TOF-SIMS Imaging of Intact Lipids in Biological Tissue with Enhanced Ion Intensity by Ionic Liquid

Abstract

Mass spectrometry imaging (MSI) is a powerful technique for visualizing the spatial distribution of chemical species in biological samples. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) enables high-spatial-resolution imaging and the detection of a broad range of molecular ions, including intact lipids. However, the observation of high-mass components remains challenging due to limitations in ionization efficiency and sample degradation. In this study, we present a novel sample preparation method for TOF-SIMS using an ionic liquid (IL) pretreatment to enhance the ionization efficiency of high-mass lipid ions. This approach increases the ionization efficiency of lipid molecules by achieving an appropriate distribution of ionic liquid to the surface of the sample. By reducing fragmentation, this method exceeds the static limit and increases the lipid ion signal by nearly 3 orders of magnitude. Furthermore, this method enabled static TOF-SIMS imaging of lipid-related ions and ion-induced electron imaging beyond the conventional static limit. These advancements significantly improve TOF-SIMS-based MSI applications, offering new opportunities for lipidomics and biological research.

1. Introduction

Mass spectrometry imaging (MSI) of biological samples is an emerging analytical technique with significant applications in biomedical research including lipidomics. − A major challenge in this field is the appropriate preparation of biological samples to facilitate the observation of relatively high-mass components while minimizing contamination, chemical degradation, and spatial distortion caused by ion beam irradiation. ,

Time-of-flight secondary ion mass spectrometry (TOF-SIMS), which can provide submicron scale spatial resolutions, enables molecular imaging at the subcellular levels, far beyond the reach of most other MSI techniques. However, its effectiveness for biological samples is limited by a substantial decline in ion intensity in the high-mass region (m/z > 200), hindering the detection of biologically important molecules such as lipids, secondary metabolites, and small drugs. In addition, surface contamination such as adsorbed water layers and hydrocarbons further complicates sample preparation.

Previous studies have explored various primary ion sources to enhance molecular ion detection in biological TOF-SIMS imaging. Early work using gold cluster ions (Au_n ⁺) demonstrated the capability of TOF-SIMS to visualize lipid distributions in biological tissues. Subsequent introduction of bismuth cluster ions (Bi_n ^m+) enabled higher spatial resolution and improved secondary-ion yields, , while fullerene ions (C₆₀ ⁺) enhanced the detection of higher-mass molecular species such as intact lipids. , More recently, gas and water cluster beams, including Ar_n ⁺ and (H₂O) _n ⁺, have provided gentler sputtering and improved secondary-ion yields from fragile biomolecules. − Among these studies, TOF-SIMS imaging of mouse brain sections has been reported using Bicluster ions for high-spatial-resolution mapping of low-mass fragments. In contrast, C₆₀ ⁺ and (H₂O) _n ⁺ cluster primary ions have been applied to detect higher-mass lipids at reduced lateral resolution. ,

One of the approaches to address this trade-off is the use of cluster ions to remove surface contamination and enable gentle depth profiling of biological samples. , For example, gas cluster ion beam (GCIB) sputtering allows in-depth observation of tissue while preserving molecular information, as demonstrated in our previous study of pyramidal cells in the mouse hippocampus. Although GCIBs reduce surface damage, the intensity of high-mass molecular ions remains limited. Hybrid systems combining TOF-SIMS with Orbitrap detection have improved mass-resolving power; and image fusion of TOF-SIMS images has been explored, yet simultaneous realization of high mass sensitivity and high spatial resolution remains difficult.

Ionic liquids (ILs) have also been investigated as matrices to enhance ionization efficiency in mass spectrometry. Walker et al. investigated the role of proton transfer in secondary ion mass spectrometry using ionic liquid matrices and systematically evaluated their effect on analyte signal enhancement using various samples. , The characteristics and ionization mechanisms of individual proton-conducting ILs have been investigated. , These ILs have been applied to TOF-SIMS analyses of biological samples, including both two-dimensional and three-dimensional imaging. − More recently, matrix-enhanced SIMS (ME-SIMS) has been actively investigated as a strategy to improve molecular-ion yields in TOF-SIMS. Matrix deposition and in situ matrix enhancement have been shown to increase lipid and biomolecular ion signals in organic films, tissue sections, and model samples, although the degree of enhancement depends strongly on the matrix material, deposition method, primary-ion species, and measurement conditions. −

Building on these findings, the present study focuses on infiltrating biological tissue with propylammonium nitrate (PAN; C₃H₁₀N⁺·NO₃ ^–) as an ionic liquid pretreatment. This approach improves the detection of high-mass ion signals while maintaining fine structural integrity, enabling microscopic visualization of tissue morphology using TOF-SIMS and ion-induced electron (IIE) imaging.

