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Multimodal imaging of chemically fixed cells in preparation for NanoSIMS Jelena Lovric, Per Malmberg, Bengt R. Johansson, John Stephen Fletcher, and Andrew G. Ewing Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02408 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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Analytical Chemistry

Multimodal Imaging of Chemically Fixed Cells in Preparation for NanoSIMS Jelena Lovrić,† ‡ Per Malmberg,† ‡ Bengt R. Johansson,¶ John S. Fletcher,§, ‡ and Andrew G. Ewing* † ‡ § ,

,

,

, , ,

†Department of Chemistry and Chemical Engineering, Chalmers University of Technology, SE-412 96, Gothenburg, Sweden ‡National Center for Imaging Mass Spectrometry, Chalmers University of Technology and Gothenburg University, SE-412 96, Gothenburg, Sweden ¶

Electron Microscopy Unit, Institute of Biomedicine, University of Gothenburg, SE-405 30, Gothenburg, Sweden

§Department of Chemistry and Molecular Biology, University of Gothenburg, SE-405 30, Gothenburg, Sweden

ABSTRACT In this work, we have employed time-of-flight secondary ion mass spectrometry (ToFSIMS) to image chemically fixed adrenal cells prepared for transmission electron microscopy (TEM) and subsequent high-spatial-resolution NanoSIMS imaging. The sample fixation methodology preserves cell morphology, allows analysis in the ultrahigh vacuum environment, and reduces topographic artifacts, thus making these samples particularly favorable for ToFSIMS analysis. ToF-SIMS imaging enables us to determine the chemistry and preservation capabilities of the chemical fixation as well as to locate specific ion species from OsO4. The OsO4 species have been localized in lysosomes of cortical cells, a type of adrenal cell present in the culture. NanoSIMS imaging of the

190

Os16O- ion specie in cortical cells reveals the same

localization as a wide range of OsO4 ions shown with ToF-SIMS. Even though, we did not use during NanoSIMS imaging the exact OsxOy- ion specie discovered with ToF-SIMS, ToF-SIMS allowed us to define the specific subcellular features with high mass spectrometric imaging. This study demonstrates the possibility for application of ToF-SIMS as a screening tool to optimize high-resolution imaging with NanoSIMS which could replace TEM for localization in ultra-high resolution imaging analyses.

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INTRODUCTION Studying changes in cells and their organelles is crucial for understanding metabolic pathways and functions related to pathological processes in neurodegenerative diseases1 dynamics of neurotransmission,2 cancer treatment and drug development,3 toxicity impact,4 etc. Cell biology has been substantially studied with transmission electron microscopy (TEM) where the technique has played, and continues to play, a key role in furthering our understanding of cell architecture and relating it to cellular function.5-7 However, due to the ultrahigh vacuum environment in the TEM column and need for ultrathin specimen sections, biological materials have to be extensively processed and fixed in some way. The most commonly applied sample preparation over the past decades has been chemical fixation, which involves initial fixation with aldehydes followed by post-fixation using osmium tetroxide, dehydration in organic solvents and embedding in epoxy-type resin at elevated temperature. The samples are sectioned and treated with heavy metals to improve contrast in the subsequent TEM images.8,9 Each of these chemical processes is critical for success and rather complicated. In spite of numerous efforts to determine the chemistry occurring during fixation procedures, to this day it is not well understood. Fixation can introduce different artifacts such as aggregation of proteins and loss of water soluble analytes and lipids.9 Therefore, understanding the chemical processes and the advantages and disadvantages of certain fixation procedures can greatly improve the reliability of the imaging results. Although TEM has nanometer and even sub-nanometer spatial resolution capabilities, it has relatively poor potential for chemical identification of specimens. Electron spectroscopic imaging can be used to extract chemical information with the spatial resolution of 1 nm but semiquantitative analysis without isotopic selectivity and poor detection limit make this technique limited for chemical imaging.10 Localization of biomolecular species is feasible but certain sample modifications have to be carried out such as immunocytochemistry. This can be a challenge due to the difficulty of finding unique labels and localization of molecules as a result of steric hindrances from labels.11 Secondary ion mass spectrometry (SIMS) can be used in the imaging mode to acquire elemental and molecular ion maps. Its mass and lateral resolution capabilities as well as detection limit in the ppm or ppb range, depending on matrix and analyte, makes this technique highly applicable for analysis of polymers,12 metals,13 geological and cosmological samples14 as well as


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biological samples.15-18 In SIMS analysis, a focused primary ion beam is used to sputter material from a sample surface and generate secondary ion species which are mass analyzed and detected as a mass spectrum for each image pixel.19,20 SIMS instruments typically use ToF21 or magnetic sector mass analyzers.22 ToF-SIMS can be used to simultaneously record atomic and molecular secondary ions formed in a wide mass range (1 to 103-104 Da.)23 with submicron spatial resolution, depending on mass analyzed. NanoSIMS instruments use coaxial primary and secondary ion beams24 that provide high spatial resolution capabilities (~ 50 nm) and very good transmission at high mass resolution but are currently limited to detection of only 7 ion species of different m/z in parallel using a magnetic sector mass analyzer. Furthermore, the NanoSIMS instrument uses atomic ion beams for analysis that result in small secondary ion fragments and elements being detected. Therefore labeling with stable or radioisotopes is often required.25 Additionally, due to the short nominal distance (400 µm) between sample and ion optical elements,14 NanoSIMS demands flat specimen surfaces, which makes TEM sectioning very suitable. Both types of SIMS instruments require an ultrahigh vacuum environment at the sample and there have been several studies to examine and validate adequate sample preparation techniques for water-rich biological samples.26-28 In this paper, we report the use of ToF-SIMS to chemically image endocrine cells originating from bovine adrenal glands such as chromaffin and cortical cells,29 prepared for TEM imaging, by scanning the sample surface over a mass range up to 1000-1500 Da. This study does not aim to evaluate sample preparation exclusively for ToF-SIMS analysis as much work has been done already on this topic already.26,27,30 The aim here is to correlate the cellular information obtained from well-established electron microscopy procedures to the chemical information extracted with ToF-SIMS in order to test the application of ToF-SIMS for evaluation of TEM sample preparation applied in NanoSIMS analysis. This work focuses on new insights into the capability of ToF-SIMS for imaging sub-cellular details and how, by probing the specimen with ToF-SIMS, possible insights into pertinent ion species can be obtained and applied for NanoSIMS imaging, which was carried out as proof of principle.



