Graphene-Coated Glass Substrate for Continuous Wave Laser

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Functional Nanostructured Materials (including low-D carbon)

Graphene-Coated Glass Substrate for Continuous Wave Laser Desorption and Atmospheric Pressure Mass Spectrometric Imaging of Live Hippocampal Tissue Jae Young Kim, Heejin Lim, Sun Young Lee, Cheol Song, JiWon Park, Hyeon Ho Shin, Dong-Kwon Lim, and Dae Won Moon ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02620 • Publication Date (Web): 11 Jun 2019 Downloaded from http://pubs.acs.org on June 12, 2019

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ACS Applied Materials & Interfaces

Graphene-Coated Glass Substrate for Continuous Wave Laser Desorption and Atmospheric Pressure Mass Spectrometric Imaging of Live Hippocampal Tissue Jae Young Kim§,†, ‡, Heejin Lim§,†, Sun Young Lee†, Cheol Song‡, Ji-Won Park∥, Hyeon Ho Shin ⊥,

Dong-Kwon Lim*,⊥, and Dae Won Moon*,†

†Department

of New Biology, Daegu Gyeongbuk Institute of Science and Technology (DGIST),

Daegu, Republic of Korea. ([email protected]) ‡Department

of Robotics Engineering, Daegu Gyeongbuk Institute of Science and Technology

(DGIST), Daegu, Republic of Korea. ∥Graduate

School of Analytical Science and Technology (GRAST), Chungnam National

University, Daejeon, Republic of Korea. ⊥KU-KIST

Graduate School of Converging Science and Technology, Korea University, 145

Anam-ro, Seongbuk-gu, Seoul, Republic of Korea. ([email protected])

KEYWORDS: Mass spectrometry imaging, Graphene-coated substrate, Laser desorption, Ambient mass spectrometry, Hippocampal tissue.

ACS Paragon Plus Environment

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ABSTRACT Atmospheric pressure mass spectrometric (AP-MS) imaging technology combined with an inverted optical microscopic system is a powerful tool for determining the presence and spatial distributions of specific biomolecules of interest in live tissues. Efficient desorption and ionization are essential to acquire mass spectrometric (MS) information in an ambient environment. In this study, we demonstrate a new and efficient desorption process using a graphene-coated glass substrate and a continuous wave (CW) laser for high-resolution AP-MS imaging of live hippocampal tissue. We found that desorption of biomolecules in a live tissue slice was possible with the aid of a graphene-coated glass substrate and indirect application of a 532 nm CW-laser on the graphene substrate. Interestingly, the desorption efficiency of live tissue on the graphene-coated substrate was strongly dependent on the number of graphene layers. Single-layer graphene was found to be the most sensitive substrate for efficient desorption and reproducible high-resolution hippocampal tissue imaging applications. The subsequent ionization process using nonthermal plasma generated sufficient amounts of molecular ions to obtain highresolution 2-dimensional MS images of the cornu ammonis (CA) and the dentate gyrus (DG) regions of the hippocampus. Therefore, graphene-coated substrates could be a promising platform to induce an efficient desorption process essential for highly reproducible ambient MS imaging.


