singlecell imaging. 6 Using plasma post-ionization, the sensitivity of AP-MALDI of some molecules was enhanced by up to three orders of magnitude. 7 Nevertheless, MALDI methods require the application of a chemical matrix which leads to a complex sample preparation. In addition, the matrix particles and ions may interfere with the MS analysis or the sample. Therefore, various matrix-free ambient methods were developed, which involve a “post-ionization” after laser desorption. The combination of laser ablation with electrospray ionization (LAESI) enabled the direct analysis of metabolites from a French marigold (Tagetes patula) seedling. 8 A cell-by-cell LAESI-MS analysis with a lateral resolution of approximately 30 μm was demonstrated on epidermal onion (Allium 3



cepa) cells, proving cyanidin as the coloring pigment in purple onion cells. 9 Ultraviolet (UV) laser ablation coupled to a flowing atmospheric-pressure afterglow (FAPA) plasma source provided a lateral resolution of 20 μm for printed substances such as caffeine. 10 Such laserplasma MSI systems require neither matrix nor solvents and are thus the closest systems to ambient conditions. A pixel size of 60 μm was reached for the detection of flavonoids baicalein and wogonin in an untreated slice of Radix scutellariae with plasma assisted laser desorption ionization (PALDI). 11 For the mass imaging of hippocampal tissue slices using a near infrared (NIR) laser for sample ablation and PALDI post-ionization, a lateral resolution of 2.9 µm was reached. 12 A lateral

resolution of 110 × 50 µm2 was reported for a laser ablation direct analysis in real time imaging mass spectrometry (LADI-MS). The functionality of the system was demonstrated by mapping the distribution of atropine and scopolamine alkaloid biosynthesis products in a seed of thornapple species

Datura leichhardtii. 13 Although the plasma probes seem very similar, they can be distinguished based on their construction, operating conditions and desorption/ionization mechanisms. 14–19 Among them a low-temperature plasma (LTP) is characterized by only a slightly increased gas bulk temperature compared to that of the environment. The LTP jet can be used for both, desorption and ionization. Such non-thermal or “cold” plasma is relatively gentle and enables a non-destructive analysis of delicate objects. For example, a direct detection of cocaine from a human finger was demonstrated. 17 The LTP could ionize a wide range of low-molecular weight compounds, including low polarity substances such as hydrocarbons, which makes it complementary to ESI and atmospheric pressure chemical ionization (APCI). 20 By modulating of the plasma temperature, compounds with different volatilities can be detected. 21 LTP jets with defined diameter can also be employed for desorption/ionization from surfaces and MSI. The analyte lifting from the surface is achieved either by momentum transfer (from the gas flow) or thermal desorption. 22 A review on ion processes, instrumental set-ups, and application examples of LTP jets in MS is presented in TrAC (2017). 23 Inspired by a report on a non-destructive analysis of paintings using LTP-MSI, 24 we created a simple “plug and play” robot and software for mapping of volatile and semi-volatile compounds in chili pepper (Capsicum annuum). 25,26 As no commercial LTP probe is available, 4


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we developed an LTP probe, which is built from three-dimensionally (3D)-printed and commercial parts. The files for the 3D printing of the LTP probe were published under a permissive Creative Commons license which allows free use and modification of the device for non-commercial use. 27 This 3D-LTP probe can generate a plasma with an apparent temperature of 28 °C. Using the 3DLTP probe an in vivo monitoring of nicotine in tobacco (Nicotiana tabacum) leaves caused only a negligible tissue damage. 28 The LTP seems to be the ideal choice for biological MSI; however, it has two major drawbacks: 1) The limited desorption of molecules/ ions from matrices and tissues and 2) the difficulty to focus the plasma jet under the operating conditions. In addition, the direct imaging of metabolites in a plant tissue is hindered by the strong cell walls. 29 Therefore, we fitted an adjustable laser unit on our 3D-LTP MSI system. Contrary to most reported MSI platforms, we use a continuous wave (CW) UV diode laser, aiming to optimize the fluence for a given laser power, instrumentation cost and analysis time. We demonstrate the functionality of the LD-LTP MSI system by the direct analysis of secondary metabolites in untreated plant tissues.

