Direct Three-Dimensional Mass Spectrometry Imaging with Laser Ablation Remote Atmospheric Pressure Photoionization/Chemical Ionization

The laser ablation remote atmospheric pressure photoionization/chemical ionization (LARAPPI/CI) platform coupled to an ultrahigh resolution quadrupole-time-of-flight (QToF) mass spectrometer was developed and employed for the first direct three-dimensional (3D) mass spectrometry imaging (MSI) of metabolites in human and plant tissues. Our solution for 3D MSI does not require sample modification or cutting into thin slices. Ablation characteristics of an optical system based on a diffraction optical element are studied and used for voxel stacking to directly remove layers of tissues. Agar gel, red radish, kiwi, human kidney cancer, and normal tissue samples were used for the tests of this new system. The 2D and 3D ion images vividly illustrate differences in the abundances of selected metabolites between cancerous and noncancerous regions of the kidney tissue and also between different parts of plant tissues. The LARAPPI/CI MSI setup is also the first example of the successful use of combined dopant-assisted atmospheric pressure photoionization (DA-APPI) and atmospheric pressure chemical ionization (APCI) ion source for mass spectrometry imaging.


■ INTRODUCTION
Mass spectrometry imaging (MSI) has emerged as an essential technology that offers the simultaneous analysis of a broad spectrum of molecular species with unparalleled chemical specificity. 1This technique excels in its ability to identify a diverse array of molecules, including endogenous and exogenous compounds, without the need for labels in a single experiment on the same tissue section. 2 It enables the detailed mapping of molecular distributions, the identification of posttranslational modifications, and the acquisition of relative quantitative data across various samples. 3−7 Three-dimensional (3D) MSI advances beyond 2D MSI by providing the ability to profile the depth of biological samples, thereby enabling the mapping of biomolecules in three dimensions within tissues and organs.It is extremely challenging for mass spectral imaging to map molecular composition in 3D.MS imaging nowadays is performed almost exclusively in 2D mode or by reconstruction of 3D objects by software-stacking of many 2D results. 8For the realization of 3D software reconstructed MSI, tissues or organs are first sectioned in series.These sections are subsequently imaged in two dimensions and reconstructed into three-dimensional images.Measurement techniques used for the abovementioned analyses are usually desorption electrospray ionization (DESI), 9 nanospray desorption electrospray ioniza-tion (Nano-DESI), 10 laser ablation electrospray ionization (LAESI), 11 and matrix-assisted laser desorption ionization (MALDI). 12ost frequently, 3D-reconstructed MSI studies are performed with the use of matrix-assisted laser desorption/ ionization mass spectrometry (MALDI MS). 13 MALDI MS has some limitations, such as a crowded chemical background in the low-mass region if low resolving power instrumentation is used 14 or not optimal ionization of neutral organic compounds. 8Additionally, commercial instruments suitable for MALDI MSI do not have the possibility of sample freezing.
There are also reports of 3D-reconstructed MSI results made with the use of DESI. 9 One of the greatest advantages of sample preparation prior to analysis.Until recently, a major limitation of DESI was its low resolution, typically ranging from tens to hundreds of micrometers. 15However, the introduction of nano-DESI allowed for achieving resolution up to 6 μm. 16Controlling the distance between the nano-DESI probe and the sample surface is critical to achieving high resolution but is also technically challenging to maintain, especially for thinner tissue sections. 17Despite significant improvements, the ionization efficiency in nano-DESI may still be insufficient for some types of analysis, particularly when analyzing complex biological matrices.A common issue with nano-DESI is the stability of solvent flow, which is dependent on the inlet vacuum of the mass spectrometer.A major limitation of DESI is the small depth of penetration of liquid into the sample, which is typically limited to a few micrometers.For example, DESI imaging did not cause physical damage to the underlying cells on the algal surfaces. 18lthough the sample removal rate of DESI is similar to that of secondary ion mass spectrometry (SIMS) 19 analytes are removed selectively, which does not allow depth profiling for three-dimensional imaging -a capability that can be achieved with SIMS 20 and LAESI. 21ethods reported in the literature as capable of direct, nonsoftware reconstructed 3D MS imaging include SIMS, 22 which uses energetic ion bombardment to erode the surface of a sample. 23SIMS-based time-of-flight (ToF) MSI is capable of depth profiling molecular content with 10 nm depth resolution.There are also reports of 60 nm depth profiling, but heavy molecular fragmentation is observed. 24,25In 2020, Zhang et al. 23 presented Cryo-OrbiSIMS for 3D molecular imaging of a frozen bacterial biofilm.It was possible to perform depth profiling by removing up to 30−50 μm of the object.With a shallow ablation depth, it is impossible to obtain a deep profile of macroscopic objects.The necessity of a high vacuum inside the system prevents the analysis of biological objects without risking deformation.Another approach was published with the use of an extreme ultraviolet (EUV) laser. 26The authors achieved submicrometer resolutions, achieving a lateral resolution of 75 nm and a depth resolution of 20 nm.