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EXPERIMENTAL SECTION Materials Adrenal medullary cell culture and treatment. Dulbecco's modified Eagle's medium-low glucose, Ham's F12 medium, fetal bovine serum, 5-fuoro-2′-deoxyuridine, cytosine β-Darabinofuranoside, Locke's solution components, penicillin-streptomycin, bovine serum albumin and percoll were purchased from Sigma-Aldrich, Sweden. Enzymatic digestion of gland tissue was done with collagenase P (from Clostridium histolyticum) obtained from Roche, Sweden. TrypLE Express, GIBCO was purchased from Fisher Scientific, Sweden and L-3,4dihydroxyphenylalanine (L-DOPA 1-13C, RING-13C6, 99%) was obtained from EURISO-TOP, France. Water was purified with Purelab Classic purification system (ELGA, Sweden). Bovine adrenal glands were kindly donated from slaughterhouse Dalsjöfors Kött AB, Dalsjöfors, Sweden. Electron microscopy. Glutaraldehyde, sodium cacodylate, osmium tetroxide and Agar 100 resin all from Agar Scientific Ltd, UK were purchased from Oxford Instruments, Sweden. Uranyl acetate (Merck, Germany) was obtained from VWR International, Sweden. Sodium azide was purchased from BDH, England and formaldehyde from Sigma-Aldrich, Sweden. Reynolds lead citrate was prepared as previously described.31 Methods Adrenal medullary cell culture and treatment. Bovine adrenal cells were prepared by collagenase digestion as described previously32 with slight modifications. Briefly, fresh glands were transported to the laboratory in sterile Locke's solution on ice. Cell isolation was done in sterile conditions. Glands were cleaned with 70% ethanol solution and trimmed of fat and connective tissue. Adrenal vein was rinsed 3 times with Locke's solution previously thermostated at 37 °C. Each gland was inflated with 4 mL 0.2% collagenase P solution and incubated for 20 min at 37 °C. After examination of glands, an additional 1 mL of collagenase P solution was introduced to the gland interior through the main vein by syringe injection in each gland and they were kept at 37 °C for the next 10 min. Glands were snipped off longitudinally and the central part of gland, digested medulla, was collected and minced with a scalpel. The resulting tissue


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suspension was filtered over a steel sieve, diluted with Locke's solution in order to reduce the activity of collagenase P and centrifuged at 300 × g for 10 min at room temperature. The pellet obtained was re-suspended in Locke's solution and filtered over sterile 100 µm nylon mesh. A crude suspension of adrenal cells was mixed gently with percoll (1:1) and centrifuged at 18600 × g (Avanti J-20XP) for 20 min at room temperature. The top dense layer was collected and filtered over 100 µm nylon mesh. Cells were diluted with Locke's solution, pelleted down at 300 × g for 10 min at room temperature and re-suspended in Locke's solution. The cell yield was approx. 4 million/mL. Cells were plated in T-75 flasks (7-8 million cells per flask) and incubated at 37 °C in a 5% CO2 environment for 1 day. Cultured cells were treated with 100 µM stable isotope labeled L-DOPA for 90 min in an incubator at conditions described above and later harvested with TrypLE Express for chemical fixation. In our work, cultured cells were treated with stable isotope labeled L-DOPA for the purpose of experiments not related to this scientific report. Electron microscopy. Cells in suspension were incubated with modified Karnovsky fixative33 containing 0.01 % sodium azide, 1 % formaldehyde and 1.25 % glutaraldehyde at 4 °C for 2-3 h. Initially fixed cells were washed with 0.15 M sodium cacodylate buffer and post-fixed with 1 % osmium tetroxide for 2 h at 4 °C and 0.5 % uranyl acetate for 1 h at room temperature in dark. Cells were dehydrated in ethanol of ascending concentrations (70%, 85%, 95% and 99.5%) and later with 100% acetone and embedded in Agar 100 resin. Sections were obtained with an ultramicrotome (Leica EM UC6) and had thickness of 70 nm for TEM imaging and 200 nm for SEM imaging. Sections for TEM imaging were post-stained with uranyl acetate and Reynolds lead citrate and visualized with Leo 912AB Omega TEM operated at 120 kV. SEM imaging was performed on sections, without post-staining, with FEI Quanta 200 ESEM and FEI Versa 3D SEM, both operated at 5 kV. ToF-SIMS analysis. SIMS imaging was done with a TOF.SIMS V (ION-TOF, GmbH) equipped with a bismuth liquid metal ion gun.34 The cells from one isolation procedure and subsequent fixation preparation were imaged. Cell sections with 500 nm and 200 nm thickness were placed onto clean silicon wafers for ToF-SIMS imaging. The images were obtained from two TEM sections, where 11 and 12 cells were imaged. In total, spectra from 23 cells were processed and they were all very similar based on visual inspection. Data were recorded in negative ion mode and spectra were acquired using Bi3+ (25 keV) primary ions. High mass resolution images from a