Experimental methods Laser desorption (LD) For the LD we used a continuous wave (CW) 405 nm ultraviolet (UV) diode laser (Fermion III Series, Micro Laser Systems Inc.), equipped with an optical fiber, a collimator (FC10, aperture 10 mm, beam size 5.5 mm, beam divergence < 0.2 mrad) and a plano-convex lens with a focal length of 50 mm and a diameter of 25 mm (Edmund Optics #32-477, New Jersey, USA).

Low-temperature plasma (LTP) probe The LTP probe was built using 3D-printed and commercial components, 27 and operated with a helium flow of 0.1 L ⋅min−1 , output voltage of 7 kV and a frequency of 10 kHz.

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Figure 1: LD-LTP MSI set-up. The 3D-LTP probe was mounted in at an angle of 45° with respect to the sample surface. The CW UV diode laser was placed with at an angle of 90° and focused using a plano-convex lens. The moving stage, lens holder and LTP-probe were developed using the RepRap technology and 3D-printed parts. The biological samples were attached to a glass slide using a double-sided adhesive tape.

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LD-LTP mass spectrometry imaging (MSI) system The 3D-LTP probe was positioned at an angle of 45°, 2 mm in front of the MS extended inlet. The collimator and plano-convex lens were mounted perpendicularly to the sampling surface. The laser focus was adjusted by modifying the distance between the sample and lens. The experimental set up is shown in Figure 1. For the sample movement we used a custom-built RepRap robot (MakerMex, León, Mexico). 30 Biological samples were fixed on the sampling plate and scanned in discrete steps with defined step width and analysis time (waiting time), following a meandering pattern. The parameters for each sample are presented in Table 2. An LCQ-Fleet ion trap MS (Thermo Scientific, USA) was used in the positive mode and range of 50-500 m/z. The spectra were acquired in a full scan and continuous mode. The capillary voltage, capillary temperature and the tube lens voltage were set to 55 V, 200 °C and 25 V, respectively. The maximum time for ion-trap injection was set to 300 ms, with 2 microscans per spectrum using the automatic gain control (AGC).

Data analyses Raw data were converted to mzML using ProteoWizard. 31 The conversion to imzML files and image processing were performed using custom software based on the statistical computing and graphics programming language R (https://www.r-project.org). The code of our imaging software RmsiGUI is freely available under the terms of the GNU General Public License, version 3 (https://www.gnu.org/licenses/), at https://bitbucket.org/lababi/rmsigui. RmsiGUI is based on MSI.R. The program allows the generation of concentration ‘topologies’ which support a better recognition of areas of differential signal intensities. 26

LD-LTP MS of coffeine with/without plant extract We prepared a dilution series of a caffeine standard solution (Sigma-Aldrich, Mexico) of 1 mg

⋅mL−1 in acetonitrile (Merck, Mexico). The series spanned from 1 ⋅10−1 to 1 ⋅10−9 mg ⋅mL−1 , and 7



included two more dilutions of 5 ⋅10−2 mg ⋅mL−1 and 5 ⋅10−3 mg ⋅mL−1 . For matrix experiments, we prepared the dilutions with a methanolic leaf extract of tomato (Solanum lycopersicum) instead of acetonitrile. 6 µL of the samples were spotted on glass slides of 0.13 mm thickness and allowed to dry. To improve the laser desorption, the glass slides were painted on the opposite side with a black permanent marker (Sharpie, USA). The LD-LTP system was operated with the parameters defined above. The laser energy was set to 404.89 MW⋅cm2 . Each spot was measured at 16 different points with a

step width of 300 µm, using the RepRap based sampling stage. For each point, 10 scans in positive mode were recorded with a scan range of 150 – 250 m/z.

Biological material A San Pedro cactus (Echinopsis pachanoi) was provided by Prof. Molina-Torres (CINVESTAV Irapuato, Mexico). The cactus was dissected with a scalpel into a cross-cut section with a thickness of 2.5 cm. The cross-section was cleaned with a paper towel prior to the analysis. To avoid dehydration during the analysis, a wet filter paper was placed underneath the cactus slice. Jimsonweed (Datura stramonium) plants were provided by Dr. Fredd Vergara (iDiv, Germany). An immature fruit was removed from the plant and dissected in half (transversally). Seeds were taken from an immature fruit and dissected in half with a scalpel (longitudinally). Tobacco (Nicotiana tabacum) seedlings were cultivated and provided by Prof. Marsch-Martínez (CINVESTAV Irapuato, Mexico). Two-week-old seedlings were removed from the soil and their roots were washed three times with deionized water. After removal of the excess soil, the roots were dried on a paper towel. The clean seedlings were deposited on a glass slide. The jimsonweed samples and tobacco seedling were attached to the glass slide using a double-sided adhesive tape.