The laser ablation remote atmospheric pressure photoionization/chemical ionization (LARAPPI/CI) 2D and 3D MSI platform presented in this work provides a solution to various problems associated with 3D MSI, such as reconstruction of 2D to 3D models, migration of metabolites, sample dehydration, evaporation of metabolites, a large volume of ablated material, and occurrence of artifacts from mechanical section preparations.Also, this allows for control shape and depth of the ablated surface, providing 3D results.The proposed solution should also be compatible with mass spectrometers that use common electrospray ionization (ESI) ion sources; it is not compatible with instruments with vacuum ion sources.The integrated 3D distance sensor acts as a surface profilometer, allowing for ablation control during the experiments.

■ RESULTS AND DISCUSSION
Direct three-dimensional MSI is considered one of the most useful analytical methods today.The possibility of detection of hundreds of compounds within a single microscopic space (voxel) on the surface (2D MSI) or inside (3D MSI) of the studied sample, and then their localization in the object, gives unlimited possibilities to biologists, biochemists, and material chemists.To perform such analysis, the method is capable of removing relatively large volumes of material in a strictly controlled manner, and also on the microscale, and quickly transferring this material to the mass spectrometer.Precise removal of entire layers of material is needed to access the lower parts or layers of the analyzed samples.
Description of the Experimental Setup.In the LARAPPI/CI MSI system, computer-controlled ablation takes place in a pressure chamber working at atmospheric pressure.The sample is placed on a sample stage (Figure 1I) with a built-in Peltier cooling plate that allows for freezing of the sample for the experiment.The temperature-controlled sample stage is mounted on a motorized high-speed XY stage.The pulsed beam from the OPO laser (Figure 1A) of 2930 nm wavelength expanded 3.75 times is redirected toward the sample stage by a gold-plated mirror (Figure 1D), goes through the diffractive optical element, and is focused onto the sample surface by a 50 mm focal length aspherical lens.
The system also contains a camera and a high-precision distance sensor acting as a profilometer.During imaging, the laser focal point remains fixed in space, while the sample (Figure 1H) is moved by the computer-controlled XY-and Zstages.A specially designed gas funnel (Figure 1E), also a focusing assembly, is placed on the ablation site.The overpressure in the chamber drives dry nitrogen gas with ablated material to the ion source.The samples are kept at subzero temperatures during analysis to keep their shape unchanged and prevent metabolites from migration.
LARAPPI/CI uses diffractive optical elements produced by HOLO/OR for the generation of a square-focused beam with a flat top profile. 27The difference in ablation crater shape for the modified beam can be seen in the laser printer paper test results shown in Supporting Information S1.
A graphical representation of the volume removed by this setup is presented in Figure 2A (blue or yellow).As can be seen, a single-voxel shape is part of a pentahedron with rounded side edges.As can be seen in Figure 2A, using laser ablation to remove these shapes leaves some material between them.Oversampling with stacked voxels as presented in Figure 2B improves material removal during multivoxel ablations.To perform ablation without the removal of upper layers due to the fixed shape of the focused laser beam, we introduced an inverted pyramid ablation scheme (Figure 2D).
