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Population Screening of Chlamydomonas reinhardtii with Single-Cell Resolution 1 Using a High-throughput Micro Scale Sample Preparation for MALDI Mass-2 Spectrometry. 3 4 5 Jasmin Krismera, Jens Sobekb, Robert F. Steinhoffa, Stephan R. Fagerera, Martin Pabsta*, 6 Renato Zenobia# 7 8 Affiliations 9 aDepartment of Chemistry and Applied Biosciences, Laboratory of Organic Chemistry, 10 ETH Zurich, Switzerland 11 bFunctional Genomics Center Zürich, ETH Zurich and University of Zurich, Switzerland 12 # Address correspondence: zenobi@org.chem.ethz.ch 13 * Present address: Martin Pabst, PolyTherics Limited, Cambridge, United Kingdom 14 Keywords: Single-cell analysis, MALDI-Mass spectrometry, High-throughput, 15 Chlamydomonas reinhardtii 16

AEM Accepted Manuscript Posted Online 5 June 2015Appl. Environ. Microbiol. doi:10.1128/AEM.01201-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract 17 Consequences of cellular heterogeneity, for instance biocide persistence, can only be 18 tackled by studying each individual in a cell population. Fluorescent tags provide tools 19 for the high-throughput analysis of genomes, RNA-transcripts or proteins on the single-20 cell level. However, the analysis of lower molecular weight compounds that elude 21 tagging is still a great challenge. Here, we describe a novel high-throughput micro-scale 22 sample preparation technique for single cells, which provides a mass spectrum of each 23 individual cell within a microbial population. The presented approach includes spotting 24 of Chlamydomonas reinhardtii cells using a non-contact microarrayer onto a specialized 25 slide and controlled lysis of cells separated on the slide. Throughout the sample 26 preparation, analytes were traced and individual steps optimized using auto-27 fluorescence detection of chlorophyll. The isolated sample lysate is subjected to a 28 direct, label-free analysis using matrix-assisted laser desorption/ionization mass 29 spectrometry. Thus we were able to differentiate individual cells of two 30 Chlamydomonas reinhardtii strains based on single cell mass spectra. Furthermore we 31 showed that only population profiles with real single cell resolution render a non-32 distorted picture of the phenotypes contained in a population. 33 34

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Introduction 35 Heterogeneity plays a pivotal role in the emergence of tolerance, persistence and 36 resistance in microbial populations towards biocides(1, 2). Also, microbial populations 37 show highly complex interactions, e.g., in the competition for nutrients or in the 38 colonization of new habitats(3). In recent years, newly developed tools for single–cell 39 analysis have greatly extended our understanding of biological variation in microbial 40 populations and its underlying mechanisms(4). These tools allow genome 41 sequencing(5) or follow transcription as well as protein synthesis on the single-cell 42 level(6). Since these techniques give a much higher resolution when observing 43 processes in given cell populations, they permit insight into inter- and intra-cellular 44 processes and the underlying mechanisms. However when it comes to high-throughput 45 measurements of small molecules, very few methods are known as of now(7). 46 Singularities of individual cells can only be fully appreciated within the context of the 47 population. Therefore, one of the most important features for single-cell methods to be 48 useful in biological applications is high-throughput capability. One of the most 49 successful high-throughput approaches to characterize heterogeneities in microbial 50 populations is flow cytometry(8). It benefits from high sensitivity and a high linear 51 dynamic range of fluorescence tagging and optical detection. However, the method is 52 strongly limited in parallelization, since excitation and emission bands overlap. Mass 53 cytometry on the other hand, which is a new approach that can be coupled to flow 54 cytometry, uses antibodies tagged with rare earth metals(9). With mass spectrometric 55 detection, over 40 features can be measured in each cell, implying that mass 56 spectrometry (MS) can boost parallelization capabilities(10). 57 Unfortunately, labeling small molecular compounds is not possible, because the 58 chemical behavior of low molecular weight compounds is heavily affected by the 59