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1. INTRODUCTION Atmospheric pressure mass spectrometric (AP-MS) imaging technology has been noteworthy as a simple, fast, and accurate chemical imaging method, and many technological developments have been achieved for its use in analyzing biological specimens.1-8 AP-MS relies on charged molecules from the desorption and ionization process in atmospheric environments (pressure and humidity) to introduce them into a mass analyzer. Because a specimen is not exposed to vacuum conditions, AP-MS can greatly reduce the possibility of deformation of its original state as compared with a vacuum-based MS instrument.3,9-13 Therefore, AP-MS has been investigated for various biotechnology research or in-situ diagnosis applications. In particular, one of the main challenges is to improve the AP-MS technique for rapidly and intuitively analyzing biological specimens containing moisture, and various engineering attempts have been made to achieve mass spectrometric (MS) imaging with high spatial information.14-18 We recently reported a micrometer resolution AP-MS imaging system for mass-based live hippocampal tissue imaging.19,20 We found that efficient desorption was possible by the use of a focused pulsed laser and rod-shaped gold nanoparticles. When coupled with a subsequent ionization process using nonthermal plasma, high-resolution hippocampal MS imaging was able to identify the chemical information of interest.19 More recently, we advanced this method with the use of a continuous wave (CW) laser, in which gold nanoparticles (AuNPs), graphene oxide (GO), or reduced graphene oxide (r-GO) solution were used to accelerate and enhance the desorption process.20 In these methods, the use of nanomaterials was found to be critical for the laser-induced desorption process. However, an incubation step for live tissue of 1.0–3.0 h to evenly distribute nanomaterials on the whole tissue surface is a time-consuming and delicate step. The failure of uniform distribution of nanomaterials on the tissue surface for any reason causes a


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failure to obtain the information of interest. In this regard, an alternative method without the incubation step with nanoparticles is required for more straightforward and universal use of an AP-MS system for various biological samples.21,22 In this work, we investigated the use of a graphene-coated substrate for AP-MS imaging of live tissues with a 532-nm CW laser. We found that the use of graphene-coated glass substrates for AP-MS imaging of live tissues is compatible with the previous method reliant upon the nanoparticle incubation step before MS imaging. Interestingly, the desorption efficiency of live tissue with a CW laser was strongly correlated with the number of graphene layers on the glass. The single-layer graphene-coated glass substrate was excellent for efficient desorption of live tissue with 200-μm thickness, which enabled micrometer spatial resolution of hippocampal live tissue imaging with the CW laser.

2. EXPERIMENTAL SECTION 2.1 Materials Polymethylmethacrylate (PMMA, average Mw ~996,000, crystalline) was purchased from Sigma Aldrich (St. Louis, MO, USA). Anisole and ammonium persulfate ((NH4)2S2O8) were purchased from Daejung Chemical & Materials Corporation (South Korea). Graphene-covered Cu foil was purchased from Graphene Platform Corporation (Japan). All materials for sucrose artificial cerebral spinal fluid (sACSF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Poly(ethylenimine) solution (50% (w/v) in H2O) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2 Preparation of single and multiple graphene layers on a glass substrate


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For the single-layer graphene-coated glass substrate, a PMMA solution (average MW ~996,000, 4.0 %(w/w) in anisole) was spin-coated onto the surface of the graphene covered Cu foil (single layer, Graphene Platform Corp., Japan) at 4,000 rpm for 45 s and dried in air. A 0.1 M aqueous solution of (NH4)2S2O8 was used to remove the Cu foil. The PMMA polymer-supported graphene was then rinsed in deionized water several times and transferred onto a glass slide. The sample was dried in air for 30 min and annealed at 180 ˚C for 3 h. The polymer was then dissolved by immersion in acetone for 30 min and rinsed in methanol for 10 min and in isopropanol for 10 min, respectively (Figure 1A).23 For the preparation of the multiple-layer graphene-coated glass substrate, the PMMA polymersupporting graphene prepared in the same manner as described above was transferred onto another graphene-covered Cu foil and dried at ~50 ˚C for 10 min. Then, a PMMA-supported graphene-layers-covered Cu foil was floated on a 0.1 M aqueous solution of (NH4)2S2O8 to remove the Cu foil. This transfer process can be repeated to control the number of graphene layers.24,25 The polymer-supported multiple-layer graphene was then rinsed in deionized water several times and transferred onto a glass slide. In the same manner, the sample was dried in air for 30 min and annealed at 180 ˚C for 3 h. The polymer was then dissolved by immersion in acetone for 30 min and rinsed with methanol for 10 min, then with isopropanol for 10 min, respectively.