Single point ablation with 20 laser pulses of agar gel enriched for opacity in titanium dioxide nanopowder produced the desired square shape with rounded edges (170 × 170 μm size) as shown in the optical photograph in Figure 2E.Optimization of oversampling (shown in Supporting Information S7) suggested that 140 μm voxel-center-to-voxel-center (please see Figure 2B−D) produced optimal results with a relatively flat bottom of 7 × 7 × 1 voxel ablation area (depth 330 μm) as judged by optical microscope observation and also profiling with a distance sensor (Figure 1F).Additional ablation results of agar gel of 11 × 7 (X × Y, top-level resolution) space are shown in Supporting Information S2. Figure 2G presents a side view of the inverted pyramid ablation scheme.As can be seen, the layers are ablated with the number of lines in the X and Y axes reduced by 1 for every ablation layer below the first one (example: first-layer resolution, 40 × 40, second 39 × 39, etc.).Additionally, the voxel pattern of each lower layer is shifted to the center of the ablated region.This ablation scheme allows avoidance of ablation of walls of upper layers, which is one of the biggest problems in 3D MSI. 11The undesirable ablation of upper layers in SIMS was partially solved by the utilization of two ion beams, the first beam ejects atoms, molecules, and secondary ions from the surface, while the second beam sputters the already analyzed surface to create a new plane for imaging the next layer. 28Theoretically, a similar solution could be possible for laser systems; however, for complete removal of material, it would still require advanced beam shaping and beam direction control, making it much more complicated and expensive than the solution presented in this work.Each 3D MSI experiment presented below was performed in an inverted pyramid mode (Figure 2D,G).To present the procedure for 2D and 3D imaging in an easily understandable form, a workflow is presented in Figure 2I.
Optimization of LARAPPI/CI Working Conditions.To provide the highest sensitivity for the detection of biological compounds in microscopic-sized ablation voxels, we have modified the Bruker VIP HESI ion source in an APCI configuration to introduce a dopant-assisted APPI ionization by using a vacuum ultraviolet (VUV) lamp producing light of a wavelength in the 110−160 nm range.This ion source irradiates a pulsed stream of ablated material from the chamber that generates low-temperature plasma in the gas phase through direct interaction with biological compounds and, most likely, with toluene vapor from the toluene-methanol mixture pumped to the APCI needle by the HPLC pump (Figure 1 bottom panel).−31 During preliminary experiments, we tested electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) ionization in both positive and negative ion detection modes.After the optimal ionization type, APCI/APPI, the liquid composition, and the flow rate were also optimized.All optimizations were carried out under conditions similar to the operating mode of the MSI system; therefore, all tests were based on laser ablation of frozen agar gel containing test compounds, each compound at a concentration of 100 μg/mL.The ablated volumes calculated for each voxel were approximately 6.6 × 10 −6 mL, which equates to 660 pg of each test compound.The test compounds were polar to medium polar biological compounds: ribose, histidine, thymidine, and uracil.The results of the tests mentioned are shown in Supporting Information S4 and S5 and Table S1.As can be concluded from the data in Table S1 and Supporting Information S5, the highest S/N ratios were obtained in the APCI/APPI negative ion mode.All compounds were detected in the APCI, APCI/ APPI positive mode, and ESI/APPI negative modes.ESI/APPI results were of lower S/N compared to APCI/APPI in both positive and negative modes, which suggests that the intensity of interactions of charged droplets emitted by the ESI needle with ablated plume material is much lower compared to interactions of plume with more abundant charged gaseous species emitted in the APCI modes.
As the results shown above suggest, the best results were obtained for APCI/APPI.Optimization of the solvent mixture pumped into our modified APCI/APPI ion source was performed in a manner similar to that in the above-described experiments using agar gel laser ablation.−38 The highest average S/N value for all four compounds was for 1% toluene in methanol at a flow rate of 200 μL/min flow rate.In conclusion, the optimal ionization conditions in our MSI setup are the combination of APCI with DA-APPI.
Examples of Three-Dimensional Results from the LARAPPI/CI MSI System.The tissue of the kiwi fruit was selected as a real-life biological test for the 3D possibilities of the LARAPPI/CI MSI system.The experiment region was selected as shown in Figure 3A,B due to the placement of the seeds under a thick layer of parenchyma.−42 In the case of kiwi fruit, direct 3D MSI was performed in 6 steps or layers.The highest level of ablation was ablated at a resolution of 140 × 140 μm resolution in a 35 × 35 (X × Y) voxel arrangement with a total depth after the six ablation steps of 1.43 mm.The total number of voxels ablated in the experiment was 6355, while the ablation time took only 3 h and 20 min plus 3 min after each level for the ablation region profiling.The entire experiment removed approximately 25−30 mm 3 , volume of tissue material, which is a unique characteristic of this system.