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tagging. Furthermore, the selectivity of tags is often determined by the strength of non-60 covalent interactions specific to an analyte, which can render labeling strategies highly 61 complex. Low molecular weight compounds often lack enough distinctive binding 62 motives to allow for specific binding in complex cellular environments. 63 On the other hand, some analytes (e.g. molecules with structural functions such as 64 lipids) are present in much higher copy numbers inside cells compared to DNA or 65 proteins, such that they are within the reach of state-of-the-art mass spectrometric 66 detection as was shown in recent years(11, 12). Matrix-assisted laser desorption-67 ionization (MALDI) imaging mass spectrometry is the method of choice for the analysis 68 of tissues(13) and new laser desorption sources render it possible to image the 69 distribution of small molecules with single-cell resolution(14, 15). Together with its 70 high-throughput capabilities single-cell MALDI-MS can give new insights into cell-to-71 cell variations of low molecular weight compounds. However the applicability of these 72 methods to microbial colonies is a big challenge due to the necessity of harsher 73 extraction conditions and the significantly smaller cell size(16). 74 Here we present a new method for single-cell sample workup of microbial cell cultures 75 and their discrete analysis using MALDI-MS. Our method targets the analysis of 76 individual cells in suspension e.g. cells of microbial cell cultures. As exemplified for the 77 alga Chlamydomonas reinhardtii, the workflow presented comprises a controlled 78 sample preparation protocol for thousands of individual cells in spatially separated 79 microarray spots, that allows the reproducible extraction and analysis of microbial cells 80 - despite their robust cell walls(17). Endogenous chlorophyll was detected by MS and 81 by fluorescence measurements. Thereby, each sample preparation step could be 82 monitored optically for optimization. Finally, MS allowed a clear differentiation of the 83 two strains of Chlamydomonas reinhardtii, based on data obtained for each single algae 84

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cell. The necessity of true high-throughput single-cell measurements is demonstrated 85 by the deterioration of characteristic signatures of the two strains when considering 86 multi-cell spots. 87 88

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MATERIALS AND METHODS 89 Chemicals. All solvents were purchased in HPCL grade quality. Acetone 90 (Chromasolv), chloroform (ReagentPlus) and isopropanol (Chromasolv) were 91 purchased from Sigma-Aldrich. Water (LiChrosolv) was purchased from Merck. The 92 MALDI-matrix 2, 5-dihydroxybenzoic acid (DHB) was purchased from Sigma-Aldrich. 93 Hutner’s trace elements were purchased from the Chlamydomonas resource center 94 (St.Paul, MN, USA). 95 Cell Culture. Chlamydomonas reinhardtii strains CC-125 (wild-type) and CC-4346 96 (chlorophyllide a oxidase mutant(18)) were obtained from the Chlamydomonas 97 Resource Center, St. Paul MN, USA. Strains were maintained on tris-acetate-phosphate 98 (TAP) agar plates. For single-cell MS single colonies of strains were inoculated in liquid 99 TAP medium(19). The cells were cultured in an incubation shaker (Minitron, Infors 100 HT) at 21°C at 110 rpm and 1200 lux continuous illumination. 101 Characterization of Chlamydomonas reinhardtii strains. The Chlamydomonas 102 reinhardtii strains were characterized on population level using UV-VIS spectrometry. 103 Absorption was measure between 350 nm and 850 nm. In UV-VIS spectra, the CAO 104 insertional mutant strain CC4346 lacks the characteristic shoulder peaks caused by 105 chlorophyll b absorption around 660 nm and 500 nm. 106 Microarrays. The Microarray for Mass Spectrometry (MAMS) targets were prepared 107 as follows. Stainless steel target slides 25 mm x 75 mm were structured using 108 picosecond laser pulses. Before usage, a slide was cleaned using a sequence of different 109 solvent conditions: 1. chloroform 2. acetone 3. isopropanol 4. 15% acetic acid in water 110 5. water. Each of the conditions was maintained for at least 60 min in a sonication bath. 111 The slides were stored under a nitrogen atmosphere until use. 112