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Alkaloid extraction and MS fragmentation Alkaloids of San Pedro parenchyma and chlorenchyma were extracted according to the methods of Harborne. 32 The final extracts were analyzed using an gas chromatograph (GC) coupled to a mass spectrometer with electron ionization GC-MS (Agilent Technologies devices 5975C and 7890A, USA). An extraction with HPLC grade methanol (Fisher Scientific, Mexico) was prepared for jim-

sonweed seeds and tobacco seedlings. MS𝑛 fragmentation patterns were measured by direct liquid injection (DLI) using an LCQ-Fleet ion trap (Thermo Scientific, USA).

Results Laser desorption

In laser desorption (LD) theory, molecules are lifted from a state 𝑥 (ground state) to a state 𝑦′

(desorbed molecule) by a photon energy of 𝐸𝜆 exceeding the energy 𝐸𝑥𝑦′ , i.e., 𝐸𝜆 > 𝐸𝑥𝑦′ (Figure 2).

Figure 2: Energy levels for desorption of molecules from a surface. 9



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The photon energy 𝐸𝜆 is dependent on its wavelength 𝜆: 33 𝐸𝜆 =

ℏ 𝜆

(1)

where ℏ = ℎ𝑐; ℎ denotes the Planck constant and 𝑐 is the speed of light. For a given laser output

power 𝑃𝜆 , the beam energy in a certain time window Δ𝑡 is: 34 𝐸 = 𝑃 Δ𝑡

(2)

By combining these equations, we can compute the total number of photons in the laser beam

in the teme window o Δ𝑡:

𝑛𝜆 =

𝐸 𝜆 𝑃 Δ𝑡 = 𝐸𝜆 ℏ

(3)

𝜆 𝑃 Δ𝑡 ℏ𝐴𝑠

(4)

When the laser is focused, the number of photons per unit area at the spot 𝑠 is 𝑛𝑠𝜆 =

This expression shows that the photon flux at the spot 𝑠, can be increased by:

1. Reducing the laser wavelength 𝜆. UV photons are more energetic than infrared (IR) photons. Peterson showed some examples of LD applications where a UV laser requires a fluence about an order of magnitude lower than that of its IR counterpart. 35

2. Increasing the laser power 𝑃 . Typically, a pulsed laser has a larger energy than that of a continuous laser, with a higher cost and increased complexity in electronic control. 3. Increasing the time window. 4. Decreasing the spot area. Ideally, the minimum spot area can be computed with the diffraction limit; however the lens material and its surface quality enlarge the real minimum spot area.

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Therefore, a UV laser requires a lower fluence than that of an IR laser with the same desorption energy. To simultanously optimize the sampling time and instrumentation costs, we employed a continuous wave (CW) diode laser with a wave length of 405 nm and nominal power of 1 W. The measured output power was up to 450 mW. With suitable laser optics a beam spot diameter of 5 μm is possible. The beam spot size was computed using Optics Software for Layout and Optimization (OSLO EDU, Lambda Research Corporation, Massachusetts, USA). Diode lasers have the advantage of long life times. In addition, the laser beam can be easily guided through glass fibers.

LD-LTP MSI system The LD-LTP MSI system is composed of a RepRap based sampling stage, continuous wave (CW) ultraviolet (UV) diode laser for laser desorption (LD), and 3D-printed low-temperature plasma (LTP) probe for post-ionization (Figure 1). The naming of the desorption/ionization combination follows the recommendations of the Analytical Methods Committee (AMC) of the Royal Society of Chemistry. 36