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100 × 100 µm2 area were obtained in high current bunched (HCB) mode35 with pulsed primary ion current of 0.4 pA, 100 µs cycle time and maximum ion dose density was 1.64 × 1012 ions/cm2. The burst alignment (BA) imaging mode35 with 100 ns ion beam pulse width was used for high lateral resolution imaging with 234 nm/pixel resolution. The fluence was 1.56 × 1013 ions/cm2 and the pulsed target current was 0.07 pA with 110 µs cycle time. SurfaceLab 6 software (v. 6.4, ION-TOF GmbH) was used for spectra recording and processing. Spectra acquired in high current bunched mode were internally calibrated to signals of [C]-, [C2]-, [CH]-, [CH2]- and [OsO3]-, whereas for spectra obtained in BA mode [C]-, [CH]-, [CH2]-, [O]-, [OH]- and [OsO3]- signals were used as calibration points. A peak search was done in all individual spectra and a mass interval list containing peak areas and centroid positions was made. In Figures 3B-F shift correction was applied based on CN- fragment m/z 26 (binned 25 of total 200 scans). Molecular Weight Calculator freeware software (v 6.49 (build 243) by Matthew Monroe, http://www.alchemistmatt.com/mwtwin.html) and Microsoft Excel were used for theoretical isotope pattern calculations. NanoSIMS analysis. A NanoSIMS 50L ion microprobe (CAMECA, France) was used to perform NanoSIMS imaging. Cell sections with 200 nm thickness were placed onto clean silicon wafers. Implantation of

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Cs+ ions prior to chemical imaging was done by scanning the area of

interest with defocused primary ion beam (aperture diaphragm: D1-1) for 1 min. High spatial resolution negative ion maps were acquired with a focused 16 keV

133

Cs+ primary ion beam.

Current at the primary ion beam Faraday cup had a value of 18 nA and after introducing aperture diaphragm D1-3, a primary ion beam current of 0.973 pA was measured at the sample, no entrance slit was used. Images with a field of view 20 × 20 µm2 were obtained with a dwell time of 5 ms/pixel containing 256 × 256 pixels, taking approximately 5 min per image layer. Ion maps were acquired for the ImageJ

plugin

12

C14N−, and

OpenMIMS

190

(v

Os16O- ion species. The images were processed using the 2.5

(rev:

713);

MIMS,

Harvard

University;

http://www.nrims.hms.harvard.edu/). Five sequential image planes were collected and drift corrected, summed and presented in arbitrary linear color scale showing lower concentrations in dark blue and higher concentrations in red and yellow. Signal filtering was done with a mean filter with 0.3 pixel radius.


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RESULTS AND DISCUSSION TEM and ToF-SIMS images of chemically fixed adrenal cells. To prepare for imaging, the initial fixation of cells was carried out with a mixture of glutaraldehyde and formaldehyde. The former is an efficient, irreversible, inter- and intramolecular cross-linker, whereas formaldehyde, being a small molecule, penetrates and reacts rapidly, both reacting with amino groups of proteins and phospholipids.8 In this work, OsO4 was employed as post-fixative. It was first introduced as a fixative and stain by M. Schultze36 in 1864. Afterwards much work was done to understand the fixation mechanism of OsO4 in biological samples.8,37,38 OsO4 is strong oxidative agent that can be used for fixation of biomolecules such as unsaturated fatty acids,39,40 components of lipid membranes, as well as for proteins.41 Although several authors have reported ToF-SIMS negative ion spectra from OsO4 treated biological samples,30,38 our first step was to identify, in more detail, the specific osmium oxide ion species located in the subcellular compartments. In the upper panel of Figure 1 excerpts of high mass resolution spectra of cells are shown. Several peaks are noticeable in mass regions m/z 220-225, m/z 236-242 and m/z 441-449 that can be attributed to OsxOy- and OsxOyHzspecies. The observed and theoretical isotope patterns are compared (Fig. 1) and specific osmium oxide ion species contributing to the osmium signal from cellular organelles have been determined. Such species are OsO2- and OsO2H- (Fig. 1A); OsO3-, OsO3H- and OsO3H2(Fig. 1B) all possibly originating from the cyclic monoester, hydrated cyclic monoester and cyclic di-ester of osmium42 and Os2O4- and Os2O4H- species (Fig. 1C) from oxo-osmium diesters.43 A group of OsO4- mass spectral peaks at m/z 252-258 (mass assignments shown in Table S-1) were also detected that did not co-localize with the osmium oxide species at m/z 220225, m/z 236-242 and m/z 441-449 (data not shown). This suggests a lower specificity of OsO4ions and its hydrated forms during fixation. Moreover, the species with m/z 253 and m/z 255 were not well resolved from peaks with lower intensity at the same m/z value which can be attributed to the presence of palmitoleic (16:1) and palmitic (16:0) fatty acids.44 In Figure 1C, it is noticeable that the relation among observed signal intensities does not correlate exactly to the theoretical pattern. Even though this can be explained by the contribution of other chemical species present in the sample or by matrix effects, the high mass accuracy (Δ ppm) for the species



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in the mass range m/z 441-449 (Table 1) and the co-localization of the same with other OsxOyand OsxOyHz- species (data not shown) suggest valid ion species assignments.