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Spotting the cells onto microarray slides. For liquid handling of nanoliter volumes a 113 spotting robot (sciFLEXARRAYER S11 , Scienion) was used. For single-cell MS, 114 Chlamydomonas reinhardtii strains were centrifuged just before spotting at 2000 x g / 115 1500 x g for 5 min (Eppendorf 5415R centrifuge) and 3 times re-suspended in 116 deionized water to reduce the quenching influence of salts contained in the TAP liquid 117 culture medium. After spotting the cells, the slide was scanned using a confocal 118 microarray scanner (LS400, Tecan). Fluorescence was excited at 633 nm and detected 119 at 670 nm. The detector gain was at kept constant for all measured slides for comparing 120 relative intensities. Scan resolution was 6 μm. 121 Cell lysis and MALDI-matrix application. To enhance extraction and co-122 crystallization of the analytes with the MALDI-matrix (2,5-dihydroxybenzoic acid) 123 while avoiding cross-contamination between microarray spots the slide was 124 submerged in liquid nitrogen. The slide was reconstituted to room temperature in a 125 desiccator to avoid condensation. A fluorescence scan of the slide was performed after 126 reconstitution to check for possible cell displacements or cross contamination. After 127 this step 2,5-dihydroxybenzoic acid (10 mg/ml in 80% aqueous acetone) was spotted 128 onto the cells to extract the analytes and co-crystallize them with the MALDI matrix. 129 The efficiency of the extraction was monitored with a confocal fluorescence scan. The 130 slide was stored under nitrogen atmosphere at -80° C until measured. 131 MALDI-FT-ICR-MS. Accurate mass spectra of single cells and cell bulk was recorded 132 using a FT-ICR mass spectrometer using the MALDI source Bruker SolariX 9.4T. 133 Additionally MS/MS spectra were recorded using collision-induced dissociation. The 134 laser was operated using a large laser focus (≈100um) and a laser power of 78% 135 arbitrary units (a.u.), with 200 laser shots over 10 scans. 136

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MALDI-TOF-MS. High-throughput single-cell MS was performed using a commercial 137 MALDI mass spectrometer (ABSciex 5800). The laser intensity was set to 4300 a.u. with 138 a laser repetition rate of 200 Hz and extraction time delay of 500 ns. A total of 70 sub-139 spectra were recorded for each spot ablating in a spiral pattern. The MALDI method 140 used had a mass resolution of 9200 (±750) for chlorophyll a, and a mass accuracy of 141 ±10ppm. 142 Differentiating strains. To generate distinct profiles for the two Chlamydomonas 143 reinhardtii strains CC-4346(18) and CC-125 the two strains were spotted from separate 144 cultures, each strain covering half of a microarray slide. Spotting a mixture of the 145 strains covered a second microarray. Using a higher cell concentration in the mixture, 146 the second microarray contained a higher number of spots with multiple cells. 147 Statistical analysis of mixed strains. The number of cells for each spot was 148 determined using fluorescence images of the microarray (ex. 670 nm / em. 630 nm). 149 The spectra were divided into groups depending on the number of cells in the spot. The 150 distribution of the chlorophyll b/ chlorophyll a ratio was fitted using a kernel density 151 estimation using the dfittool from MATLAB version 2014a (normal, automated 152 bandwidth). The probability density function was then fitted with Gaussian 153 distributions accounting for the contribution of the wild-type strain CC125. Since the 154 chlorophyll b/a ratio in strain CC4346 is zero due to the complete absence of 155 chlorophyll b, the averaged signal of the two strains is fitted with a Gaussian 156 distribution. 157 158