2.3 Mouse hippocampal tissue preparation The live tissue slice has been a well-established experimental model in various research areas such as neurophysiology, pathophysiology, and electrophysiology. In particular, hippocampal tissue slices have been utilized for studying stretch-activated ion channels (SACs) and


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microelectrode arrays (MEAs) of various organotypic tissues. Live hippocampal tissue slices for our study were prepared with the sample preparation protocol used in the SAC and MEA studies.26,27 Male 7-week-old C57BL/6 mice were purchased from Koatech (Pyeongtaek, South Korea). We followed the guidelines for animal use and care in the Daegu Gyeongbuk Institute of Science & Technology (DGIST, South Korea). The primary purpose of developing ambient MS imaging is to observe live or fresh tissues with a minimal sample deformation. Immediately after sacrifice, the brain was extracted, and the isolated hippocampus was sliced transversely with a tissue chopper (Mcllwain Tissue Chopper, Cavey Laboratory Engineering, UK) at a thickness of 200 to 500 µm according to the experimental protocol. It is very difficult to obtain hippocampal tissue slices of less than 200 μm without freezing or paraffin-fixation. In addition, a thickness of more than 200 μm is also required to maintain a short-term living tissue state. Oxygenated sucrose artificial cerebral spinal fluid (sACSF) was used to aerate the hippocampal tissue slices (sACSF preparation: a solution containing 124 mM NaCl, 2.5 mM KCl, 24 mM NaHCO3, 5 mM HEPES, 2 mM MgSO4, 2 mM CaCl2, 12.5 mM D-glucose, and 4 mM sucrose was bubbled with 95 % O2/5% CO2 using an aquarium bubbler at 32 °C). After 1 h of aeration, the hippocampal tissue slice was washed five times and placed on an O2 plasma-pretreated graphene-coated substrate for tissue adhesion. For AuNP-treated hippocampal slices, the aerated slices were submerged in sACSF solution (5 mL) with citrate-AuNPs (0.5 nM, 20 nm) for 3 h. Then, the hippocampal slice was washed ten times and placed on an O2 plasma-treated graphene-coated glass slide (Figure 1B).

2.4 AP-MS imaging system setup The previously developed AP-MS imaging system was used in this study with a change of the optical path from the laser source.19,20 The AP-MS imaging system consisted of a mass


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spectrometer, a 532-nm CW laser, a scanning stage, an atmospheric pressure (AP) plasma device, and airflow-assisted ion transport equipment, as shown in Figure 2 (schematics) and Figure S1 (picture). The mass spectrometer is a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). (MS parameters for the full scan: positive ion mode, mass range (100– 1000 m/z), mass resolution (35,000 FWHM), maximum injection time (100 ms), one microscan, automatic gain control (106), capillary temperature (350 °C)). (MS parameters for the tandem scan: mass resolution (17,500 FWHM), high-energy collisional dissociation with normalized collision energy (20 to 80), isolation window (0.4 m/z), automatic gain control (5.0 × 104), maximum injection time (200 ms)). The diode-pumped solid-state laser system (MGL-III-532, CNI Optoelectronics Tech, China) was used to generate CW laser light (532 nm, 300 mW). A graduated neutral density (ND) filter was used to control the input laser power. A dichroic beam splitter (NFD01-532-25x36, Semrock, USA) was used to monitor and to induce the desorption of a biological specimen simultaneously. The inverted optical microscope (IX73, Olympus, Japan) with an objective lens (20×, 0.45 NA) was used to determine the region of interest and to guide the laser light for desorption. A motorized XY scanning stage (AS-MIX73-C, iNexus, South Korea) was used to scan a tissue slice. The motion control program of the sample stage was used to control the scanning stage, the mass analyzer, and the optical shutter for lasers simultaneously, as shown in Figure 2.19,20 The AP plasma jet device consisted of a dielectric tube having two tubular electrodes, which was a typical dielectric barrier discharge device with a small discharge current.19,20 The tube was made of quartz glass with an inner diameter of 2 mm and an outer diameter of 3 mm, surrounded