It should be noted that the experiment examples shown in each figure with MSI results (Figures 3 to 5) contain ion distributions of just three ions out of hundreds for the sake of clarity.The search provides information that kiwi fruit tissue was studied by MALDI MSI that provided information on differences in compound distribution in pulp, skin, and seeds. 43n the case of our experiment, one of the 3D-imaged compounds in this fruit was malic acid (Figure 3D,E, green color), widely present as a side product of carbohydrate metabolism in various tissues. 44The methyl salicylate found in the seeds of kiwi fruit (Figure 3D,E, blue color) was previously found in blended kiwi pulp. 45It is said to play a role in signaling, and when demethylated to salicylic acid, exhibits anti-inflammatory properties. 46,47Zeatin, found in the locule walls of the kiwi fruit (Figure 3D,E, red color), is a cytokinin responsible for the regulation of plant growth. 48D MSI of a cross section of red radish is the next example of possibilities of the LARAPPI/CI MSI platform.The compounds identified in the root of the radish stem originate from LC-MS and MS/MS analyses conducted by our team (Supporting Information S6).Oxalic acid (4D,E, purple) is a compound commonly found in almost all plants.The most prominent function suggested for oxalic acid in plants is the retention of ions, specifically calcium, that form calcium oxalate crystals. 49It has also been associated with plant growth, development regulation, and stress responses. 505,7-Dimethoxycoumarin (Figure 4D,E, red color) has been previously identified in the epidermis of various plants, such as Citrus aurantifolia peel, and has been reported to possess antioxidant properties. 51Glutamic acid (Figure 4D,E, green color) was found in plants to promote callus formation, 52 regulate nitrogen metabolism, serve as a precursor to other amino acids, and is also a building block for proteins. 53D MSI of a red radish root (Raphanus sativus) was conducted in 2014 by Seaman et al. by MALDI-MS imaging.The research was focused on the metabolism of nitrogen into amino acids. 54A different work from 2015 utilized laser ablation and solvent capture by aspiration (LASCA) with an electrospray ion source.The ablation process with an ablation depth of 10−25 μm and volume of approximately 1 nl per pixel allowed differentiation of the spatial distributions in varying structures of the root proved possible, however, the images were still two-dimensional, as stated previously. 55uman kidney tissue with visible cancer and normal regions was studied with a 3D MSI system.The identification of compounds found in kidney tissue was performed using previous research.Arachidonic acid (Figure 5D,E, red color) is linked to pro-inflammatory activity in kidney tissue, being released as a result of cell stress.Arachidonic acid then acts as a precursor to bioactive mediators that can lead to renal dysfunction. 56,57The ways taurine levels (Figure 5D,E, blue color) influence renal processes are many, such as ion reabsorption, antioxidant properties, impact of blood flow, cell apoptosis, and more. 58,59The taurine transporter gene is down-regulated by the chemotherapeutic agent cisplatin. 60lic acid (Figure 5D,E, green color), widely present as a side product of carbohydrate metabolism in various tissues, has also been previously identified in kidney tissue. 61,62D mass spectrometry imaging of both normal tissue and cancerous tissue of the human kidney has previously been performed for lipidomic, drug, and drug metabolite spatial distribution or proteomic analyses.Zhang et al. have used desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to study metabolic profiles of renal oncocytoma, renal cell carcinoma (RCC), and healthy tissue to utilize the data obtained data for building prediction models. 63DESI-MSI has also been used as a potential prognostic tool for RCC by Vijayalakshmi et al. 64 Other methods, such as MALDI-MSI for protein and lipid profiling 65 and SALDI-MSI for cancer biomarker identification, 66 have also been performed.Even despite successful results of 2D MSI, two-dimensional methods of profiling can prove unreliable in a tissue of highly varied structure and, therefore, chemical composition, which can only become known by three-dimensional analysis.