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RESULTS 159 Single-cell sample preparation of microbial cells. Extracting the small amount of 160 molecules from a microbial cell, which is surrounded by tough cell walls, is challenging. 161 Most importantly the extraction has to reproducibly extract a representative fraction of 162 the contents of each cell. A schematic and fluorescence scans of the key steps during 163 sample preparations are presented in Figure 1. To minimize cross-contamination 164 between adjacent spots on the microarray the liquid handling was carried out using a 165 piezo-driven contactless spotting device as described in the experimental section. The 166 microarray slide has 1430 spots of 300 μm diameter each and 720 μm center-to-center 167 distance. A detailed protocol of the spotting procedure can be found in TableS1. A very 168 low number (2/1430) of false positive spectra, i.e. spots featuring a mass spectrometric 169 signal reminiscent of a cell without a cell being present, indicated that cross-170 contamination is indeed negligible. Thanks to the orthogonal fluorescence detection of 171 the auto-fluorescent chlorophyll it can be visualized throughout the sample preparation 172 process. Fluorescence detection allowed the monitoring of the lysis and extraction of 173 the cells. Moreover, contamination caused by early lysis in the medium or during the 174 analyte extraction can be optically traced back. Our improved lysis procedure not only 175 increased the percentage of successful single-cell measurements in MALDI-TOF-MS to 176 about 95% for the wild-type CC125, but also boosted the signal-to-noise value of 177 chlorophyll a in the spectra to a median value of 540. 178 High-resolution MALDI-MS for peak identification. To assign the signals obtained in 179 single-cell measurements using MALDI- (TOF-) MS on Chlamydomonas reinhardtii, we 180 used MALDI-FT-MS to determine their accurate mass (≤ 0.5 ppm mass deviation) and 181 performed population level MS/MS measurements (Figure S1) which were matched 182 with Chlamydomonas reinhardtii literature(20, 21) allowing for high confidence signal 183

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identification. A list of assigned compounds can be found in Table S2. Using the MALDI 184 matrix DHB in the positive ion mode, the peaks identified from single-cell spectra can 185 be assigned to different lipid classes such as monogalactosyl-diacylglycerols (MGDG), 186 digalactosyl-diacylglycerols (DGDG), homoserine lipids (DGTS) or to chlorophylls. 187 Chlorophylls are detected in the form of pheophytins due to the acidification of the 188 extract during crystallization with the acidic DHB. The acidification leads to complete 189 dissociation of Mg2+ from the porphyrin ring and therefore to the formation of the 190 corresponding pheophytins(22). No intact chlorophylls are detected. Other major 191 thylakoid lipid constituents such as sulfo- as well as phospholipids can be detected on 192 the single-cell level in negative mode spectra using 9-aminoacridine (data not shown). 193 Single-cell mass spectra show the same peak composition as spectra collected from a 194 bulk of cells but they differ in relative composition (FIGURE 1). 195 Characterization and identification of strains on the single-cell level. We used the 196 new technique to distinguish two different strains of Chlamydomonas reinhardtii at the 197 single-cell level. The distinction between the two strains was based on the presence and 198 absence of a single compound, chlorophyll b: the wild-type strain CC-125 contains both 199 chlorophyll a and b, while the chlorophyllide-a-oxidase insertional mutant CC-4346 200 contains chlorophyll a but lacks chlorophyll b(18). The absence of chlorophyll b in the 201 mutant strains was verified both by MALDI-MS and by UV-VIS measurements on the 202 population level (Figure S2). Single-cell spectra were recorded producing a population 203 profile for both strains (Figure S3). The cells were spotted and lysed on the array slide 204 as described above. 715 spots of each cell suspension were spotted. In 541 spots a 205 single cell from strain CC125 was found by fluorescence detection, 511 of these spots 206 showed chlorophyll a signals in MALDI-MS. Single cells of strain CC-4346 were present 207 in 332 spots with 283 of these showing a signals of chlorophyll a in MALDI-MS. The raw 208

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spectra are plotted as a heat map (MATLAB, msheatmap function, midpointValue = 209 0.99) in FIGURE 2. The background peaks, caused by the MALDI matrix or 210 contaminants are stable throughout the 1430 spectra, as clearly visible from the heat 211 map. Furthermore, the strains can be easily distinguished based on the absence of 212 chlorophyll b in CC4346. 213 Deterioration of strain-specific phenotypes in multi-cell mass spectra. A key 214 advantage of any single-cell resolved technique is the distinct information it can supply 215 in contrast to population measurements. Therefore we performed an experiment with 216 an up concentrated cell suspension to see how the population profile differs when 217 looking at single cells compared to multi-cell spots. Interestingly, single-cell 218 measurements from this run could be assigned to one of the two strains solely based on 219 the mass spectrum. This is the first report on strain identification in single cells based 220 on MS. Furthermore, spots that contain more than one cell either show distinct or 221 mixed responses that can be attributed to contributions wither one or both strains. The 222 more cells per spot the higher is the chance for a superimposed readout as shown in 223 Figure 4. This emphasizes the ability of the method to perform real single-cell 224 measurements and the detrimental effect of even few cell measurements on the 225 observed population heterogeneity. 226 227 228

