Transmission Soft X-ray Microscopy Research Articles

Observation of mammalian living cells with femtosecond single pulse illumination generated by a soft X-ray free electron laser

Soft X-ray transmission microscopy is a powerful tool for visualizing cellular structures due to the natural contrast between organic material and water, but radiation damage has hindered its application to living cells. We have developed a soft X-ray microscope using femtosecond pulse illumination generated by a soft X-ray free electron laser, with which structural change of cells caused by radiation damage is negligible. Employing Wolter mirrors for illumination and objective optics allowed us to perform soft X-ray imaging with a large field of view, enabling observation of mammalian cells. We successfully captured images of living cells in a culture medium visualizing their carbon distribution. The broad wavelength-tunability of soft X-ray free electron lasers, in conjunction with the achromaticity of Wolter mirrors, enabled wavelength resolved cellular imaging.

Direct Observation of Room-Temperature Magnetic Skyrmion Motion Driven by Ultra-Low Current Density in Van Der Waals Ferromagnets.

The recent discovery of room-temperature ferromagnetism in 2D van der Waals (vdW) materials, such as Fe3GaTe2 (FGaT), has garnered significant interest in offering a robust platform for 2D spintronic applications. Various fundamental operations essential for the realization of 2D spintronics devices are experimentally confirmed using these materials at room temperature, such as current-induced magnetization switching or tunneling magnetoresistance. Nevertheless, the potential applications of magnetic skyrmions in FGaT systems at room temperature remain unexplored. In this work, the current-induced generation of magnetic skyrmions in FGaT flakes employing high-resolution magnetic transmission soft X-ray microscopy is introduced, supported by a feasible mechanism based on thermal effects. Furthermore, direct observation of the current-induced magnetic skyrmion motion at room temperature in FGaT flakes is presented with ultra-low threshold current density. This work highlights the potential of FGaT as a foundation for room-temperature-operating 2D skyrmion device applications.

In Situ Studies of Copper-Based CO2 Reduction Electrocatalysts by Scanning Transmission Soft X-ray Microscopy.

A microfluidic-enabled electrochemical device has been developed to investigate electrochemically active nanomaterials under reaction conditions using in situ scanning transmission soft X-ray microscopy (STXM). In situ STXM measurements were conducted on electrodeposited Cu catalysts under electrochemical CO2 reduction (CO2R) conditions. The study provides detailed, quantitative results about the changes in the morphology and chemical structure of the catalytic nanoparticles as a function of applied potentials. The deposited Cu nanoparticles initially contain both Cu(0) and Cu(I). As an increasingly cathodic potential is applied, the Cu(I) species gradually convert to Cu(0) over the potential range of +0.4 to 0 V versus the reversible hydrogen electrode (VRHE). During this process, Cu(I) particles of various sizes are converted to metallic Cu at different reaction rates and at slightly different potentials, indicating a degree of heterogeneity in the electrochemical response of discrete particles. At CO2R relevant potentials, only metallic Cu is observed, and the morphology of the particles is fairly stable within the spatial resolution limits of STXM (∼40 nm). We also report in situ STXM studies of a working electrode with relatively thick Cu-based electrodeposits. The spatially resolved chemical analysis identifies that Cu-oxide species can persist under CO2R conditions, but only when the catalytic nanoparticles are electronically isolated from the working electrode and therefore are catalytically irrelevant. In summary, in situ STXM is presented as a technique to gain advanced morphological and spatially resolved chemical structure insights into electrochemically active nanomaterials, which was used to provide improved understanding regarding Cu nanomaterial catalysts under CO2 reduction conditions.

Mapping Heterogeneity of Pristine and Aged Li- and N-Mnhcf Cathode by Synchrotron-Based Energy-Dependent Full Field Transmission X-ray Microscopy.

Manganese hexacyanoferrate is a promising cathode material for lithium and sodium ion batteries, however, it suffers of capacity fading during the cycling process. To access the structural and functional characteristics at the nanometer scale, fresh and cycled electrodes are extracted and investigated by transmission soft X-ray microscopy, which allows chemical characterization with spatial resolution from position-dependent x-ray spectra at the Mn L-, Fe L- and N K-edges. Furthermore, soft X-rays prove to show superior sensitivity toward Fe, compare to hard X-rays. Inhomogeneities within the samples are identified, increasing in the aged electrodes, more dramatically in the Li-ion system, which explains the poorer cycle life as Li-ion cathode material. Local spectra, revealing different oxidation states over the sample with strong correlation between the Fe L-edge, Mn L-edge, and N K-edge, imply a coupling between redox centers and an electron delocalization over the host framework.