To test the analytical performance of the LD-LTP MS system for the detection of plant metabolites, we placed a dilution series of caffeine on a glass slide. To mimic the matrix effect of plant compounds, we prepared the same concentrations with a tomato (Solanum lycopersicum) leaf extract. As illustrated in Figure 3, the signal intensity increments with the caffeine content in a certain area. Although some outliers were recorded, a fair spot-to-spot repeatibility can be stated for the 16 individual measurements of each preparation. With plant extract, the detection limit was drastically augmented and the sensibility reduced. Such matrix effects are well known in mass spectrometry. Reliable signals were obtained from a content of 20 ng ⋅mm2 . Altogether, the results of this ex-

ploratory experiment demonstrates the possibility to estimate the abundance of plant compounds by LD-LTP MSI. To verify the lateral resolution of the system, we screened the surface of a Schefflera arboricola 11



Figure 3: LD-LTP MS of dried caffeine standards on a painted glass slide (16 measurements per spot). The addition of a plant extract affects the analytical sensibility for the detection of caffeine. The spot-to-spot repeatibility is acceptable for mass spectrometry imaging (MSI).

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leaf with the UV laser, using typical parameters for LD-LTP imaging: 208.6 MW⋅cm−2 for 500 ms, with a step width of 100 µm.

Figure 4 shows the uniform spacings between the spots in the sampled area. The coloring of the tissue in the affected region indicates a thermal desorption mechanism rather than laser ablation. We explored suitable LTP parameters and defined them for all experiments (see Experimental methods). At the chosen LTP operating conditions, the desorption of molecules from the plant tissue by the LTP jet is very limited owing to the ambient gas/plasma bulk temperature (30 °C) and

low gas flow rate (0.1 L⋅min−1 ). Therefore, the desorption is mostly controlled by the UV laser. Our analysis shows that a time of 500 ms is sufficient for desorption and post-ionization and thus used this setting for all experiments. We adjusted the laser power in the individual experiments to optimize the signal-to-noise ratio (summarized in Table 2). The data acquisition speed is discussed in a separate section below. Further, we demonstrate the utility of the LD-LTP MSI system by imaging secondary metabolites in untreated plant samples.

Distribution of mescaline in Echinopsis pachanoi cactus Mescaline is a hallucinogenic drug from the peyote cactus (Lophophora williamsii), endemic to Mexico. As peyote is an endangered species, we probed the distribution of mescaline on a crosssection of a cultivated Andean San Pedro cactus (Echinopsis pachanoi), known to produce the same alkaloid.

The signal at 212.14 m/z, corresponding to the [M+H]+ ion of mescaline, was enhanced in the

outer layer of the stem (chlorenchyma, Figure 5A). The measurement was performed with a step

width of 300 µm. An MS2 fragmentation of a chlorenchyma extract and subsequent comparison

with spectra from a reference standard confirmed the identity of mescaline. To demonstrate the localization of mescaline, we dissected the parenchyma and chlorenchyma, extracted the alkaloids and performed a GC-MS analysis. The total ion count (TIC) chromatograms demonstrated the 13



Figure 4: System suitability test using a Schefflera arboricola leaf. The tissue coloring after the UV laser application with typical imaging operating parameters indicates a thermal desorption mechanism. The equidistant spacings of the spots demonstrate the uniform sampling at a lateral resolution of 100 µm.

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= 212.13 m/z) and other unidentiFigure 5: A) Anatomical distribution of mescaline ([M+H]+ 𝑡ℎ𝑒𝑜𝑟. fied ions on the cross-section of Echinopsis pachanoi. The white scale bar represents 1 mm, while the bar scale shown at the right part of each image represents the relative abundance of the ions. B) GC-MS analyses performed on the parenchyma and chlorenchyma extracts demonstrate the identity and differential content of mescaline in the parenchyma and chlorenchyma.

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enrichment in mescaline in the chlorenchyma area; the fragmentation patterns were compared with the National Institute of Standards and Technology (NIST version 2011) database, confirming the identity of mescaline (Figure 5B). Although the lateral resolution of the MSI experiment was moderate, the results were sufficient to correctly determine the anatomical region of the mescaline enrichment. Furthermore, a large, untreated tissue section with dimensions of 41.70 × 42.00 mm2 was analyzed for only 164 min.