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with two copper electrodes with a length of 6 mm. The distance between the inner edges of two electrodes was 7 mm, and the electrode on the downstream side was 5 mm apart from the tube orifice. The plasma plume was generated by applying helium gas (99.999%, flow rate: 0.5 slm) and a sinusoidal voltage (5 kVp and 27 kHz frequency). The helium plasma jet was applied to the sample with a 30° angle of incidence. The desorbed neutral molecules produced by the focused laser essentially collide with the helium plasma. The metastable helium species in the plasma readily react with water, oxygen, or other components in the air to generate reactive protonated water clusters. A possible mechanism for ionization is that the desorbed biomolecules with proton affinities greater than water can be ionized by these protonated water clusters.28,29 The airflow-assisted ion transport equipment requires a gas pumping system coupled with the mass spectrometer. The equipment consisted of an ion transfer tube, a chamber, and a diaphragm pump designed to transfer the ionized molecules to the mass analyzer. Even though a considerable number of biomolecules desorbed by the focused laser were directed to the MS inlet using the ion transport equipment, only a small fraction of the desorbed biomolecules was ionized by reactive protonated water clusters and metastable helium species. Therefore, carryover is not a significant problem in this AP-MS system.

2.5 Instruments for Raman and helium ion microscopy (HIM) Raman spectra were acquired using an inverted Raman microscope (NOST, South Korea) with a 60× objective (NA 0.7) (Olympus, Tokyo, Japan). The sample was excited using a 532-nm laser to characterize the graphene substrate. The scattered Raman signal was detected with a confocal motorized pinhole (100 μm) directed to a spectrometer (FEX-MD, NOST, South Korea)


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(600 g mm-1 grating). EzScan (NOST, South Korea) software was used for the spectra acquisition and analysis. Helium ion microscopy (HIM) (Orion NanoFab, Carl Zeiss, USA) was used to obtain the surface morphology of the tissue slices. No conductive coating was required for HIM analysis. (Instrument setting for HIM: beam energy: 30 keV, beam current: 1.0–1.98 pA, average scan number: 16, working distance: 10.20 mm, gas field ion sources (GFIS) aperture size: 20 μm, scan dwell time: 2 μs, field-of-view: 700 μm).

3. RESULTS AND DISCUSSION 3.1 Preparation and characterization of graphene coated glass substrate The typical transfer method for graphene onto the glass substrate is suitable to prepare a controlled number of graphene layers on a glass substrate.23-25 The Raman analysis showed a characteristic Raman spectrum of graphene on glass as shown in Figure 3A. The G peak at 1,580 cm-1 is the signature peak produced by the in-plane phonon mode of all graphite materials.25 The intensity of the G peak is proportional to the number of graphene layers (< 18 layers). 25 The D peak at 1,350 cm-1 is related to a defect in the graphene. The shape of the 2D peak at 2,700 cm-1 can be varied with an increasing number of graphene layers because of the increased inelastic scattering by phonon. As shown in Figure 3A, the Raman spectra of the single graphene layer showed no structural defects and matched well with published results. 23-25 The 2D/G ratio of the Raman spectra of graphene layers was sharply decreased with an increased number of graphene layers. The 2D/G ratios of the Raman spectra of one, two, three, five, and ten graphene layers were found to be 2.48, 1.47, 0.84, 0.61, and 0.53, respectively.