Ionic liquid functionalized tin halide perovskite investigated by STXM and spectro-ptychography

The structural and compositional properties of tin halide perovskites (CH(NH2)2SnI3, FASnI3) with and without an ionic liquid of 1-Ethyl-3-methylimidazolium bis(trifluormethylsulfonyl) imide (EMITFSI) were investigated using the scanning transmission soft X-ray microscopy (STXM) and spectro-ptychography techniques at the Sn M5,4-edge, I M5,4-edge, O K-edge, C K-edge, and N K-edge. The study offers a comprehensive interpretation of the spectral characteristics of main components (C, N, Sn, and I) in Sn-based perovskites through STXM-based techniques and further reveals differences between the perovskite samples with and without adding EMITFSI ionic liquid. The chemical imaging of Sn and I shows that the perovskite sample without ionic liquid has Sn-rich regions in iodine cavities, while the ionic liquid-treated sample exhibits a more homogeneous perovskite phase. Furthermore, the C and N K-edge XANES resonance features support the interaction between the non-volatile ionic liquid and FA+, thus playing a protective role in the formation and stabilization of perovskite phase.

(Digital Presentation) Post Mortem Microscopical and Selective Analysis of Manganese Hexacyanoferrate Cathode Material By Transmission Soft X-Ray Microscopy

Prussian Blue (PB) and its analogues (PBAs) are a large family of transition metal hexacyanoferrates with general formula of AxM[Fe(CN)6]γ□1-γ∙zH2O. They have open framework structure, redox-active sites and strong structural stability, which makes them perspective electrode materials, as PBAs are able to perform the rapid insertion and extraction of the ions with the lowest possible lattice strain. In addition, they are safe, non-toxic and relatively inexpensive. Among simple PBAs, manganese hexacyanoferrate (MnHCF) displays a high specific capacity and redox plateaus at a high voltage against lithium and sodium in LIBs and SIBs [1]. The synthesis of MnHCF is possible by simple co-precipitation method. In our group we synthesized and investigated its structure and electrochemical performance as a cathode material. For the characterization of MnHCF several conventional and synchrotron-based techniques were used.Generally, after cycling inside the battery some changes might occur in the cathode material, including changing the charge states of the different elements. For the investigation of this phenomenon synchrotron techniques are particularly informative, such as energy-dependent full field transmission soft X-ray microscopy (TXM), which is able to give a full picture at the nanometre scale of the chemical state and spatial distribution of the elements [2]. By using X‐rays in the “soft” energy region (<3 KeV), it is possible to access transitions from the core levels of the light elements, such as the K‐edge of nitrogen, oxygen, fluorine, as well as L- and M- edges of heavier elements [3, 4]. TXM provides pixel-by-pixel absorption spectrum, making it possible to select groups of pixels and map regions with the similar spectral features [2]. Therefore, TXM studies were conducted on our material before and after cycling, and the special protocol of the data treatment was developed and applied to the results of the analysis in order to enhance the representation of the heterogeneities, if any, and then to extract the most relevant spectra.Indeed, the inhomogeneities within the samples were identified, especially in the aged electrodes. It was observed that the distribution of the heterogeneities were different for the electrodes extracted from the Li-ion system, compared to Na. Characteristic spectra from the entire samples as well as particular regions with different oxidation states were obtained and compared to one another, which made it evident, that the treated results from the iron L-edge and manganese L-edge expressed strong correlation, however, while in the Fe K-edge the distribution of heterogeneities looked quite random, a weak bulk-border effect was observed in case of the Mn K-edge.[1] A. Mullaliu, J. Asenbauer, G. Aquilanti, S. Passerini, M. Giorgetti, Small Methods 4, (2019) 1900529.[2] A. Sorrentino, L. Simonelli, A. Kazzazi, N. Laszczynski, A. Birrozzi, A. Mullaliu, E. Pereiro, S. Passerini, M. Giorgetti, D. Tonti, Appl. Sci., no. 11, p. 2791, 2021.[3] D. Tonti, M. Olivares-Marín, A. Sorrentino, E. Pereiro, L. P. Mehdi Khodaei, Ed., IntechOpen, 22 March, 2017.[1] A. Mullaliu, J. Asenbauer, G. Aquilanti, S. Passerini, M. Giorgetti, Small Methods 4, (2019) 1900529.[2] A. Sorrentino, L. Simonelli, A. Kazzazi, N. Laszczynski, A. Birrozzi, A. Mullaliu, E. Pereiro, S. Passerini, M. Giorgetti, D. Tonti, Appl. Sci., no. 11, p. 2791, 2021.[4] D. Attwood, Cambridge: Cambridge University Press, 1999. Figure 1