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DISCUSSION 229 MALDI-MS of single cells allows the detection of abundant low molecular weight 230 compounds with polar character. The analytes identified from single-cell 231 measurements using a widely used matrix (DHB) were mainly lipids belonging to 232 different classes such thylakoid membrane lipids, membrane lipids, or pigments. The 233 selectivity for these compounds can be attributed to their abundance, polarity and 234 solubility in the extractive solvent. Application of the MALDI matrix, which is dissolved 235 in a solvent mixture, represents a liquid extraction from the cracked cells and helps to 236 reduce the complexity of the biological background. Changing the extracting solvent or 237 the matrix therefore allows access to different analytes and changes their relative 238 contribution to the obtained cell spectra. 239 For maintaining single-cell resolution preventing cross-contamination between spots is 240 essential. Both the non-contact microarrayer and the spacing of the microarray help to 241 avoid cross-contamination. Only 2 of 453 empty wells showed MS signals reminiscent 242 of cells in their spectra. 243 Another aspect of utmost importance for successful single-cell analysis is cell lysis. If 244 the lysis process is not effectively controlled, the developmental state or nature of the 245 cell wall will strongly influence the extraction. Thus, a bias for cells with a weakened 246 cell wall due to changes in cell wall composition, during, e.g., the cell cycle or aging 247 would be introduced. Incomplete lysis can result in poor or irreproducible mass 248 spectrometric readout, since the MALDI favors the ionization of the fraction “co-249 crystallized” with the matrix. Our system gives experimental evidence of this 250 phenomenon, since fluorescence scanning after MALDI-MS shows significant 251 fluorescence intensity for non-lysed cells (Figure S4). The orthogonal fluorescence 252

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information thus not only helped in optimizing the method but also helps to retrace the 253 sources of errors. 254 The optimized lysis procedure also improves spectral quality by increasing the 255 percentage of single cells that yield a spectrum, the signal-to-noise values and the 256 number of detected analytes. 257 In many applications, MALDI-MS performs best giving qualitative information. 258 However, relative quantitative information can be obtained when biological matrix 259 background is reproducible. Since our method is label-free, it delivers direct access to 260 the phenotype of individual cells. The composition of small molecules in individual cells 261 of microbial populations can be used, e.g., to determine the penetrance of a mutation on 262 the single-cell level. We therefore used the methods to differentiate cells of different 263 strains based on single-cell mass spectra. Chlamydomonas reinhardtii mutant CC4346 264 that is incapable of chlorophyll b biosynthesis(18) can be differentiated from the wild-265 type by the absence of chlorophyll b. As predicted by population level MALDI-MS as 266 well as UV-VIS spectroscopy, there were no detectable levels of chlorophyll b in the 267 knockout even when analyzed with single-cell resolution. Mixing the wild-type and the 268 chlorophyll-b-less mutant strain still showed distinct phenotypes in a mixed 269 population. This was underlined by analyzing multi-cell measurements of the mixture 270 of the two strains. Mixing the two strains caused a rapid deterioration of the distinct 271 spectral response from the two strains with increasing numbers of cells measured, 272 since both strains contribute to the spectra. This is an important validation of the 273 experimental data that highlights the importance of real single-cell spectra for the 274 characterization of molecular phenotypes present within a population. Since our 275 experimental system represents the an extreme case where the distinction is based on 276 the presence or absence of a signal, this effect is expected to be more severe if the 277

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differences of the two phenotypes in the population are less pronounced i.e. when 278 distinctions are based on different amounts of a compound. 279 While recent publications still included few-cell spectra(12), the newly developed high-280 throughput sample-preparation method, allows to measure pure single-cell profiles 281 from microbial populations and their mixtures for the very first time. The quality and 282 the flexibility of the single-cell measurements are highly promising concerning future 283 applications of MALDI-MS for single-cell resolved population analysis in microbiology. 284 285 286