Tropane alkaloids in jimsonweed (Datura stramonium) fruit and seeds Datura stramonium, also known as ‘thornapple’ or ‘jimsonweed’ is a plant originated in Mexico. 37 It is used as a model system in chemical ecology owing to its production of tropane alkaloids which are also of pharmaceutical relevance. 38–40 Figure 6A shows the distribution of several ions on the cross-section of the jimsonweed fruit,

measured with a step width of 200 µm. The expected [M+H]+ ion of atropine at 290.33 m/z was enriched only in the seeds. Therefore, an individual seed cross-section was analyzed with a higher lateral resolution of 50 µm. The mass spectrum in Figure 6C shows the increased abundances of the ions with 290.33 and 304.12 m/z in the seed coat. These signals are consistent with the

[M+H]+ ions of the main tropane alkaloids in Datura stramonium, atropine or its isobar littorine, and scopolamine, respectively. Their identity was further confirmed by the spatial distribution of expected transition ions (Figure 6B, http://mona.fiehnlab.ucdavis.edu/ 41 ). 42–44 The low signal/noise ration is characteristic for ambient ionization MSI. The streaking of the mass trace 110.08 m/z in Figure 6B is most likely an artifact caused by ions which are not eliminated quickly enough from the analyzer. Since other ion maps are well-defined, we exclude the carry-over of material on the sample.

MS2 fragmentation experiments on seed extractions and the spectra comparison with reference standards confirmed the identity of atropine/littorine and scopolamine. It is well known that these natural products cause negative effects on many herbivores, particularly insects. 38–40 The localization of these toxic compounds on the interface of the seed with possible 16


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Figure 6: A) Anatomical distribution of different ions on the cross-section of a complete jimsonweed (Datura stramonium) fruit. B) Distribution of the ions at 290.33 m/z and 304.12 m/z corresponding to the [M+H]+ ions of the tropane alkaloids atropine and scopolamine, respectively. Fragment ions of these two alkaloids co-localize with the same nonuniform anatomical distribution. The ions inside the red box represent precursors of alkaloids. In both images, the white scale bar represents 500 µm, while the color bar represents the relative abundance of each ion. C) Individual mass spectrum of the seed analysis, indicating the presence of the tropane alkaloids atropine/littorine ([M+H]+ =290.33 m/z) and scopolamine ([M+H]+ =304.12 m/z). 17



predators underlines their ecological function in plant defense. Using reference standards, we identified fragments corresponding to the alkaloids. The red box in Figure 6B indicates three ions, consistent with a previous study on distribution of precursors of tropane alkaloids in Datura leichhardtii seeds. 13 The signal at 175.11 m/z corresponds to the

[M+H]+ ion of arginine. The signals at 140.10 and 142.12 m/z correspond to [M+H]+ ions of

tropine and tropinone, respectively. The latter three compounds were identified only in the embryo tissue of the D. stramonium seed. The differential anatomical distribution in the two MSI data sets suggests that the tropane alkaloid synthesis occurs in the embryo tissue, whereas the final products accumulate in the seed coat.

MSI of a complete tobacco (Nicotiana tabacum) seedling In wild tobacco (Nicotiana sylvestris) biosynthesis of insecticide nicotine occurs in the roots, from where the alkaloid is transported to the leaves. 45 The accumulation of nicotine is a part of the response against plant wounding. 46 Recently, we reported an in vivo monitoring of nicotine content in commercial tobacco (Nicotiana tabacum) leaves after mechanical and chemical treatments, using a 3D-LTP probe. 28 To study the distribution of nicotine in the whole plant, we performed a LD-LTP MSI on a tobacco seedling. As shown in Figure 7A, the ion at 163.18 m/z was enriched in the root and pe-

ripheral section of the leaves. The identity of nicotine was confirmed by an MS2 fragmentation

and comparison with reference spectra. Figure 7B shows an accumulated spectrum during 60 s of analysis from a representative plant section; the peak at 163.18 m/z was the most intense signal.

In addition, the adduct ion [2M+H]+ of nicotine is detected at 325.08 m/z. 3D-LTP measurements

without laser application showed strong signals at 271.3 m/z and 289.2 m/z indicating the release of labdanoids from trichomes without leaf destruction. 28 Therefore, the spectrum in Figure 7B indicates the rupture of cell structures and release of leaf contents. Figure 7C shows examples of non-uniformly distributed ions. The ion signal at 123.08 m/z could be attributed to nicotinamide, while that at 80.00 m/z corresponds to its fragment (C5H6N+ ). Nicotinamide is known as a pre18


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Figure 7: A) Anatomical distribution of nicotine ([M+H]+ = 163.18 m/z) in a Nicotiana tabacum seedling. The white scale bar represents 500 µm, while the color bar represents the relative abundance of each ion. B) 1 min average spectrum of a representative analyzed zone. C) Examples of other ions with nonuniform distribution.