Magnetic anisotropy in permalloy antidot square lattice

Magnetic anisotropy of Permalloy (Py) antidot square lattice was investigated by torquemetry method using Rotation Magneto-Optic Kerr Effect (ROTMOKE). We find that there exists a field-dependent 4-fold magnetic anisotropy with the easy magnetization axis along the [11] axis of the antidot square lattice. In addition, there also exists an artifact of a uniaxial magnetic anisotropy in ROTMOKE result. We show that both results are due to the period wiggling of the magnetization in space which was confirmed by magnetic imaging using magnetic transmission soft x-ray microscopy (MTXM). Micromagnetic simulation from MuMax3 supports the wiggling structure of the magnetization, as well as reproduces ROTMOKE result. A simplified model was developed based on the periodic wiggling of the magnetization and successfully explored the physical origin of the field-dependent 4-fold anisotropy and the artifact of the uniaxial anisotropy.

X-ray Microspectroscopy and Ptychography on Nanoscale Structures in Rock Varnish

X-ray microspectroscopy is a powerful analytical method in geoscientific and environmental research as it provides a unique combination of nanoscale imaging with high spectroscopic sensitivity at relatively low beam-related sample damage. In this study, “classical” scanning transmission soft X-ray microscopy (STXM) with X-ray absorption spectroscopy and the recently established soft X-ray ptychography are applied to the analysis of selected rock varnish samples from urban and arid desert environments. X-ray ptychography enhances the spatial resolution relative to STXM by up to 1 order of magnitude. With its high chemical sensitivity, it can resolve nanoscale differences in valence states of the key varnish elements manganese (Mn) and iron. Our results emphasize the complex nanoarchitecture of rock varnish as well as the diverse mineralogy of the Mn oxy–hydroxide matrix and its embedded dust grains. In contrast to the fast-growing urban varnish, the slow-growing arid desert varnish revealed a remarkable nanoscale stratification of alternating Mn valence states, providing hints on the layer-wise and still enigmatic growth process.

Magnetic textures and singularities in ferri/ferromagnetic multilayers

The stacking of ferrimagnetic and ferromagnetic films leads to a competition between magnetic interactions that can stabilize Bloch points and other singularities at the interfaces. In this work, GdxCo1-x/NdCo5/GdyCo1-y trilayers, with different thicknesses, have been prepared. By tuning the stoichiometry of the Gd-Co outer layers, the Co magnetic sublattice dominates in one layer, whereas the opposite side is Gd dominated. In this way, in-depth magnetization domain walls can be created due to the balance between exchange and magnetostatic interactions. In addition, the weak perpendicular magnetic anisotropy of the Nd-Co central layer induces a stripe domain pattern, supporting the formation of Bloch points, meron-like textures, and vortex-antivortex pairs. The interaction between both types of structures, in-depth domain walls and magnetic singularities, has been studied by a combination of macroscopic Magneto-Optical Kerr Effect and Vibrating Sample Magnetometry measurements with microscopic Magnetic Force Microscopy and element selective magnetic Transmission soft X-ray Microscopy imaging. The results confirm marked changes in the magnetic properties of the trilayers, as compared with those single NdCo5 films, and the formation of complex magnetic textures, associated with partial magnetization reversal, where magnetic singularities are formed.