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3.2 Graphene layer dependence on the desorption efficiency of a transmission-mode CW laser The uniform desorption of live tissue slices is critical for reliable MS imaging. To demonstrate the possibility of combining a transmission-mode CW laser and a graphene layer for efficient desorption, single-line scanning was performed on the hippocampal tissue slice (200 μm thickness) placed on a graphene-coated glass substrate by applying a 532 nm CW laser (20×, 0.45 NA objective lens) with varying power input (50–300 mW). The actual laser power on the sample was measured to be 50 % of the input power, and the diameter of the laser beam focused on the sample was calculated to be 1.442 μm using airy disc size. The input power of 50, 75, 100, 200, and 300 mW of the CW laser corresponded to the laser power density of 15, 23, 31, 61, and 92 mW/μm2, respectively. Figure 3B shows the summarized results for the line width of hippocampal tissue slice ablated by laser line scanning on the different numbers of graphene layers and laser power. The line width represents the mean value of the desorbed line from HIM images in Figure 3C-3H. The HIM images clearly showed the line of the desorbed mouse hippocampal tissue slice depending on the laser power and the number of graphene layers. No desorption was found in the absence of graphene layers on the glass substrate (Figure 3C), even in the case of applying the highest laser input of 300 mW (92 mW/μm2). However, the tissue slice on a single-layer graphene-coated substrate showed a highly uniform and narrow desorption pattern when the CW laser was applied with an input power higher than 75 mW (Figure 3D). However, non-uniform desorption patterns were observed in the case of applying the laser with a power lower than 75 mW (23 mW/μm2), as shown in Figure 3D (75 and 50 mW). The tissue slices on bi- and tri-layered graphene substrates showed reproducibly uniform desorption patterns when applied with a 200-mW input


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laser (61 mW/μm2) (Figure 3E and 3F). However, low laser power input (< 200 mW) caused irreproducible desorption patterns. As the number of graphene layers increases, higher laser power is required for successful desorption. As shown in HIM images obtained with five- or tenlayer graphene-coated substrates (Figure 3G and 3H), high laser power (at least 200 mW) is required for successful desorption of hippocampal tissues. These results indicate the desorption process for tissue samples does not solely rely on light absorption by graphene but also relies on the energy transfer process from the graphene layer to the tissue sample. This finding is probably because the transmittance of the visible light decreases,30-32 as the number of graphene layers increases and laser light with higher energy is required for sufficient desorption of the biological specimen. Therefore, it is confirmed that a single-layer graphene coated substrate is the most effective and desirable substrate for ambient laser-induced desorption.

3.3 Desorption characteristics (graphene coated glass versus AuNPs) To investigate the differences in desorption behaviors by the use of AuNPs or a graphene-coated substrate, we performed single line scanning with a CW laser (200 mW) for the tissue slice (200 μm thickness) after incubation with AuNPs or a fresh tissue slice on the single layer graphenecoated substrate. The HIM and the confocal laser microscope image in Figure 4A and 4B showed desorbed lines with narrow bottom widths (5.4, 4.8 μm) and top widths (8.4, 7.2 μm) (Figure 4B). The scanned lines of the hippocampal tissue on single-layer graphene also showed a successful result of desorption with a micrometer resolution (bottom widths 7.9, 8.8 μm, top widths 11.3, 15.0 μm) (Figure 4C and 4D). The line width of the tissue slice obtained from the graphenecoated glass was wider than that obtained with AuNP treatment. This results from the direction of the desorption process. The graphene layer lying in the bottom of the tissue slice first absorbs


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the laser light and transfers thermal energy to the adjacent tissue area, the tissue at hundreds of micrometers thickness was ablated upward, and the ablated pattern was rough, as shown in Figure 4C and 4D. For this reason, the desorption efficiency can be significantly affected by tissue slice thickness. To investigate the thickness and laser power input dependence on the desorption efficiency, tissue slices with four different thicknesses (200, 300, 400, and 500 μm) were prepared and single line scanning performed on a single-layer graphene-coated substrate with varying CW laser power inputs (50, 75, 100, 200, and 300 mW (15, 23, 31, 61, and 92 mW/μm2)). The 200 μm thick slice showed successful desorption with the 75 mW input laser (Figure 5A), but the tissue slices of 300 or 400 μm thickness required 100 mW of laser power to obtain a uniform and narrow desorption efficiency (Figure 5B, 5C). The 500 μm thick tissue slices required at least 200 mW of laser power input for successful desorption (Figure 5D). Therefore, we posited that a 200 μm tissue slice thickness and a 100 mW laser power input are the most desirable conditions for an ambient MS imaging system. Compared with previously reported MS analysis studies using a laser absorption layer as a sample substrate,33-37 the transmission-mode CW laser configuration and the use of a graphene-layer substrate showed greatly improved laser desorption performance.