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cursor of nicotine; its presence in the roots is consistent with the synthesis localization. 47 We also detected several non-uniformely distributed ions which could not be identified. However, their concentrations in specific zones such as leaves or roots hints suggest their biosynthesis or involvement in tissue-specific physiological processes (Figure 7). The distribution of nicotine on the tobacco seedling is in agreement with its biosynthesis in the roots and subsequent transport to the leaves. 45 Fast LD-LTP MSI analyses of complete seedlings also enable the fast screening of mutant libraries and untargeted, space-resolved, study of the plant metabolism.

Discussion Desoption and ionization characteristics Table 1 presents common ambient MSI methods with their characteristics. The desorption of analytes with a solvent spray (ESI methods) or a gas stream (LTP) allows the sampling of surfaces without exposing the objects to thermal stress. 3,17,28 Although a spatial resolution of 35 µm has

been reported for DESI, 48 the pixel size of such methods is usually limited to 100 × 100 µm2 pixel size due to the difficulty to focus a fluid flow.

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Table 1: Comparison of ambient MSI methods. MSI

sampling

ionization

method

AP-

UV laser abla-

MALDI

tion

DESI

solvent spray

MALDI

organic

chemical heat

pixel

solvent

matrix

size

yes

yes

ref.

[µm2 ] yes

1.4 ×

6

1.4 ESI

yes

no

no

35 ×

3–5

35 nano-DESI

solvent

ex- ESI

yes

no

no

traction ELDI/

UV/IR

LAESI

ablation

LA-FAPA/

UV/IR

PALDI/

ablation

27 ×

5

150 laser

ESI

no

no

yes

30 ×

8,9,49

30 laser

plasma

no

no

yes

2.9 ×

10–13

5

LADI LD-LTP

CW-UV laser

LTP

no

no

yes

desorption LTP

gas flow

50 ×

here

50 non-

no

no

thermal

no

250 ×

24–26

250

plasma Infrared-laser (IR) ablation and UV-laser desorption/ionization are different in how the material is liberated from the sample. UV light is absorbed by a broad range of plant pigments and secondary products, which leads to the desorption and possible ionization of molecules. 50 In contrast, IR-laser light of 2,940 nm wavelength is absorbed by the O-H bonds of water, which leads to a two-step process: First, water-containing material of the surface is heated and vaporized. If the

21



applied laser energy cannot be dissipated, biological tissues are disrupted by an explosive vaporization, which leads to the ejection and volatilization of analytes. 51,52 Laser-based MSI methods are affected by the sample surface topology, but it is possible to compensate the roughness of plant surfaces for improving the lateral resolution by either or laser triangulation 53,54 or profilometry. 55 The objects analyzed in this study were all non-flat, but the LD-LTP MSI demonstrated to be insensitive against small variations in height. However, analyzing rough surfaces with high lateral resolution would require to implement an automated regulation of the z-axis. 55 The identified ions correspond to protonated molecules and their adducts, as usually observed in LTP ionization. 23 The presence of few fragmentated species indicates a low contribution of the laser to ion activation. This is congruent with the minimal fragmentation of organic molecules with dielectric barrier discharge postionization, following high-repetition rate laser ablation. 56 The mean kinetic energy of energetic LTP particles, which are responsible for the ionization, corresponds only to a few eV. 57 Therefore, the LD-LTP system can be classified as a ’soft’ ambient MSI ionization source.

Imaging performance The analysis time scales linearly with the pixel size and sample object dimensions. Therefore, a high scanning speed is crucial in MSI. The total measuring time for a single pixel sums up from the stage movement, desorption/ionization and MS data acquisition. Table 2 summarizes the LD-LTP MSI experiments. Depending on the object size and analytical question, an adequate lateral resolution was chosen. In the case of the San Pedro cactus (Echinopsis

pachanoi) with a tissue dimensions of 41.70 × 42.00 mm2 , a resolution of 300 µm was sufficient to correctly elucidate the anatomical region of accumulation. This experiment lasted 164 min which is acceptable for such a large imaging area.