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Acknowledgements 287 This work was financially supported by ETH Zurich. Many thanks go to Rolf 288 Brönnimann and Dr. Konstanins Jesimovs for the micro structuring of the microarray 289 slides, Prof. Dr. Niklaus Amrhein and Prof. Dr. Stefan Hörtensteiner for their expertise 290 and advice concerning Chlamydomonas reinhardtii and chlorophyll metabolism, Rolf 291 Häfliger and Dr. Xiangyang Zhang for their help with the FT-MS measurements and to 292 Dr. Carolin Blum, Dr. Lothar Opilik, Jacek Szcerbinski, Dr. Simon Weidmann, Dr. Jan-293 Christoph Wolf for their support during data analysis and writing. 294 295 Author contributions statement 296 J.K and J.S conducted the experiments. J.K., R.F.S., S.R.F. and M.P. analyzed the results. 297 298

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299 References 300 1. Dhar N, McKinney JD. 2007. Microbial phenotypic heterogeneity and 301

antibiotic tolerance. Current Opinion in Microbiology 10:30-38. 302 2. Brehm-Stecher BF, Johnson EA. 2004. Single-Cell Microbiology: Tools, 303

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3. Buckling A, Harrison F, Vos M, Brockhurst MA, Gardner A, West SA, 306 Griffin A. 2007. Siderophore-mediated cooperation and virulence in 307 Pseudomonas aeruginosa. FEMS Microbiol Ecol 62:135-141. 308

4. Lidstrom ME, Konopka MC. 2010. The role of physiological heterogeneity in 309 microbial population behavior. Nat Chem Biol 6:705-712. 310

5. Blainey PC. 2013. The future is now: single-cell genomics of bacteria and 311 archaea. FEMS Microbiol Rev 37:407-427. 312

6. Eberwine J, Sul J-Y, Bartfai T, Kim J. 2014. The promise of single-cell 313 sequencing. Nat Meth 11:25-27. 314

7. Zenobi R. 2013. Single-cell metabolomics: analytical and biological 315 perspectives. Science 342:1243259. 316

8. Davey HM, Winson MK. 2003. Using flow cytometry to quantify microbial 317 heterogeneity. Curr Issues Mol Biol 5:9-15. 318

9. Tanner S, Baranov V, Ornatsky O, Bandura D, George T. 2013. An 319 introduction to mass cytometry: fundamentals and applications. Cancer 320 Immunology, Immunotherapy 62:955-965. 321

10. Nolan GP. 2011. Flow cytometry in the post fluorescence era. Best Practice 322 & Research Clinical Haematology 24:505-508. 323

11. Amantonico A, Urban PL, Fagerer SR, Balabin RM, Zenobi R. 2010. 324 Single-Cell MALDI-MS as an Analytical Tool for Studying Intrapopulation 325 Metabolic Heterogeneity of Unicellular Organisms. Analytical Chemistry 326 82:7394-7400. 327

12. Ibáñez AJ, Fagerer SR, Schmidt AM, Urban PL, Jefimovs K, Geiger P, 328 Dechant R, Heinemann M, Zenobi R. 2013. Mass spectrometry-based 329 metabolomics of single yeast cells. Proceedings of the National Academy of 330 Sciences 110:8790-8794. 331

13. Römpp A, Spengler B. 2013. Mass spectrometry imaging with high 332 resolution in mass and space. Histochemistry and Cell Biology 139:759-783. 333

14. Zavalin A, Todd EM, Rawhouser PD, Yang J, Norris JL, Caprioli RM. 334 2012. Direct imaging of single cells and tissue at sub-cellular spatial 335 resolution using transmission geometry MALDI MS. J Mass Spectrom 336 47:1473-1481. 337

15. Schober Y, Guenther S, Spengler B, Römpp A. 2012. Single Cell Matrix-338 Assisted Laser Desorption/Ionization Mass Spectrometry Imaging. Analytical 339 Chemistry 84:6293-6297. 340