Topology-dependent stability of vortex-antivortex structures

The non-trivial topology of magnetic structures such as vortices and skyrmions is considered as a key concept to explain the stability of those structures. The stability, dictated by non-trivial topology, provides great potential for device applications. Although it is a very critical scientific and technological issue, it is elusive to experimentally study the topology-dependent stability owing to the difficulties in establishing stably formed magnetic structures with different topologies. Here, we establish a platform for vortex-antivortex structures with different topological charges within Ni80Fe20 rectangular elements thick enough to stabilize a unique three-dimensional magnetic structure with non-uniform magnetization along the thickness of the elements. The detailed magnetization configurations of the three-dimensional vortex-antivortex structures and their annihilations during their field-driven motions are investigated by utilizing magnetic transmission soft x-ray microscopy and micromagnetic simulation. We demonstrate that the stability of vortex-antivortex structures significantly depends on their topologies and the topology-dependent stability is associated with their different annihilation mechanisms. We believe that this work provides in-depth insight into the stability of magnetic structures and its topology dependence.

Three-dimensional fast elemental mapping by soft X-ray dual-energy focal stacks imaging.

The three-dimensional (3D) dual-energy focal stacks (FS) imaging method has been developed to quickly obtain the spatial distribution of an element of interest in a sample; it is a combination of the 3D FS imaging method and two-dimensional (2D) dual-energy contrast imaging based on scanning transmission soft X-ray microscopy (STXM). A simulation was firstly performed to verify the feasibility of the 3D elemental reconstruction method. Then, a sample of composite nanofibers, polystyrene doped with ferric acetylacetonate [Fe(acac)3], was further investigated to quickly reveal the spatial distribution of Fe(acac)3 in the sample. Furthermore, the data acquisition time was less than that for STXM nanotomography under similar resolution conditions and did not require any complicated sample preparation. The novel approach of 3D dual-energy FS imaging, which allows fast 3D elemental mapping, is expected to provide invaluable information for biomedicine and materials science.

Calculating absorption dose when X-ray irradiation modifies material quantity and chemistry.

X-ray absorption is a sensitive and versatile tool for chemical speciation. However, when high doses are used, the absorbed energy can change the composition, amount and structure of the native material, thereby changing the aspects of the absorption process on which speciation is based. How can one calculate the dose when X-ray irradiation affects the chemistry and changes the amount of the material? This paper presents an assumption-free approach whichcan retrieve from the experimental data all dose-sensitive parameters -absorption coefficients, composition (elemental molecular units), material densities - which can then be used to calculate accurate doses as a function of irradiation. This approach is illustrated using X-ray damage to a solid film of aperfluorosulfonic acid fluoropolymer in a scanning transmission soft X-ray microscope. This new approach is compared against existing dose models which calculate the dose by making simplifying assumptions regarding the material quantity, density and chemistry. While the detailed measurements used in thisapproach go beyond typical methods to experimental analytical X-ray absorption, they provide a more accurate quantitation of radiation dose, and help to understand mechanisms of radiation damage.

Soft X-ray Transmission Microscopy on Lithium-Rich Layered-Oxide Cathode Materials

Energy-dependent full field transmission soft X-ray microscopy (TXM) is able to give a full picture at the nanometer scale of the chemical state and spatial distribution of oxygen and other elements relevant for battery materials, providing pixel-by-pixel absorption spectrum. We show different methods to localize chemical inhomogeneities in Li1.2Mn0.56Ni0.16Co0.08O2 particles with and without VOx coating extracted from electrodes at different states of charge. Considering the 3d(Mn,Ni)-2p(O) hybridization, it has been possible to discriminate the chemical state of Mn and Ni in addition to the one of O. Different oxidation states correspond to specific features in the O-K spectra. To localize sample regions with specific compositions we apply two different methods. In the first, the pixel-by-pixel ratios of images collected at different key energies clearly highlight local inhomogeneities. In the second, introduced here for the first time, we directly correlate corresponding pixels of the two images on a xy scatter plot that we call phase map, where we can visualize the distributions as function of thickness as well as absorption artifacts. We can select groups of pixels, and then map regions with similar spectral features. Core-shell distributions of composition are clearly shown in these samples. The coating appears in part to frustrate some of the usual chemical evolution. In addition, we could directly observe several further aspects, such as: distribution of conducting carbon; inhomogeneous state of charge within the electrode; molecular oxygen profiles within a particle. The latter suggests a surface loss with respect to the bulk but an accumulation layer at intermediate depth that could be assigned to retained O2.