22


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Table 2: Summary of biological MSI experiments. pixel size sample San Pedro

[µm]

[pixel]

[MW⋅cm−2 ]

[min]

19,072

317.86

164.00

200 x 200

18.00 × 15.30

6,885

298.00

58.23

50 x 50

4.25 × 4.00

6,800

278.13

58.19

100 x 100

9.20 × 15.00

14,043

298.00

117.00

seed tobacco

time

41.70 × 42.00

fruit jimsonweed

area [mm2 ]

laser intensity

300 x 300

cactus jimsonweed

area

seedling The jimsonweed (Datura stramonium) fruit was investigated in two steps: First, the cross-

section of a complete fruit with an object size of 18.00 × 15.30 mm was measured with a relatively

low lateral resolution of 200 µm. Second, a region of interest (seed with a size of 4.25 × 4.00 mm2 )

was subjected to a more detailed study at a resolution of 50 µm. In the two MSI steps, approximately the same numbers of scans were acquired; each analysis was completed in a time period shorter than 1h. The rapid turnover of samples enables an intuitive exploration of regions of interest. A complete tobacco (Nicotiana tabacum) seedling was analyzed for a time periode shorter than 2 h at a lateral resolution of 100 µm. 500 to 516 ms were required for the measurement of a single image pixel. This high imaging speed enables the screening of collections, MSI replicates, and time course experiments. In addition, a tissue pre-treatment is not necessary, thus reducing the risk of chemical contamination and preserving the anatomical integrities of the samples. 58

23



Conclusions Continuous wave UV laser desorption (LD), coupled to low-temperature plasma (LTP) ionization enables the fast mass spectrometry based imaging (MSI) of metabolites from native plant materials. LD-LTP MSI is robust against sample unevenness which permits for example the analysis of intact plant seedlings. The LD-LTP MSI is based on RepRap/3D-printed components, which is a low-cost approach, enabling customization by other laboratories. The technical lateral resolution of the system is defined by the movement stage and laser focus. With the current set-up, a theoretical resolution of 12.5 µm and maximum sample size of 16 x 16

cm2 could be achieved. Depending on the sample size, the experiments in this study where per-

fomed with a pixel size of 50 x 50 µm2 to 300 x 300 µm2 . These parameters are sufficient for most studies on macroscopic objects, considering an MSI experiment as a compromise between lateral resolution, signal intensity/sensitivity, image dimensions, analysis time, data/information ratio and cost. We mainly identified alkaloids, but LTP ionization is capable to ionize a wide range of natural products. 23 To improve the detection of molecules of interest, also ’reactive’ LTP strategies should be considered. 59,60 Further studies on the underlying desorption/ionization mechanisms and possible applications of LD-LTP-MS(I) are ongoing. For all three studied objects, San Pedro cactus (Echinopsis pachanoi) cross-sections, jimsonweed (Datura stramonium) fruits and seeds, and tobacco (Nicotiana tabacum) seedlings, alkaloids were enriched at tissue regions interfacing with possible pests. Their location is congruent with their biological functions in plant defense, and exemplify the practical use of the LD-LTP MSI system in real-life studies.

24


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Author contributions AMP and HGA built the 3D-LTP MSI platform and performed the measurements, IRR and COV designed and optimized the laser system, IRR developed the robot/MS automation, DDR and NMM provided the tobacco seedlings and performed stereoscopic analyses, JCC and JMT grew San Pedro cactus and performed GC-MS analyses, FV cultivated jimsonweed, RW designed the project and edited the paper. All authors participated in the writing and approved the final manuscript.

Conflict of interest statement The authors declare the following competing financial interest(s): SMJ and RW are inventors of the patent application “Non-thermal plasma jet device as source of spatial ionization for ambient mass spectrometry and method of application” (WO 2014/057409). RW is co-owner of the company KUTURABI SA de CV.

Acknowledgments We thank Dr. Maria Teresa Carrillo Rayas, and Maria Isabel Cristina Elizarraraz Anaya, Thermo and Waters Mexico for excellent technical support, as well as M. Sc. Raúl Alcalde Vázquez for help with microscopic photography. The project was funded by the CONACyT Fronteras project 2015-2/814 and the bilateral grant CONACyT-DFG 2016/277850. AMP, DDR, HGA, JCC and COV acknowledge their CONACyT scholarships and COV his CONACyT Cátedra project 809.

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