2. Materials and Methods

2.1. Sample Preparation

Mouse brain samples (Funakoshi Co., Ltd., Tokyo, Japan) were pretreated to obtain tissue sections according to the method described in ref In this study, 100% (KANTO Chemical, Tokyo, Japan) and 100 mM PAN prepared in spectroscopic-grade ethanol were selected as the IL because their nonhalogenated composition minimizes the risk of residual halogen contamination in the TOF-SIMS instrument. Although a fluorinated IL, N,N-diethyl-N-methyl-N-(2-methoxyethyl) ammonium trifluoromethanesulfonate (demaTfO, C₉H₂₂NO⁺·CF₃SO₃ ^–), showed a similar initial increase in ion intensities, it was not further examined due to the potential for fluorine contamination. Other halogen-containing ILs were excluded for the same reason.

The improved sample preparation method used in this study is schematically shown in Figure . First, a frozen mouse brain was immersed in a 0.5% glutaraldehyde/ethanol solution at −80 °C to chemically fix tissue. After sufficient dehydration, the sample was transferred to spectroscopic-grade ethanol at −80 °C. The brain was cut into 1.5–2.0 mm-thick slices using a mouse brain slicer (RWD Brain Slicer, BrainMatrix) and a razor blade (BrainMatrix). The dissection was performed inside a cooler box maintained at approximately 0 °C using a cooling pack (HYOUTENKA PACK GT – 16 °C HARD, LOGOS). The mouse brain slicer, razors, and high purity silicon wafers (AS ONE Corp., Osaka, Japan) were precooled on coolant packs.

1.

Scheme of improved sample preparation used in this study.

The cut section was placed on a clean silicon wafer, filled around with a 50% propylammonium nitrate (PAN)/ethanol solution, and sandwiched between the silicon wafers. This sandwich preparation was intended to reduce surface topography, which can interfere with TOF-SIMS imaging, and to suppress excessive accumulation of PAN on the sample surface. The section was gently dried overnight in a lyophilizer and then loaded into the TOF-SIMS instrument.

2.2. TOF-SIMS Measurements

TOF-SIMS analysis was performed using a PHI TRIFT V nanoTOF instrument (ULVAC-PHI, Japan). A 60 keV Bi₃ ²⁺ liquid metal ion beam, suitable for detecting relatively large ions at high spatial resolution, was used as the primary ion source. Ion etching was performed with Ar₂₅₀₀ ⁺ GCIB. Experimental parameters such as spatial resolution and ion current were optimized to obtain high-quality ion images under various conditions. The average pulsed Bi₃ ²⁺ primary ion current used for dose estimation was estimated to be 0.03 pA. The value was calculated from the total beam current (0.25 nA) using the pulse width (13.6 ns), the assumed Bi₃ ²⁺ fraction in the beam (12%), and the charge state of Bi₃ ²⁺. Because the Bi₃ ²⁺ fraction was not measured directly with a Faraday cup under the pulsed imaging conditions, the calculated dose should be regarded as an estimate. All the spectra and images were acquired automatically using WinCadence software (ULVAC-PHI).

2.2.1. TOF-SIMS Spectra

Positive ion mass spectra were acquired from a 50 μm × 50 μm area (256 × 256 pixels). The spot diameter of the primary ion beam was approximately 500 nm, and the ion current (before pulsing) applied to the sample was set at 2 nA. For mass calibration, C₂H₃ ⁺ (m/z 27.0235), C₃H₅ ⁺ (m/z 41.0391), and C₄H₇ ⁺ (m/z 55.0547) ions were used as internal low-mass standards. Further calibration was performed using m/z 184.1074 (C₅H₁₅NO₄P⁺ choline headgroup) and m/z 369.4352 (C₂₇H₄₅ ⁺, cholesterol) as high-mass references. The m/z values of the assigned lipid species matched the theoretical values within m/z 0.1. For peak assignment in the TOF-SIMS spectra, mass calibration was performed using the accurate masses of the calibration ions under the high mass resolution measurement condition.