3.4 High spatial resolution mass spectrometric imaging of live hippocampal tissue In the hippocampal tissue, pyramidal cells in the cornu ammonis (CA) and granule cells in the dentate gyrus (DG) are two major neuronal cell types with different lipid and metabolite compositions.38,39 Thus, CA1 and DG regions of the mouse hippocampus were analyzed as a model for MS imaging. The CW laser was applied on an area of 600 μm × 500 μm (433 × 100 pixels) (single pixel size: 1.4 μm × 5.0 μm). The total acquisition time was 117 min. Since the


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nonthermal AP-plasma device was used as an ionization source, the plasma background spectra should be removed from the entire mass spectra after obtaining the mass spectra from the tissue slice as shown in Figure 6. The Human Metabolome Database (www.hmdb.ca/) was used to identify the obtained mass spectra from the tissue slice. The tentative assignment of ion peaks was displayed in the supporting information (Table 1). This ion assignment was also confirmed by a comparison of tandem MS data with the corresponding standard materials (Figure S2). Several monoacylglycerol (MAG) ions, MAG 16:1, MAG 16:0, MAG 18:1, MAG 18:0, and MAG 18:2, and adenine, sphingosine, sphinganine, and ceramide 18:0 could be identified based on their chemical formulas and confirmed in the form of [M+H]+ and [M+H-H2O]+. As is wellknown, cholesterol ions were found in the form of [M-H]+ and [M+H-H2O]+.40,41 This is probably related to the ionization mechanism producing the reactive protonated water clusters and the experimental environment. It should be noted that the ambient environment and the wet state of the tissue slice in this MS imaging. The two-dimensional MS images of the hippocampal tissue were obtained by transforming the MS data using BioMAP software (Novartis Institutes for BioMedical Research, Cambridge, USA) as shown in Figure 7. Figure 7A–C shows selected ion images obtained from a hippocampal tissue slice on the graphene-coated substrate and Figure 7D–F shows ion images from a hippocampal tissue slice following treatment of AuNPs. To obtain the hippocampal tissue ion images, a 200 mW (61 mW/μm2) CW laser was used for both MS analyses shown in Figure 7. After MS analysis with the CW laser, the scanned area showed some destruction, indicating that an efficient desorption process occurred in this area (Figure 7A, 7D). The intensity of selected ion-species such as monoacylglycerols (16:1, 16:0, 18:1, 18:0), adenine, cholesterol, sphinganine, and ceramide 18:0 from each pixel were used to construct the MS images. In both


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cases, the ion image of adenine (m/z = 136.061) can be divided into the apical and basal dendritic areas in the CA1, which were located on the inside and outside of the soma location, respectively (Figure 7B, 7E). In the CA1 region, most of the lipid ions except for adenine were mainly distributed in the apical and basal dendrites, and the distribution of adenine is dominant in the cell body area. Such a distribution tendency was more clearly observed in the DG (Figure 7C, 7F), where lipid ions are distributed in lesser amounts in the cell bodies and mainly distributed in dendrites. These micrometer resolution molecular ion images can be used to study the molecular composition and distribution between the CA1 and DG regions at the tissue level. To investigate the spatial resolution of the AP-MS image, we used a zebrafish (Danio rerio) caudal fin consisting of structurally separated bony rays, blood vessels, and inter-ray mesenchymal tissue (Figure S3). When the mass spectrometry analysis is performed with the caudal fin on the graphene-coated substrate, an MS image based on the adenine peak (m/z = 136.061) showed a very distinct boundary around the bone ray area (Figure S3-A inset). The xaxis line profile of intensity from the MS image enabled determination of the lateral spatial resolution. Spatial resolution can be defined as the distance across the signal change from 16% to 84% of the maximum intensity.42,43 As a result, the lateral resolution is measured to 2.8–3.5 μm when the x-axis direction velocity is 10 μm/s.