Creation and observation of Hopfions in magnetic multilayer systems

Among topological solitons, magnetic skyrmions are two-dimensional particle-like objects with a continuous winding of the magnetization, and magnetic Hopfions are three-dimensional objects that can be formed from a closed loop of twisted skyrmion strings. Theoretical models suggest that magnetic Hopfions can be stabilized in frustrated or chiral magnetic systems, and target skymions can be transformed into Hopfions by adapting their perpendicular magnetic anisotropy, but their experimental verification has been elusive so far. Here, we present an experimental study of magnetic Hopfions that are created in Ir/Co/Pt multilayers shaped into nanoscale disks, known to host target skyrmions. To characterize three-dimensional spin textures that distinguish Hopfions from target skyrmions magnetic images are recorded with surface-sensitive X-ray photoemission electron microscopy and bulk-sensitive soft X-ray transmission microscopy using element-specific X-ray magnetic circular dichroism effects as magnetic contrast. These results could stimulate further investigations of Hopfions and their potential application in three-dimensional spintronics devices.

Megapixel scanning transmission soft X-ray microscopy imaging coupled with compressive sensing X-ray fluorescence for fast investigation of large biological tissues.

Soft X-ray microscopy coupled with low energy X-ray fluorescence is a powerful tool for investigating complex biological systems like cells and tissues. Due to certain characteristics of X-ray sources, sample stage motors, and detectors, the examination of large areas at high resolutions is very time consuming, often confining the analysis only to a restricted number of pre-selected representative regions. Here we propose and demonstrate a compressive sensing method that provides an alternative approach for overcoming such limitations and can be applied to different kinds of samples and other microscopy and analytical techniques.

Revealing 3D magnetization of thin films with soft X-ray tomography: magnetic singularities and topological charges

The knowledge of how magnetization looks inside a ferromagnet is often hindered by the limitations of the available experimental methods which are sensitive only to the surface regions or limited in spatial resolution. Here we report a vector tomographic reconstruction based on soft X-ray transmission microscopy and magnetic dichroism data, which has allowed visualizing the three-dimensional magnetization in a ferromagnetic thin film heterostructure. Different non-trivial topological textures have been resolved and the determination of their topological charge has allowed us to identify a Bloch point and a meron-like texture. Our method relies only on experimental data and might be of wide application and interest in 3D nanomagnetism.

Laboratory Soft X-Ray Microscopy with an Integrated Visible-Light Microscope-Correlative Workflow for Faster 3D Cell Imaging.

Laboratory transmission soft X-ray microscopy (L-TXM) has emerged as a complementary tool to synchrotron-based TXM and high-resolution biomedical 3D imaging in general in recent years. However, two major operational challenges in L-TXM still need to be addressed: a small field of view and a potentially misaligned rotation stage. As it is not possible to alter the magnification during operation, the field of view in L-TXM is usually limited to a few tens of micrometers. This complicates locating areas and objects of interest in the sample. Additionally, if the rotation axis of the sample stage cannot be adjusted prior to the experiments, an efficient workflow for tomographic imaging cannot be established, as refocusing and sample repositioning will become necessary after each recorded projection. Both these limitations have been overcome with the integration of a visible-light microscope (VLM) into the L-TXM system. Here, we describe the calibration procedure of the goniometer sample stage and the integrated VLM and present the resulting 3D imaging of a test sample. In addition, utilizing this newly integrated VLM, the extracellular matrix of cryofixed THP-1 cells (human acute monocytic leukemia cells) was visualized by L-TXM for the first time in the context of an ongoing biomedical research project.

Significant Enhancement in Light Output of Photonic-Crystal-Based YAG:Ce Scintillator for Soft X-Ray Detectors