2.2.2. IIE Images and TOF-SIMS Images

IIE microscopy images were acquired from areas of 600 μm × 600 μm, 200 μm × 200 μm, and 50 μm × 50 μm (256 × 256 pixels) for 15 s, respectively. The spot diameter of the primary ion beam was 100 nm, and the ion current (before pulsing) was 0.25 nA. The primary ion doses were 3.2 × 10¹² ions cm^–2, 2.9 × 10¹³ ions cm^–2, and 4.7 × 10¹⁴ ions cm^–2, respectively.

2.2.3. Ion Etching

GCIB sputtering was performed using a 20 keV Ar₂₅₀₀ ⁺ beam following an initial surface measurement by TOF-SIMS. During sputtering, an ion current of 6 nA was applied to the sample. The sputtering process was conducted over a 2000 μm × 2000 μm area in 20 s. The sputter etching and TOF-SIMS measurements were repeated several times. The measurement time for each cycle was 180 min, and the primary ion dose was ca. 1.0 × 10¹³ ions cm^–2/cycle. The lipid imaging measurements were performed under conditions optimized for spatial resolution rather than mass resolution; therefore, the lipid ion images are described using nominal m/z values.

3. Results

The Results section follows the stepwise development of the optimized PAN-assisted TOF-SIMS method. We begin by assessing PAN effects using lipid standards, then evaluate PAN treatment on biological tissue. Next, we introduce an improved PAN pretreatment that balances ionization enhancement and spatial preservation. Finally, we tested the method by GCIB etching for sample-internal lipid imaging.

3.1. Improvement of Lipid Ion Sensitivity by IL Addition

In order to evaluate the effect of IL on the sensitivity of TOF-SIMS for lipids, TOF-SIMS measurements were conducted on lipid standards, DPPC (dipalmitoyl-phosphatidylcholine) treated with an IL (propylammonium nitrate (PAN)). Figure shows the ion intensity for m/z 184 (phosphocholine headgroup), 735 (protonated DPPC molecule ion), and 1468 (protonated DPPC dimer ion) with respect to ion fluence up to approximately 4 × 10¹³ ions cm^–2. A comparison of Figure A,B shows a significant enhancement in the signal intensity of lipid-derived ions with PAN treatment. Even within the static limit dose range of 10¹² ions cm^–2, the sensitivity increased approximately an order of magnitude. Furthermore, secondary ions of lipid molecules continued to be produced even beyond the static limit, indicating sustained ion production without significant signal degradation over time. Even after a cumulative ion fluence of approximately 4 × 10¹³ ions cm^–2 during continuous TOF-SIMS measurements, the ion intensity remained stable, indicating prolonged signal retention.

2.

TOF-SIMS ion intensity profile of total ion, m/z 735 (DPPC), m/z 39 (K), m/z 184 (phosphocholine headgroup) and m/z 1468­(DPPC Dimer) ions, with respect to Bi₃ ⁺² primary ion fluence. (A) Intact DPPC reference and (B) DPPC (1 mg/mol) added in PAN.

The TOF-SIMS positive ion spectra of PAN under the similar measurement conditions are presented in Figure S1. While PAN-derived peaks were detected, their intensities were deemed negligible and unlikely to influence the interpretation of lipid peaks.

3.2. Improvement of Lipid Ion Sensitivity of Biological Samples by Addition of PAN

The PAN pretreatment was applied to the conventionally treated biological samples (sliced mouse brain). Slices from similar locations were used for three different conditions: without PAN, with 100% PAN spray, and with 100 mM PAN spray. The 100 mM PAN spray condition was selected as a diluted PAN treatment to reduce the amount of PAN distributed on the sample surface. This experiment was performed to evaluate the balance between lipid-ion signal enhancement and preservation of image contrast when PAN was applied from the sample surface. The PAN spray was applied sufficiently to cover the entire sample surface. The TOF-SIMS ion images obtained from these samples are shown in Figure .

3.

TOF-SIMS dose profiles and positive ion images. The profile and images are shown for m/z 735 (DPPC), m/z 39 (K), m/z 184 (phosphocholine headgroup), and total positive ion. (A) Untreated Mouse brain section (B) 100% PAN sprayed on the mouse brain section, and (C) 100 mM PAN sprayed on the mouse brain. The scale bars in the images represent 10 μ m.