Figure 1. Comparison of observed (upper panel) and theoretical (lower panel) isotope patterns of OsxOy- and OsxOyHz- species. Theoretical isotope patterns were determined based on observed pattern in high mass resolution spectrum by designating contributions of specific osmium oxide species in following ratios: A. OsO2- : OsO2H- = 1 : 0.9 B. OsO3- : OsO3H- : OsO3H2- = 3 : 1 : 1.3 C. Os2O4- : Os2O4H- = 2 : 1. Peaks labeled with an asterisk in A and B originate from unassigned ions.

Table 1 summarizes the ion species used for image generation. The isotopic patterns of osmium oxide species shown in Figure 1 and their mass accuracy (Table 1) give strong support for the correct assignment of the peaks. One could argue that signals m/z 223 and m/z 241 originate from inositol bisphosphate (IP2),45 however, the observed mass deviates from the predicted mass of IP2 (Table S-1). Strong peak intensities are observed in the low mass region (m/z < 100) from fragments such as CN- (m/z 26), CNO- (m/z 42), PO2- (m/z 63) and PO3- (m/z 79). The CN- and CNO- fragments might be distinctive for nitrogen-containing molecules, including but not limited to proteins and nucleic acids, whereas the ions PO2- and PO3- could originate from phosphate containing molecules such as nucleic acids and proteins. Although phosphate fragments can originate from phospholipids, ethanol and subsequent acetone dehydration during sample preparation likely extract the lipids from the cells.30,46 Uranyl acetate has been used as a heavy metal stain in fixation sample preparation.47,48 Several ion signals are detected that can be attributed to the uranyl acetate. Even though the peak at m/z 303 also could be assigned to IP249 or arachidonic acid,45 we show in Table S-1 that the high Δ ppm for these ion species rather indicates that it should be attributed to UO4-.


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Table 1. Summary of ToF-SIMS secondary ions used for negative ion image generation (only peaks with mass accuracy of < ± 60 ppm are included). Observed Theoretical m/z (Da) m/z (Da) 26.005

26.004

62.966

62.964

78.962

78.959

286.034 287.041

286.034 287.041

Δ ppm

Observed m/z (Da)

Ion

CN

34.6 31.8 34.2

-

PO 2

-

PO 3

-

UO3

1.2

235.948 236.949

-

-1.7

UO3 H

303.036

303.036

-1.3

572.039

572.060

-36.8

U2 O6

220.954

220.950

19.9

189

220.957

-15.5

188

OsO2 H

221.950

15.3

190

OsO2 -

221.957

-19.9

189

OsO2 H

46.6

190

-

OsO2

222.960

222.950

-51.5

186

18.9

189

-14.1

188

-47.1

187

OsO3 H2

237.945

10.5

190

OsO3

237.952

-22.4

189

OsO3 H

237.947

-

OsO2

237.960

-55.3

188

OsO3 H2

239.945

10.4

192

OsO3

239.952

-22.3

191

OsO3 H-

239.960

-54.8

190

OsO3 H2

-1.4

192

-

OsO3

236.945 236.960

-

-

239.947

-

OsO2 H -

240.952

Observed m/z (Da)

Ion

-18.4

235.952

236.952 -

Δ ppm 187

235.960

UO4

221.953

Theoretical m/z (Da)

240.952

-

OsO3 H

OsO3 H2 OsO3

440.902 -

-

441.895

OsO3 H

442.900

-

443.898 -

-

444.889 -

445.901

OsO3 H

Ion

22.9 5.1

188

Os2 O4 H

7.0

189

Os2 O4

441.900

-10.7

188

442.892

18.2

440.892 441.892

442.900

0.6

443.892

13.7

443.900

-3.9

444.892

-6.6

444.900

-24.1

445.892

20.4

445.900

2.8

223.952

223.950

10.7

192

241.945

43.4

194

446.906

446.900

14.0

224.959

224.957

7.0

192

OsO2 H-

241.952

11.0

193

OsO3 H-

447.898

447.892

13.6

235.948

235.945

14.8

188

OsO3 -

241.960

-21.3

192

OsO3 H2 -

448.906

448.900

13.9

241.955

-

Δ ppm

188/189

440.900

-

Theoretical m/z (Da)

Os2 O4

-

-

-

Os2 O4 H189/190 Os2 O4 189 Os2 O4 H 190 Os2 O4 189 Os2 O4 H 190/191 Os2 O4 190 Os2 O4 H191 Os2 O4 190 Os2 O4 H 191 Os2 O4 H 192 Os2 O4 192 Os2 O4 H-

TEM images of chemically fixed chromaffin cells are shown in Figures 2A and 2B, whereas Figure 2C shows an image of a cortical cell. Although it is expected that the isolated medullary cell culture used in these experiments should have only chromaffin cells, it had a fraction of cortical cells as well. Cortex tissue nearest to medulla is often present in the tissue sample used for cell isolation.29 Chromaffin cells are catecholamine producing cells and depending on the type of catecholamine secreted, they can be distinguished as adrenergic, synthesizing adrenaline and noradrenergic producing noradrenaline, both stored in large dense core vesicles (LDCVs). The chromogranin proteins (Cgs) are the main component of LDCVs, tightly packed in the core.50 This core appears dense in osmium stained TEM samples due to the aggregation of soluble components (beside the major fraction of Cgs, there are neurotransmitters, nucleotides, Ca2+, enzymes etc.) via electrostatic interactions. Cortical cells present in the sample originate from the zona reticularis, innermost layer of cortex, closest to adrenal medulla. They secrete glucocorticoids, and some sex steroids and are rich in mitochondria, smooth endoplasmic reticulum and lysosomes.29,51



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Figure 2. TEM images of chemically fixed adrenal cells. A. Adrenergic chromaffin cell (scale bar 2 µm); B. Noradrenergic chromaffin cell (scale bar 2 µm); C. Cortical cell (scale bar 5 µm).