16. Watrous JD, Dorrestein PC. 2011. Imaging mass spectrometry in 341 microbiology. Nat Rev Micro 9:683-694. 342

17. Goodenough UW, Heuser JE. 1985. The Chlamydomonas cell wall and its 343 constituent glycoproteins analyzed by the quick-freeze, deep-etch technique. 344 J Cell Biol 101:1550-1568. 345

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18. Tanaka A, Ito H, Tanaka R, Tanaka NK, Yoshida K, Okada K. 1998. 346 Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from 347 chlorophyll a. Proc Natl Acad Sci U S A 95:12719-12723. 348

19. Gorman DS, Levine RP. 1965. Cytochrome f and plastocyanin: their 349 sequence in the photosynthetic electron transport chain of Chlamydomonas 350 reinhardi. Proceedings of the National Academy of Sciences 54:1665-1669. 351

20. Giroud C, Gerber A, Eichenberger W. 1988. Lipids of Chlamydomonas 352 reinhardtii. Analysis of Molecular Species and Intracellular Site(s) of 353 Biosynthesis. Plant and Cell Physiology 29:587-595. 354

21. Riekhof WR, Benning C. 2009. Chapter 2 - Glycerolipid Biosynthesis, p 41-355 68. In Witman EHHBSB (ed), The Chlamydomonas Sourcebook (Second 356 Edition) doi:http://dx.doi.org/10.1016/B978-0-12-370873-1.00010-1. 357 Academic Press, London. 358

22. Lorenzen CJ. 1967. DETERMINATION OF CHLOROPHYLL AND PHEO-359 PIGMENTS: SPECTROPHOTOMETRIC EQUATIONS1. Limnology and 360 Oceanography 12:343-346. 361

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Figure Captions 365 FIGURE 1. High-throughput micro-scale sample preparation for single-cell MALDI-MS. A-D Schematic describing the 366 individual steps of the workflow. E-H Confocal fluorescence scans of 9 wells on the microarray. A/E The cleaned 367 empty microarray slide has wells where the stainless steel surface is roughened up. B/F Cells are spotted from cell 368 suspensions. Chlorophyll auto-fluorescence makes the cells clearly visible. C/G Fast freezing of the cells in liquid 369 nitrogen leads to damages in the cell walls. D/H Chlorophyll and other soluble analytes are extracted from the cells 370 by the matrix solution (10 mg/ml DHB in aqueous acetone). Evaporation of the solvent leads to “co-crystallization” of 371 the extracted analytes with the MALDI matrix. 372 FIGURE 1 MALDI mass spectra obtained from a single cell (upper spectrum) and from a bulk Chlamydomonas 373 reinhardtii (lower spectrum) on a FT-MS instrument. The single-cell mass spectrum is background corrected for the 374 purpose of better visibility of the peaks in the lower mass region. This was achieved by subtracting the spectrum of 375 an empty spot from the single-cell spectrum. 376 FIGURE 2 Single-cell mass spectra obtained from one measurement series. Spectra originate from two different 377 strains of Chlamydomonas reinhardtii. CC-125 is a wild-type strain while CC4346 is a chlorophyll b deficient mutant 378 strain reflected by the absence of the pheophytin b signal at 885.6 m/z. The visualization was achieved by using the 379 msheatmap function in MATLAB. The midpointValue was chosen as 0.99 which means that only the 1 % most intense 380 signal values appear in the representation. 381 FIGURE 4 Probability densities of the chlorophyll b/chlorophyll a peak area ratios for spots containing 1-5 cells. 382 Single-cell distributions are averaged out as the number of cells per measured spot increases. Measuring single cells 383 of chlorophyll b deficient strain CC4346 (red) the chlorophyll b/ chlorophyll a peak ratio is zero while wild-type 384 strain CC125 (green) shows a ratio around 0.1. The two strains can therefore easily be distinguished on the single-385 cell level. Measuring spots with more than one cell from mixed cultures leads to an intermediate response due to 386 averaging of the two strain specific responses. The effect is more pronounced the more cells are measured in one 387 spot. 388 389

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