■ MATERIALS AND METHODS
All chemicals were analytical reagent grade.All solvents were of LC-MS purity, except water (18 MΩcm water produced locally).Steel plates of 4.5 cm × 3.5 cm size used as sample plates were machined from H17 stainless steel of 0.8 mm thickness.Optical photographs and size/depth measurement results of ablation shapes were obtained with a motorized microscope built locally with three motorized stages in XYZ configuration (ThorLabs MTS50), DeltaOptical USB 3.0 camera (DLT Cam Pro 14 MPix) with a 2.5x InfiniFlex HD Compact Lens, and microscope light ring.Syringe filters (PTFA membrane, 0.2 μm-pore) were purchased from Merck Poland.
LARAPPI/CI System Setup.A Nd/YAG-pumped, OPO laser (Opolette HE 2940 model, factory tuned to 2930 ± 1 nm); Opotek, Carlsbad, CA, USA) with a pulse length shorter than 7 ns generated mid-IR laser pulses with a maximum repetition frequency of 20 Hz.The pulse energy measured before the diffractive element was 3.5 mJ (measured using a pyroelectric energy meter, PE25-SH-V2; Ophir, Logan, UT, USA).
The LARAPPI/CI system is based on an airtight chamber, as shown in Figure 1.The chamber (C) is pressurized with nitrogen gas to produce a nitrogen stream of 10 L/min.The sample is placed on a 50 × 50 mm sample stage (I) made of aircraft-grade aluminum alloy; under it, there is a Peltier cooling plate (TE-127−1.4−1.5;TE Technology, Traverse City, MI, USA) that maintains the sample at temperatures as low as −18 °C.The heat generated from the Peltier element is removed using circulating water and an external radiator (not shown in Figure 1).
The temperature-controlled sample stage is mounted on a motorized high-speed XY-stage (Figure 1J; MLS203, Thorlabs, Sweden).The pulsed beam from the OPO laser (Figure 1A) enters the sample chamber (Figure 1C) through a 1″ sapphire window (Figure 1B) with both sides with AR-coatings (Thorlabs, Sweden), is then expanded with CaF 2 planoconcave lens of f = −40.0mm, and collimated with CaF 2 plano-convex lens of f = 150 mm, both are AR-coated for 2−5 μm (not shown in Figure 1).The expanded laser beam is redirected toward the sample stage by a 1 in.gold-plated mirror (Figure 1D; Thorlabs, Sweden) and goes through a diffractive optical element (HM-396, Holo-OR Ltd.Israel), and is focused onto the sample surface by a 50 mm focal length aspherical ZnSe lens with AR coatings (both in Figure 1E, Thorlabs, Sweden).
The optical assembly and also the camera (Figure 1F; FLIR Blackfly S, Color Camera, 6 MPix, Sony IMX178 sensor) with a lens (12 mm C Series Fixed Focal Length Lens, Edmund Optics, UK) and distance sensor (Baumer OM70; Figure 1G) are mounted on locally machined precision aluminum rails and are in a fixed configuration; the only moving parts are XY (Figure 1J) and Z (Figure 1K) stages that are mounted on a precision aluminum 15 mm plate mounted at the bottom of the pressure chamber.During imaging, the laser focal point remains fixed in space, whereas the sample (Figure 1H) is moved by the computer-controlled XY-and Z-stages.A specially designed gas funnel (Figure 1E) is also a focusing assembly and is connected to a 6/4 mm (O.D/I.D.) PTFE tube.The gas funnel bottom surface is placed over (∼4 mm) the laser ablation site.The overpressure in the chamber drives a 10 L/min nitrogen gas flow through the tube.The nitrogen, chamber lighting, and cooling systems are connected through relays and controlled by a control program.
The laser ablation plumes are entrained into the gas and transported to the modified ion source (Bruker VIP HESI in the APCI configuration) of the Bruker Impact II mass spectrometer.The outlet end of the PTFE tube is mounted at an angle of 30°to the axis of the spectrometer inlet.The ion source also has a VUV source (Hamamatsu L12542) mounted axially to the MS sampling cone inside the ion source.A binary HPLC pump (Agilent G1312A) provided a steady flow of a solvent mixture (1% toluene in methanol; 200 μL/min) to the APCI needle.