TEM images (Fig. 2) show cell morphology similar to those imaged with ToF-SIMS (Fig. 3). Based on the morphology of the cells visible in the TEM images above (Fig. 2), we observe that the quality of the cell culture and the chemical fixation of the samples are suitable for ToFSIMS imaging. Even though some authors have reported the importance of imaging with high spatial and high mass resolution at the same time,30,35,52-54 much early work has been done by assigning the formulas to peaks with high mass resolution in high-current-bunched mode and acquiring high spatial resolution secondary ion images in burst-alignment mode.55-58 As we did not observe overlapping peaks in close proximity to selected m/z values (Table 1) when using the high mass resolution mode, we used the burst alignment mode for acquiring ion images with higher spatial resolution (Fig. 3).


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Figure 3. Negative ion ToF-SIMS images of fixed adrenal cells. A. Localization of OsxOy- and OsxOyHz- ions; B. Distribution of PO2- and PO3- ion species. Red arrows indicate potential chromaffin cells; C. Localization of CN- fragment; D. Color overlay of OsxOy- and OsxOyHz- ion species (red), PO2and PO3- fragments (green) and CN- ion (blue); E. Localization of UOx- and UOxHy- ions; F. Color overlay of UOx- and UOxHy- ions (red), OsxOy- and OsxOyHz- ion species (green) and CN- fragment (blue). Field of view: 60 × 60 µm2. The m/z values of used fragments are summarized in Table 1.

The functional protein dense matrix is observed as a dark electron rich area in TEM images of the LDCVs as OsO4 binds mostly to vesicular proteins (Figs. 2A-B). Even though OsO4 reacts with unsaturated lipids and has a role in their fixation, the OsxOy- and OsxOyHz- signals (Fig. 3A), are unlikely to originate from bonded lipids. Lipids are extracted during the dehydration steps and we did not find significant signals from lipid fragments, which is in accordance with the literature.30 Localization of OsxOy- and OsxOyHz- fragments can be observed in Figure 3A. The intense signals from osmium oxide ions are visible in areas surrounding the nuclei of certain cells. Based on previous studies, OsO4 is known to act as a stabilizer mainly of proteins and unsaturated fatty acids.59,60 Our first assumption was that the intense signal of OsxOy- and OsxOyHz- ions originates from proteins of chromaffin LDCVs. In Figures 3A-F, it is possible to distinguish two cell types in the secondary ion images, where one cell type, labeled with red arrows in Figure 3B, shows a less intense signal for OsxOy-, OsxOyHz- (Fig. 3A), POx- (Fig. 3B) and UOx-, UOxHy- (Fig. 3E) ion species across the cells. In order to validate our speculation that chromaffin cells show the most



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intense signal of the targeted ion species, we carried out secondary electron microscopy (SEM) imaging of the cell sections to identify cell types and perform consequent ToF-SIMS imaging. Figure S-1 shows an SEM image revealing cortical cells marked with red arrows compared to the other chromaffin cells (adrenergic and noradrenergic). However, SEM imaging produced significant electron damage that made subsequent ToF-SIMS analysis difficult due to the profound blurriness in SIMS images for OsxOy-, OsxOyHz- and CN- signals (Fig. S-2). Therefore, we approached the analysis in the reverse order, and performed ToF-SIMS analysis first and subsequently carried out SEM imaging (Fig. S-3). Cortical cells from the zona reticularis in the adrenal gland, have significant amounts of lysosomes. Lysosomes are organelles representing intracellular digestive system. They are rich in acidic hydrolytic enzymes, active only at low pH value present in lysosomes and degrading a wide range of biomolecules.61,62 In Figure S-4 lysosomes in cortical cells are shown, which in the SEM image correspond to bright cellular features and in the TEM image to darker organelles. When the SEM image (Fig. S-3A) and ToFSIMS ion map of OsxOy- and OsxOyHz- signals of the same area (Fig. S-3B) are compared, it is evident that a strong signal from the osmium oxide ion species localizes in the cortical cells and specifically in their lysosomes. The tendency of OsxOy- and OsxOyHz- ion species to be confined in lysosomes could be explained by bonding of OsO4 to diverse biomolecules present in these organelles, especially to different hydrolytic enzymes such as proteolytic enzymes, glucosidases, nucleases, phosphatases, phospholipase, sulfatases, and therefore having as well, a large amount of proteins, carbohydrates, nucleotides, lipids and products of their enzymatic degradation.61,62 Figure 3B shows an interesting cell morphological feature. If we compare the TEM images and the ion images of the cell nucleus and nucleolus (Fig. 2 and 3B), it is possible to make correlations between them. It is known that the eukaryotic nucleus and nucleolus have heterochromatin,63 a tightly packed form of DNA, RNA, and protein complexes rich in phosphates.64 Therefore it is possible to observe phosphate signals localized in specific areas of the nucleus in the ion image (Fig. 3B). These correspond to electron dense regions from the OsO4 fixative (proteins) and uranyl acetate stain (proteins and nucleic acids) in the TEM images of the nucleus (Fig. 2). The ion image of CN- at m/z 26 (Fig. 3C) revealed a similar localization trend as the phosphate species observed in cortical cells but was more abundant. The CN- fragment can be assigned to nitrogen-containing molecules such as proteins and nucleotides, the building blocks of DNA and RNA. Its higher intensity in assigned cortical cells suggests that they are richer in