Because of its high light output, Gd <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> S:Tb (P43) phosphor powder is widely used in scintillator detectors for scanning transmission soft X-ray microscopy. However, the long decay time (>1 ms) of P43 phosphor powder limits the image scanning speed. Inorganic scintillators such as YAG:Ce are very suitable for fast scanning imaging due to their short decay time (tens of nanoseconds). Nevertheless, its light output is lower than that of P43 phosphor powder. Patterning photonic crystals (PhCs) on the output surface of an inorganic scintillator is a simple and efficient method to enhance its light output. In this article, we use a soft X-ray interference lithography technique to produce a large-area pattern of nanoscale PhCs on a 200-μm -thickness and 1-in-diameter YAG:Ce scintillator. Measurement of the enhancement ratio was performed on the soft X-ray beamline (BL08U1A) at the Shanghai Synchrotron Radiation Facility. The results show that the light output of the PhC-patterned YAG:Ce scintillator with air coupling is approximately 2.8 times higher than that of the unpatterned YAG:Ce scintillator. Meanwhile, the time-resolved emission with optical excitation showed that the PhC structures did not affect the spontaneous emission rate in the internal of scintillator crystal. This enhancement of the light output indicates that the PhC-patterned YAG:Ce scintillator is a good candidate for soft X-ray detection.

Magnetite magnetosome biomineralization in Magnetospirillum magneticum strain AMB-1: A time course study

Magnetotactic bacteria are a highly studied group of diverse prokaryotes that biomineralize chains of magnetosomes, single domain, single crystal magnetic nanoparticles of magnetite or greigite, enclosed by a lipid bilayer membrane whose synthesis is under strict genetic control. In addition to characterizing the genetics and physicochemical properties of both cultured and uncultured environmental species, there have been a number of investigations using a time course approach to determine the chemical pathway of magnetite biomineralization in these organisms. In time course studies, cells of MTB are typically grown in the absence of iron so they cannot make magnetite, and then provided with iron in culture medium which initiates the biomineralization of magnetosome chains over a subsequent time period. Results from previous time course studies are not consistent with one another, differing with regard to the nature of chemical intermediates and the rate of establishment of magnetosome chains. In this work we report a time course study of Magnetospirillum magneticum strain AMB-1 over a 48h (hour) period, using transmission electron microscopy (TEM) and soft X-ray scanning transmission X-ray microscopy (STXM) at the Fe L-edge. STXM provides capability to measure X-ray absorption spectra (XAS) and map chemical species with ∼25nm spatial resolution and thus gives detailed results on the chemistry of individual particles in single cells. An evolution of the iron oxide speciation, from a more Fe(III)-rich species, possibly α-hematite (Fe2O3), to magnetite (Fe3O4), was observed in the early stages, with evidence for the presence of the Fe(III)-rich character persisting up to 24 h. The spectromicroscopy (X-ray absorption, XAS and X-ray magnetic circular dichroism, XMCD) and TEM results show that biomineralization occurs in a stepwise fashion. First, very small particles, with no measurable magnetization, are produced at different sites in the cell without significant chain formation. The Fe L3 spectra of these early stage particles typically differ from magnetite with an additional signal at 708.4 eV that is consistent with α-hematite. By 6–8h the particles are more numerous, partial chain formation is evident, and the L3 spectrum is very similar to that of magnetite. By 24 h particles-in-chains are the dominant motif and magnetism with the moment along the chain is established. By 48 h the cells are essentially the same as cells grown in Fe-rich medium.

Quantitative Distribution of DNA, RNA, Histone and Proteins Other than Histone in Mammalian Cells, Nuclei and a Chromosome at High Resolution Observed by Scanning Transmission Soft X-Ray Microscopy (STXM).

Soft X-ray microscopy was applied to study the quantitative distribution of DNA, RNA, histone, and proteins other than histone (represented by BSA) in mammalian cells, apoptotic nuclei, and a chromosome at spatial resolutions of 100 to 400 nm. The relative distribution of closely related molecules, such as DNA and RNA, was discriminated by the singular value decomposition (SVD) method using aXis2000 software. Quantities of nucleic acids and proteins were evaluated using characteristic absorption properties due to the 1s–π * transition of N=C in nucleic acids and amide in proteins, respectively, in the absorption spectra at the nitrogen K absorption edge. The results showed that DNA and histone were located in the nucleus. By contrast, RNA was clearly discriminated and found mainly in the cytoplasm. Interestingly, in a chromosome image, DNA and histone were found in the center, surrounded by RNA and proteins other than histone. The amount of DNA in the chromosome was estimated to be 0.73 pg, and the content of RNA, histone, and proteins other than histone, relative to DNA, was 0.48, 0.28, and 4.04, respectively. The method we present in this study could be a powerful approach for the quantitative molecular mapping of biological samples at high resolution.