In the absence of PAN (Figure A), brain cell images were visible in the ion images of potassium and other fragments, while intact lipid ions such as protonated DPPC exhibited very small intensity. In contrast, the sample treated with 100% PAN spray (Figure B) showed continuous detection of high-mass lipid ions even at ion doses exceeding the static limit. However, the ion images of all ions showed reduced spatial contrast at the micrometer level.

When a diluted PAN solution (100 mM) was applied (Figure C), the ion intensity of intact lipid ions increased relative to the untreated sample, although the signal level did not exceed the static limit as in the 100% PAN condition.

Table shows the number of DPPC and POPC (palmitoyl-oleoyl-phosphocholine) molecules detected at dose levels above the static limit, extracted from the dose profile shown in Figure . The 100% PAN-treated sample showed a three-order of magnitude increase in the number of intact lipid ions compared to the untreated sample. However, as shown in Figure , the visibility of morphological features in the associated ion images was reduced.

1. Detected Positive-Ion Counts of Intact DPPC and POPC Signals Obtained From Mouse Brain Tissue Sections under Ion Doses Exceeding the Conventional Static Limit, Extracted From the Dose Profile Shown in Figure .

	DPPC		POPC
	counts	cf. Intact	counts	cf. Intact
100% PAN	399,909	×652	197,568	×549
100 mM PAN	2203	×4	1974	×5
untreated	613	-	360	-

^a4 × 1013 Ions cm^–2 for untreated, 100% PAN-sprayed, and 100 mM PAN-sprayed mouse brain tissue sections for m/z 734.57 (protonated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) and m/z 760.59 (protonated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC).

3.3. Improved Sample Preparation Methods for TOF-SIMS Imaging

The use of PAN successfully increased the ionization efficiency in TOF-SIMS measurements. However, simple application methods such as drop deposition obscured the original lipid ion distribution, preventing clear visualization, likely due to the presence of excessive or overly thick PAN layers on the surface, resulting in blurred images. To achieve both ionization and high-resolution imaging, the processing conditions would need to be optimized.

As the next step, we implemented the improved sample pretreatment method described in the materials and methods section. This approach was intended to introduce PAN into the tissue during dehydration and drying with the PAN/ethanol solution, while avoiding excessive PAN accumulation on the outermost surface, thereby facilitating ionization without substantially degrading imaging resolution. Mouse brain slices prepared with the new method were able to maintain a strong lipid parent ion signal even when ion irradiation was performed beyond the static limit.

Figure shows TOF-SIMS measurement results of a mouse brain section treated with improved PAN pretreatment. This figure shows that PAN treatment maintains lipid ion signal intensity while preserving structural details. As shown in Figure A,B, comparison of positive-ion spectra acquired before and after IIE images observation reveals no change in the surface ion intensity, and the ion intensity of intact lipid ions remained unaffected. The primary ion doses were 3.2 × 10¹² ions cm^–2, 2.9 × 10¹³ ions cm^–2, and 4.7 × 10¹⁴ ions cm^–2 for IIE acquisition.

4.

TOF-SIMS and IIE imaging results for mouse brain tissue prepared with the improved PAN pretreatment. (A) IIE images, (B) TOF-SIMS spectra acquired before and after IIE imaging, and (C) corresponding TOF-SIMS ion images. The scale bars represent 50 μm.

Minimizing ion beam-induced damage to the specimen enabled the acquisition of IIE images. Based on the comparison of TOF-SIMS images acquired before and after IIE observation shown as Figure C, there were no significant changes in strength or distribution.

3.4. GCIB Ion Etching of the Sample Surface

As shown in Figure , ion etching of the sample surface was performed using a 20 keV Ar∼₂₅₀₀ ⁺ GCIB in the same TOF-SIMS instrument after the TOF-SIMS measurements in Figure . The DC current irradiated to the sample during sputtering was 5 nA. Ion etching was repeated 14 times on a square area of approximately 2000 μm × 2000 μm in each 20 s. The sputtering rate was estimated to be 15.2 nm/min, based on our previous study, and corresponding to an incremental etching depth approximately 5 nm per cycle, with a total depth of approximately 70 nm after 14 cycles. The profile shows no significant changes in the intensity of fragment and intact ion signals during TOF-SIMS measurement.

5.