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proteins and nucleotides. The difference in protein content between cortical and chromaffin cells possibly, but not exclusively, explains the strong CN- intensity in the former. In order to confirm the protein distribution across the cell, one might consider analysis of amino acid fragments in the positive ion mode and correlate these fragments to the CN- and CNO- ion distribution, but this was not within the scope of our study. Figure 3E shows the ion map of UOx- and UOxHy- species, with a tendency to localize in cortical cells. Uranyl acetate as a stain has great affinity towards proteins and nucleic acids, especially towards negatively charged groups such as phosphates and carboxyl groups.65 Again, it might be possible that cortical cells are richer in these biomolecular species that is also suggested from the imaging data of the CN- ion. However, in order to confirm this, a more detailed study should be done. Based on these results, we suggest that ToF-SIMS imaging is capable of distinguishing chemically fixed adrenal cell types. NanoSIMS imaging with ToF-SIMS support. Combined studies with TEM and NanoSIMS techniques allow correlation between the structural and chemical information of the sample66,67 where no further preparation step between TEM and NanoSIMS acquisition is necessary. However, due to the limitation of detecting only 7 masses in parallel with the magnetic sector mass analyzer of the NanoSIMS instrument, pre-selection of fragments of interest with ToF-SIMS analysis could be an advantage. In this study, the sample preparation for both SIMS imaging sub-techniques is the same and therefore the sample can be pre-investigated with ToF-SIMS before proceeding to NanoSIMS analysis. Overlaying TEM images and ion images obtained by NanoSIMS is a well-established approach for precise correlation between structural and chemical information. However, even though TEM grids used for NanoSIMS acquisition are coated with a supporting film,67 especially in high-spatial resolution imaging, the delicate nature of thin TEM section exposed to the NanoSIMS primary ion beam, could lead to artifacts when constructing the overlaying image. Therefore, in order to exclude the use of TEM images for sub-cellular orientation, another approach could be proposed. If one correlates subcellular features in TEM images by localizing the areas in cells where the electron density is the highest originating from heavy metals used as fixatives and staining agents, and investigating the heavy metal fragments with ToF-SIMS, it might be possible to preselect certain heavy metal ion fragments and analyze these with NanoSIMS. In this case, the ion image of heavy metal fragments can be used with the same purpose as a TEM image and the overlay can be done between ion images only. Therefore, we imaged cortical cells with the high resolution NanoSIMS



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50L mass spectrometer and acquired, in parallel, chemical maps for

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12

C14N- and 190Os16O- ions

(Fig. 4). The cellular features were observed by imaging 12C14N- (Fig. 4A) where it is possible to locate several cellular organelles such as the nucleus and nucleolus, lipid droplets, and probably lysosomes. Acquisition of

190

Os16O- ion images shows they are localized in cortical cells (Figs.

4B-C) in a similar manner as observed in the ToF-SIMS images of OsxOy- and OsxOyHz- ion species (Figs. 3A and S-3B). The color overlay in Figure 4D displays the distribution of the 190

Os16O- ion species around the nucleus in a similar pattern to the localization of lysosomes in

the SEM image of cortical cells (Fig. S-1).

Figure 4. Negative ion NanoSIMS images showing: A. Localization of 12C14N- ion specie; B. Distribution of the 190Os16O- ion; C. Distribution of the 190Os16O- ion (mean filter); D. Color overlay of 12 14 C N (green) and 190Os16O- (red, mean filter) ion species. Scale bar: 5 µm.

The localization of 190Os16O- in lysosomes confirms the conclusions from the ToF-SIMS data that OsO4 binds to the biomolecule diverse lysosomal environment. NanoSIMS imaging of the 190

Os16O- ion specie showed the same localization as OsxOy- and OsxOyHz- ions obtained with

ToF-SIMS. When using ToF-SIMS, the signal from the OsO- specie was too weak to be used for


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image acquisition (data not shown). This inconsistency might be explained due to the lower rate of fragmentation when using the Bi3+ primary ions68 used in ToF-SIMS acquisition and thus the peak intensity was lower for the diatomic OsO- ion specie. The fragmentation effect from use of the Cs+ primary ion probe is more profound69 and therefore with NanoSIMS it is not possible to image osmium oxide ion species with 3 or more atoms, but the OsO- specie gives a strong signal.

CONCLUSIONS ToF-SIMS has been used to generate ion images of chemically fixed samples prepared for TEM analysis and subsequent NanoSIMS imaging. The data provide insights into the selective properties of specific fixatives and stains towards biomolecules present in adrenal cells. We demonstrate the potential of ToF-SIMS imaging for distinguishing two types of adrenal cells, cortical and chromaffin cells, based on their chemical composition. Furthermore, ToF-SIMS provides insights in specific ion species related to chemical fixatives and their localization in lysosomes in cortical cells. Finally, to the best of our knowledge, the localization of the osmium oxide ion species using high-spatial resolution with NanoSIMS imaging, revealing the same distribution in cells as detected with ToF-SIMS is unique. An approach to preselect desirable ion fragments, characteristic for certain biomolecules or organelles, using ToF-SIMS, followed by subsequent imaging with NanoSIMS, might simplify the use of this high-resolution imaging technique. Thus, we suggest the application of ToF-SIMS a useful discovery tool to evaluate the preservation capabilities of different fixation methods for biological samples and as a stepping stone to advance the ultrahigh resolution imaging workflow.