3.5 Graphene-coated slide substrate for highly reproducible MS imaging With the use of a graphene-coated substrate, the sample preparation time can be shortened because the additional 3 h incubation process required for AuNPs is unnecessary. After the sacrifice of the adult mouse, the hippocampal tissue is extracted and sliced, and the MS analysis can be started immediately after aeration (to remove dead cells), which allowed MS imaging of the fresh tissue slice. The optical images of the tissue slice incubated with AuNPs clearly showed


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the formation of cracks between the CA1 and DG regions as shown in Figure 7D. Although the formation of cracks following the AuNP incubation step does not always occur, this phenomenon can increase the failure rate of sample preparation. In addition, when AuNPs are used as a light-absorbing material for lasers, it is necessary to ensure the uniform distribution of AuNPs on the tissue slice to obtain a successful MS image.19 However, when a graphene-coated substrate is used as the light-absorbing layer, the potentially confounding distribution of AuNPs on the tissue slice is not an issue. Moreover, the graphenecoated substrate can be applied to various samples that cannot take up nanomaterials, such as dried and fixed tissue samples. The standard material solution for MS identification can also be analyzed using this proposed AP-MS method. The desorption patterns of the tissue slice on the graphene-coated substrate were rough compared with the AuNP-treated specimens (Figure 7A, 7D), whose MS images were similar to each other and not particularly coarse. The quality of the MS image, such as pixel size and spatial resolution, is mainly determined by the scan setting, including the rastering speed of the focused laser, the spacing between the x-scan lines, and the internal scan rate of the mass spectrometer. Therefore, the roughness of the microscopic desorption pattern did not affect the quality of the MS image. In both cases, it can be observed that the tissue slice successfully absorbs the applied laser light, and then an effective thermal desorption process occurs.

4. CONCLUSIONS Our results show that the efficient desorption of molecules is possible by the use of a single-layer graphene-coated substrate. Compared with the previously reported method relying on the nanomaterial incubation step with tissue slices, the current method is simpler and reliable for various ambient mass-based imaging applications. The graphene-coated substrate can be


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prepared by a simple transfer procedure in the laboratory and is ready-to-use as soon as the tissue slice is prepared, which can greatly decrease the overall analysis time without inducing any variation in imaging quality. The use of a graphene-coated substrate can avoid potential problems such as the formation of cracks on the tissue slice following the incubation step required for AuNPs. The inverted microscopy system with a scanning stage is also an important part of the current ambient mass imaging system. The focused laser light is directly applied to the laser absorbing layer (graphene) rather than the tissue slice, which can greatly increase the desorption efficiency. Thus, even if the tissue slice is prepared at a 500-μm thickness, it can be sufficiently desorbed at a laser output of 200 mW. Moreover, using a graphene-coated substrate, it is possible to analyze various tissue slices of interest that are difficult to pretreat with nanoparticles, thereby enabling wide applicability of the proposed AP-MS method.


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Figure 1. Fabrication of the graphene-coated glass substrate and the CW-laser-based MS system for live hippocampal tissue imaging. (A) Experimental procedures to prepare the graphenecoated glass substrate for hippocampal tissue imaging. (B) Schematic description for the ambient MS system combined with an inverted optical microscopic system equipped with a focused (20×, 0.45 NA objective lens) CW laser (532 nm) for desorption and AP helium plasma for ionization.


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Figure 2. Schematic description of the AP-MS imaging system used in this study. The AP-MS imaging system consists of (i) a mass spectrometer, (ii) a scanning stage, (iii) a 532-nm CW laser, (iv) an AP plasma device, and (v) airflow-assisted ion transport equipment.