(A)­TOF-SIMS intensity profiles for selected fragment ions and intact lipid ions of PAN-treated mouse brain tissue during sequential GCIB sputtering. (B) Representative positive-ion mass spectra obtained during the sputtering cycles.

A set of IIE images was obtained as shown in Figure and S3, and although the spatial distribution of each ion changed with etching, images could be acquired continuously. Intact lipid (DPPC, POPC) derived ion images were obtained, and the distribution was different from inorganic ions, indicating that lipid-specified distributions are observed. Furthermore, the lipid-derived ionic intensity was maintained even after repeated sputtering and long-term ion dosing (1.0 × 10¹³ ions cm^–2).

6.

Selected TOF-SIMS ion images of intact lipids (m/z 735­(DPPC) and 761­(POPC)), normalized to total positive ions, together with the total positive-ion images acquired during GCIB sputtering of PAN-treated mouse brain tissue. Images correspond to the same region used for the profiles shown in Figure . The scale bars represent 10 μm.

The TOF-SIMS positive ion spectra before and during GCIB sputtering are shown in Figure S4, and although PAN-derived peak was obtained. The ionic intensity was maintained but very low, presumably had no effect on the discussion of lipid peak intensities.

4. Discussion

The results presented here demonstrate that optimized ionic-liquid (IL) pretreatment enables TOF-SIMS imaging of biological lipids with both high spatial resolution and enhanced sensitivity. Below, we discuss the key findings in relation to ionization efficiency, spatial integrity, and depth-resolved chemical imaging.

4.1. PAN-Enhanced Ionization of Intact Lipids

The signal enhancement observed in the present PAN-treated samples is broadly consistent with the general trend reported in recent ME-SIMS studies, in which matrix addition or in situ matrix enhancement improved lipid and biomolecular ion yields in model films and tissue sections. − However, direct quantitative comparison is not straightforward because the matrix chemistry, sample preparation, primary-ion species, and measurement conditions differ among studies. In both lipid standards and mouse brain tissue, application of PAN markedly increased the yield of protonated high-mass lipid ions and stabilized their signals under prolonged ion irradiation. ^, These observations align with previous reports on protic ILs − ,− and suggest that PAN facilitates proton transfer, thereby improving the yield of protonated intact lipid ions. The ability to detect intact lipid ions well beyond the conventional static limit represents a particularly notable outcome, indicating that PAN not only promotes softer ionization but also suppresses the rapid signal decay typically associated with cumulative primary-ion exposure.

The dose-dependent behavior observed in Figures and supports this interpretation. Table further shows that samples treated with higher amounts of PAN yielded greater numbers of intact DPPC and POPC ions also under static-limit-exceeding conditions as same as sample surface. The collective results indicate that PAN plays a dual role: promoting protonation and stabilizing the surface against ion-induced degradation.

4.2. Optimized PAN Infiltration Preserves Morphology while Enhancing Ionization

While increasing PAN content improves intact-lipid ion yields, the results in Figure B demonstrate that excessive amounts of IL on the surface reduce spatial image contrast at the micrometer scale. This loss of morphological detail may be related to excess PAN accumulated on the surface, which could promote migration or redistribution of lipid molecules and thereby obscure fine structural features. Thus, although PAN enhances ionization efficiency, its surface distribution must be carefully controlled to maintain spatial fidelity.

The intermediate condition using 100 mM PAN (Figure C) provides additional insight. In this case, intact lipid ion intensities increased relative to untreated samples without a substantial loss of spatial detail. Together with Table , these results suggest that an optimal amount of PAN exists where ionization is enhanced while the morphological integrity of the tissue is preserved. Establishing this balance is essential for applying IL-assisted TOF-SIMS to biological samples in which both high mass molecular sensitivity and spatial resolution are critical.

4.3. Morphological Measurements after GCIB Ion Etching of the Sample Surface

The improved PAN pretreatment also enhanced the robustness of TOF-SIMS imaging after GCIB ion etching. As shown in Figure , intact lipid ions such as DPPC and POPC maintain stable intensities across multiple sputtering cycles, while fragment ions exhibit comparatively minor variation. This behavior indicates that PAN reduces the effects of primary-ion implantation not only on the surface but also inside the sample.