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ASSOCIATED CONTENT Supporting Information Supporting Information is available free of charge via the Internet.

AUTHOR INFORMATION Corresponding Author * Mailing address: Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Kemivägen 10, SE-412 96 Gothenburg, Sweden. E-mail: [email protected] & [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT We would like to thank Ms. Yvonne Josefsson from Electron Microscopy Unit, Institute of Biomedicine, University of Gothenburg, Sweden, for technical assistance in sample preparation for electron microscopy and Johanna Höög from Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden for valuable insights and comments during article writing. Dalsjöfors Kött AB, Dalsjöfors, Sweden is gratefully acknowledged for donation of bovine adrenal glands. This research was funded by the Joint Chalmers-GU Center for Bioanalytical Chemistry, the Swedish Research Council (VR), European Research Council (ERC), Wallenberg Foundation, and the USA National Institutes of Health (NIH). The SIMS experiments were conducted at the National Center for Imaging Mass Spectrometry (NCIMS), Gothenburg, Sweden.


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REFERENCES (1) Quintana, C.; Wu, T. D.; Delatour, B.; Dhenain, M.; Guerquin-Kern, J. L.; Croisy, A. Microsc. Res. Tech. 2007, 70, 281-95. (2) Sombers, L. A.; Maxson, M. M.; Ewing, A. G. J. Neurochem. 2005, 93, 1122-31. (3) Wedlock, L. E.; Berners-Price, S. J. Aust. J. Chem. 2011, 64, 692-704. (4) Moore, K. L.; Lombi, E.; Zhao, F. J.; Grovenor, C. R. M. Anal. Bioanal. Chem. 2012, 402, 3263-73. (5) Preston, R. D. Nature 1958, 182, 1402-05. (6) Echlin, P.; Fendley, J. A. Nature 1973, 244, 409-14. (7) Muller, S. A.; Aebi, U.; Engel, A. J. Struct. Biol. 2008, 163, 235-45. (8) Hayat, M. A. Basic techniques for transmission electron microscopy; Academic Press Inc.: San Diego, 1986. (9) Ayache, J. B., L.; Boumendil, J.; Ehret, G.; Laub, D. Sample preparation handbook for transmission electron microscopy. Methodology; Springer: New York, 2010. (10) Lobinski, R.; Moulin, C.; Ortega, R. Biochimie 2006, 88, 1591-1604. (11) Malecki, M.; Hsu, A.; Truong, L.; Sanchez, S. Proc. Natl. Acad. Sci. 2002, 99, 213-18. (12) Briggs, D.; Fletcher, I. W.; Reichlmaier, S.; Agulo-Sanchez, J. L.; Short, R. D. Surf. Interface Anal. 1996, 24, 419-21. (13) Nygren, H.; Eriksson, C.; Hederstierna, K.; Malmberg, P. Appl. Surf. Sci. 2008, 255, 1092-95. (14) Hoppe, P.; Cohen, S.; Meibom, A. Geostand. Geoanal. Res. 2013, 37, 111-54. (15) Brunelle, A.; Touboul, D.; Laprevote, O. J. Mass Spectrom. 2005, 40, 985-99. (16) Lechene, C.; Hillion, F.; McMahon, G.; Benson, D.; Kleinfeld, A. M.; Kampf, J. P.; Distel, D.; Luyten, Y.; Bonventre, J.; Hentschel, D.; Park, K. M.; Ito, S.; Schwartz, M.; Benichou, G.; Slodzian, G. J. Biol. 2006, 5, 1-30. (17) Fletcher, J. S. Analyst 2009, 134, 2204-15. (18) Fletcher, J. S.; Vickerman, J. C.; Winograd, N. Curr. Opin. Chem. Biol. 2011, 15, 733-40. (19) Benninghoven, A. Surf. Sci. 1973, 35, 427-57. (20) Pacholski, M. L.; Winograd, N. Chem. Rev. 1999, 99, 2977-3006. (21) Benninghoven, A. Angew. Chem., Int. Ed. Engl. 1994, 33, 1023-43. (22) R. Castaing, G. S. J. Microsc. 1962, 1, 395–410. (23) Belu, A. M.; Graham, D. J.; Castner, D. G. Biomaterials 2003, 24, 3635-53. (24) Slodzian, G.; Daigne, B.; Girard, F.; Boust, F.; Hillion, F. Biol. Cell 1992, 74, 43-50. (25) Boxer, S. G.; Kraft, M. L.; Weber, P. K. Annu. Rev. Biophys. 2009, 38, 53-74. (26) Robinson, M. A.; Castner, D. G. Biointerphases 2013, 8, 15. (27) Fletcher, J. S.; Rabbani, S.; Henderson, A.; Lockyer, N. P.; Vickerman, J. C. Rapid Commun. Mass Spectrom. 2011, 25, 925-32. (28) Grovenor, C. R. M.; Smart, K. E.; Kilburn, M. R.; Shore, B.; Dilworth, J. R.; Martin, B.; Hawes, C.; Rickaby, R. E. M. Appl. Surf. Sci. 2006, 252, 6917-24. (29) Bornstein, S.; Ehrhart-Bornstein, M.; Usadel, H.; Böckmann, M.; Scherbaum, W. Cell Tissue Res. 1991, 265, 1-9. (30) Malm, J.; Giannaras, D.; Riehle, M. O.; Gadegaard, N.; Sjovall, P. Anal. Chem. 2009, 81, 7197-205. (31) Reynolds, E. S. J Cell Biol. 1963, 17, 208–12. (32) O'Connor, D. T.; Mahata, S. K.; Mahata, M.; Jiang, Q.; Hook, V. Y.; Taupenot, L. Nat. Protoc. 2007, 2, 1248-53.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 20