Samples are kept at −18 °C during analysis by the Peltier module.The spatial resolution is typically 140 μm with applied oversampling (Figure 2).Each pixel/voxel in 3D MSI experiments was exposed to the laser for 500 ms, at a laser pulse repetition rate of 20 Hz.The delays between pixels were 1000 ms.Between pixels, the sample stage moved at a speed of 50 mm/s.The time delay between lines was 3 s.Timesynchronization of laser pulses with signals recorded by the mass spectrometer is aided by an ethernet-based time server.
Each 3D experiment was carried out in an inverted pyramid scheme (Figure 2D) with the following procedure (Figure 2H): 1. Calibration of the MS instrument.2. Sample scanning with a distance sensor, generation, and user-analysis of the 2D and 3D profile of the object's top side shape.3. Setting the ablation region with the camera image.4. Setting the Z level for the first ablation level.5. Ablation of the layer with the recording of MS or MS + bbCID data.6. Profiling of ablated object with a distance sensor; visual inspection with the camera image.7. Setting the Z level for the next ablation, etc.It must be noted that the XY ablation area of the lower layers is set automatically by software with the same resolution as the first layer but with an ablation area smaller by one X and one Y row and also centered.

■ CONCLUSIONS
We created a laser ablation remote atmospheric pressure photoionization/chemical ionization (LARAPPI/CI) platform coupled to an ultrahigh resolution quadrupole-time-of-flight (QToF) mass spectrometer.The optics of this system is based on a mid-IR laser and diffractive optical element.We have shown a novel approach, the inverted pyramid ablation scheme that is suitable for the removal of the layers required for threedimensional MSI.Various optimizations of the MSI system are shown, and the most important one is the modified APCI/ dopant-assisted-APPI ion source.The MSI solution was used for the direct three-dimensional (3D) mass spectrometry imaging (MSI) of metabolites in human and plant tissues.
■ ASSOCIATED CONTENT

Figure 1 .
Figure1.Schematic of the LARAPPI/CI MSI system.The upper panel presents a perspective view of the system without a mass spectrometer; the lower panel contains a simplified top view of the whole system.(A) OPO laser, (B) sapphire window, (C) pressure chamber, (D) gold mirror assembly, (E) laser focus assembly, (F) camera, (G) distance sensor, (H) sample, (I) sample stage with Peltier module and water block, (J) XY high-speed stage, (K) Z vertical stage, (L) diffractive optical element, (M) aspherical ZnSe lens, and (N) ablated material port connected via PTFE tubing to the ion source.

Figure 2 .
Figure 2. Renderings of 3D models of ablated voxel spaces: (A) not optimal arrangement of two voxels (notice that the lower part of space between voxels is not matching); (B) oversampling of four voxels allows for complete ablation of space; (C) example of oversampling arrangement for one layer of 8 × 8 voxels array; (D) inverted pyramid arrangement for oversampled voxels for three-dimensional ablation of material.The lower panel presents: (E) optical microscope image of a single voxel ablated in an agar gel with 20 laser pulses; (F) profilometer scan result of the ablated space of agar gel for a 7 × 7 × 1 voxel array with 140 × 140 μm resolution (20 laser pulses per voxel).(G) simplified inverted pyramid arrangement of voxels in kiwi and red radish 3D MSI experiments as seen from the side of the object; (H) 2D (steps 1−5) and 3D (steps 1−8) MSI workflow.

Figure 3 .
Figure 3. Photographs of the object studied (kiwi fruit cross section) and selected 3D MSI results.(A) Optical photographs of the starting object and analysis region marked with a white dashed line; (B) optical photographs of the postanalysis object; (C) side view of the object; (D) 3D MSI ion images for three ions represented by different colors−top views; (E) side views of 3D ion images of ions of different colors.The intensity of an ion signal is represented by the opacity of the 3D cloud within a given color.The bottom panel contains optical photographs of the preablation region and after the ablation steps.The Z-value was measured with the precision distance sensor.

Figure 5 .
Figure 5. Photographs of the object studied (human kidney tissue) and selected 3D MSI results.(A) Optical photographs of the starting object and analysis region marked with a white dashed line; (B) optical photographs of the postanalysis object; (C) side view of the object; (D) 3D MSI ion images for three ions represented by different colors−top views; (C, D) side views of 3D ion images of ions of different colors.The intensity of an ion signal is represented by the opacity of the 3D cloud within a given color.