Figure further illustrates the depth-resolved chemical behavior of lipids, showing consistent spatial distributions across sequential sputtering cycles. These images demonstrate that PAN-treated tissues preserve molecular integrity within the shallow subsurface region, enabling chemically specific imaging with high lateral resolution. Such damage suppression is consistent with earlier observations that protic ionic liquids enhance secondary-ion yields while minimizing molecular degradation when used as primary-ion sources. ,

This suggests that the intrinsic physicochemical properties of PAN contribute to both enhanced ionization and reduced damage accumulation by primary-ion implantation, even inside the sample. However, further comparison with untreated samples during depth profiling will be necessary to clarify the depth-dependent contribution of PAN in more detail. Such stability may be critical for extending TOF-SIMS to surface and three-dimensional structural biochemical analyses of biological tissues.

4.4. Compatibility of TOF-SIMS with IIE Imaging

The ability to obtain IIE images from the same region without measurable degradation of TOF-SIMS signals (Figure ) indicates that the optimized PAN pretreatment is compatible with correlative morphological and molecular imaging under the present measurement conditions. Combined with the structural detail revealed in Figure S2, this compatibility enables the correlative use of morphological and molecular imaging within a single instrument, strengthening the utility of PAN-assisted TOF-SIMS for biological studies.

4.5. Outlook and Mechanistic Considerations

While the present results establish a practical framework for IL-assisted TOF-SIMS imaging, the underlying mechanismsparticularly the sustained lipid ion emission beyond the static limitremain to be fully elucidated. These results suggest that PAN may reduce the accumulation of sputter-cascade-induced molecular damage in the near-surface region. In addition, lipid molecules in the PAN-containing medium may replenish the analyzed surface as molecules are removed by sputtering; however, whether such molecular mobility affects the lateral resolution of TOF-SIMS imaging remains to be examined. Direct comparison with previous brain-tissue imaging studies is not straightforward because of differences in sample preparation, ion species, polarity, and spatial scale, although the observation of phospholipid-related signals in the mouse hippocampal tissue is broadly consistent with previous reports. ,, Although GCIB etching was performed in increments of 5 nm, previous studies have shown that Bi3² irradiation in organic films can induce both ten nanometer-scale implantation and measurable surface erosion of comparable magnitude. Nevertheless, the maintained detection of lipid ions even at doses exceeding the static limit indicates that, in the PAN-infiltrated samples, the accumulation of primary-ion -induced damage is suppressed, resulting in sustained secondary-ion intensities.

Further work should clarify how PAN distribution within tissue affects ion trajectories, sputter dynamics, and proton-transfer pathways. Integration with advanced 3D TOF-SIMS and OrbiSIMS methodologies may broaden the applicability of this approach to more complex biological and materials systems.

5. Conclusion

In this study, we demonstrated enhanced ionization efficiency and improved TOF-SIMS imaging of intact lipids in biological tissue using an IL (PAN) pretreatment. The optimized PAN pretreatment method significantly improved the detection of high-mass lipid ions while preserving structural integrity, enabling high-resolution chemical imaging beyond the static limit.

Our findings highlight three key advantages of PAN-assisted TOF-SIMS.

• 1. Enhanced Ionization Efficiency: PAN application improved the detection of high-mass ions by reducing fragmentation, thereby enhancing lipid ion sensitivity.
• 2. Observation of the internal sample structure: Using GCIB sputtering on a PAN-pretreated sample, chemically specific signals could be obtained from subsurface regions with high sensitivity.
• 3 Preservation of Sample Morphology: the optimized pretreatment method, allowing for IIE imaging with minimal structural distortion.

These improvements may help bridge the gap between ionization efficiency and high-spatial-resolution analysis.

At the same time, several limitations and open questions remain. This method cannot be applied to samples that are impermeable to ionic liquids. In addition, it remains unclear to what extent the application of PAN pretreatment preserves the native chemical distribution within biological tissues. These limitations should be carefully considered when interpreting PAN-assisted TOF-SIMS images.

Addressing these issues will be essential for the broader application and optimization of IL-based sample preparation methods in TOF-SIMS lipidomics and bioimaging.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c03014.

• TOF-SIMS positive ion spectra of PAN, additional ion images during GCIB sputtering, and supplementary depth profiles (PDF)

†.

FURUKAWA ELECTRIC CO., LTD

‡.

InR Labo., 4–20–12 Ooka, Minami-ku, Yokohama, Kanagawa 232–0051, Japan.

The authors declare no competing financial interest.