(33) Karnovsky, M. J. J. Cell Biol. 1965, 27, 137-38A. (34) Kollmer, F. Appl. Surf. Sci. 2004, 231–232, 153-58. (35) Sodhi, R. N. S. Analyst 2004, 129, 483-87. (36) Schultze, M. Verh. Naturhist. Verein. Rheinlande, 1864, 2, 61. (37) Palade, G. E. J. Exp. Med. 1952, 95, 285-98. (38) Belazi, D.; Sole-Domenech, S.; Johansson, B.; Schalling, M.; Sjovall, P. Histochem. Cell Biol. 2009, 132, 105-15. (39) Collin, R. J.; Griffith, W. P. J. Histochem. Cytochem 1974, 22, 992-93. (40) Bahr, G. F. Exp. Cell Res. 1955, 9, 277-85. (41) Nielson, A. J.; Griffith, W. P. J. Histochem. Cytochem. 1979, 27, 997-99. (42) Riemersma, J. C. In Some Biological Techniques in Electron Microscopy, Parsons, D. F., Ed.; Academic Press: New York, 1970. (43) Griffith, W. P.; Rossetti, R. J. Chem. Soc., Dalton Trans. 1972, 14, 1449-53. (44) Malmberg, P.; Nygren, H.; Richter, K.; Chen, Y.; Dangardt, F.; Friberg, P.; Magnusson, Y. Microsc. Res. Tech. 2007, 70, 828-35. (45) Touboul, D.; Brunelle, A.; Halgand, F.; De La Porte, S.; Laprévote, O. J. Lipid Res. 2005, 46, 138895. (46) Maunsbach, A. B.; Afzelius, B. A. Biomedical Electron Microscopy: Illustrated Methods and Interpretations; Academic Press: Burlington, 1998. (47) Kellenberger, E.; Ryter, A.; Sechaud, J. J. Biophys. Biochem. Cytol. 1958, 4, 671-78. (48) Terzakis, J. A. J. Ultrastruct. Res. 1968, 22, 168-84. (49) Hsu, F.-F.; Turk, J. J. Am. Soc. Mass Spectrom. 2000, 11, 986-99. (50) Winkler, H. Neuroscience 1976, 1, 65-80. (51) Rosol, T. J.; Yarrington, J. T.; Latendresse, J.; Capen, C. C. Toxicol. Pathol. 2001, 29, 41-48. (52) Rabbani, S.; Fletcher, J. S.; Lockyer, N. P.; Vickerman, J. C. Surf. Interface Anal. 2011, 43, 380-4. (53) Fletcher, J. S.; Rabbani, S.; Henderson, A.; Blenkinsopp, P.; Thompson, S. P.; Lockyer, N. P.; Vickerman, J. C. Anal. Chem. 2008, 80, 9058-64. (54) Brison, J.; Robinson, M. A.; Benoit, D. S. W.; Muramoto, S.; Stayton, P. S.; Castner, D. G. Anal. Chem. 2013, 85, 10869-77. (55) Nygren, H.; Börner, K.; Malmberg, P.; Tallarek, E.; Hagenhoff, B. Microsc. Res. Tech. 2005, 68, 329-34. (56) Malmberg, P.; Börner, K.; Chen, Y.; Friberg, P.; Hagenhoff, B.; Månsson, J.-E.; Nygren, H. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2007, 1771, 185-95. (57) Börner, K.; Malmberg, P.; Månsson, J.-E.; Nygren, H., Int. J. Mass Spectrom. 2007, 260, 128-36. (58) Lanekoff, I.; Kurczy, M. E.; Hill, R.; Fletcher, J. S.; Vickerman, J. C.; Winograd, N.; Sjövall, P.; Ewing, A. G. Anal. Chem. 2010, 82, 6652-59. (59) Riemersma, J. C. Biochim. Biophys. Acta, Lipids Lipid Metab. 1968, 152, 718-27. (60) Wigglesworth, V. B. Proc. R. Soc. London, Ser. B 1957, 147, 185-99. (61) Fawcett, D. W. The Cell, 2nd ed.; Saunders WB: Philadelphia, 1981. (62) Cooper, G.M. The Cell: A Molecular Approach, 2nd ed.; Sinauer Associates: Sunderland, 2000. (63) Heitz, E., Jb. Wiss. Bot. 1928, 69, 762-818. (64) Adolph, K. W.; Kreisman, L. R.; Kuehn, R. L. Biophys. J. 1986, 49, 221-31. (65) Tzaphlidou, M.; Chapman, J. A.; Al-Samman, M. H. Micron 1982, 13, 133-45. (66) McMahon, G.; Glassner, B. J.; Lechene, C. P. Appl. Surf. Sci. 2006, 252, 6895-906.


Page 19 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(67) Audinot, J.-N.; Georgantzopoulou, A.; Piret, J.-P.; Gutleb, A. C.; Dowsett, D.; Migeon, H. N.; Hoffmann, L. Surf. Interface Anal. 2013, 45, 230-33. (68) Shishido, R.; Fujii, M.; Seki, T.; Aoki, T.; Matsuo, J.; Suzuki, S. Rapid Commun. Mass Spectrom. 2016, 30, 476-82. (69) Appelhans, A. D.; Delmore, J. E. Anal. Chem. 1989, 61, 1087-93.



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