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Figure 3. Raman analysis of graphene-coated glass substrates and desorption efficiency depends on the number of graphene layers and the applied CW laser power (50-300 mW). (A) Raman spectrum of the graphene-coated glass substrate. (B) The summarized line width of desorbed hippocampal tissue obtained from HIM images in (C-H). (C-H) HIM images of hippocampal tissues after applying the CW laser to hippocampal tissue on a glass substrate without a graphene layer. (C) A single graphene layer (D), a graphene bi-layer, (E), a graphene tri-layer (F), fivelayered graphene (G), and ten-layered graphene (H). (Scale bar in C-H is 100 μm, the input CW laser powers of 50, 75, 100, 200, and 300 mW correspond to 15, 23, 31, 61, and 92 mW/μm2).


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Figure 4. Helium ion microscopy (HIM) image (A) of AuNP-treated hippocampal tissue and its confocal fluorescence image (B) after line scanning with a 532-nm CW laser. Helium ion microscopy (HIM) image (C) of hippocampal tissue on single-layer graphene (SLG) substrate and its confocal fluorescence image (D) after line scanning with a 532-nm CW laser. (A confocal microscope (LSM-700, Carl Zeiss, Germany) was used to obtain images). The scale bar in A, C is 10 μm.


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Figure 5. Helium ion microscopy (HIM) images of hippocampal tissue on a single-layer graphene-coated substrate. (A) 200 μm, (B) 300 μm, (C) 400 μm, and (D) 500 μm hippocampal tissue thickness. Scale bar=100 μm. Input CW laser powers of 50, 75, 100, 200, and 300 mW correspond to power densities of 15, 23, 31, 61, and 92 mW/μm2.


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Figure 6. Positive ion mode AP-mass spectra from a mouse hippocampal tissue slice. (A) APmass spectra from hippocampal tissue with a helium plasma background in the range of m/z = 100–500. (B) Plasma background-subtracted mass spectra from hippocampal tissue.


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Figure 7. Micrometer-resolution MS imaging of adult mouse hippocampal tissue slices. (A) Photo of adult mouse hippocampal tissue on the graphene-coated substrate before and after MS analysis. Selective ion images for the CA1 region (B) and the DG region (C) of the hippocampus (A). (D) The photo of adult mouse hippocampal tissue before and after MS analysis with the treatment of gold nanoparticles. Selective ion images for the CA1 region (E) and the DG region (F) of the hippocampus (D). Scale bar = 500 μm.


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ASSOCIATED CONTENT Supporting Information. Illustration of the AP-MS imaging system; Tandem MS validations of representative ion peaks and corresponding standard materials; Lateral resolution test of AP-MS imaging; Tentatively assigned ions from mouse hippocampal tissue

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] Author Contributions §J.Y.K.

and H.J.L. contributed equally to this work. The manuscript was written with

contributions from all authors. All authors have confirmed approval of the final version of the manuscript. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the Basic Science Research Programs through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2016R1A6A3A11930198, NRF-2016R1A2B3013825, 2018R1A2A3075499), the Ministry of


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Trade, Industry, & Energy (MOTIE, Korea), the Ministry of Science & ICT (MSIT, Korea), and the Ministry of Health & Welfare (MOHW, Korea) under the Technology Development Program for AI-Bio-Robot-Medicine Convergence (20001533) and the KU-KIST school project.

ABBREVIATIONS AP, atmospheric pressure; AP-MS, atmospheric pressure mass spectrometry; AuNP, gold particles; CA, cornu ammonis; CW, continuous wave; DG, dentate gyrus; GFIS, gas field ion sources; GO, graphene oxide; HIM, helium ion microscopy; MEA, multielectrode array; MS, mass spectrometry; ND, neutral density; PMMA, polymethylmethacrylate; r-GO, reduced graphene oxide; SAC, stretch-activated ion channel; sACSF, sucrose artificial cerebral spinal fluid; SLG, single-layer graphene

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Graphene-Coated Glass Substrate for Continuous Wave Laser

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