3D Mass Spectrometry Imaging Research Articles

Spatial distribution of active compounds in stratum corneum—partitioning between corneocytes and lipid matrix

The interaction of active substances with molecular structures in stratum corneum (SC) is crucial for the efficacy and safety of cosmetic formulations and topical drugs. However, the molecular architecture of SC is highly complex and methods to unambiguously localize exogenous molecules within SC are lacking. Consequently, little is known about the distribution of actives within SC, and proposed penetration mechanisms through SC are typically limited to simple diffusion via a tortuous (lipid only) or transverse (across corneocytes and lipid matrix) pathway. In this work, 3D mass spectrometry imaging is used to determine the spatial distributions of four active substances at subcellular resolution in SC, including partitioning between the corneocytes and the intercellular lipid matrix. The results indicate that caffeine, 2-methyl resorcinol and oxybenzone are homogeneously distributed in the corneocytes but largely absent in the lipid matrix, despite considerable differences in lipophilicity. In contrast, the distribution- of jasmonic acid derivative is more inhomogeneous and indicates considerable localization to both the lipid phase and the corneocytes.

High-resolution 3D spatial distribution of complex microbial colonies revealed by mass spectrometry imaging

IntroductionBacterial living states and the distribution of microbial colony signaling molecules are widely studied using mass spectrometry imaging (MSI). However, current approaches often treat 3D colonies as flat 2D disks, inadvertently omitting valuable details. The challenge of achieving 3D MSI in biofilms persists due to the unique properties of microbial samples. ObjectivesThe study aimed to develop a new biofilm sample preparation method that can realize high-resolution 3D MSI of bacterial colonies to reveal the spatial organization of bacterial colonies. MethodsThis article introduces the moisture-assisted cryo-section (MACS) method, enabling embedding-free sectioning parallel to the growth plane. The MACS method secures intact sections by controlling ambient humidity and slice thickness, preventing molecular delocalization. ResultsCombined with matrix-assisted laser desorption ionization mass spectrometry (MALDI)-MSI, the MACS method provides high-resolution insights into endogenic and exogenous molecule distributions in Pseudomonas aeruginosa (P. aeruginosa) biofilms, including isomeric pairs. Moreover, analyzed colonies are revived into 3D models, vividly depicting molecular distribution from inner to outer layers. Additionally, we investigated metabolite spatiotemporal dynamics in multiple colonies, observing changes over time and distinct patterns in single versus merged colonies. These findings shed light on the repel-merge process for multi-colony formation. Furthermore, our study monitored chemical responses inside biofilms after antibiotic treatment, showing increased antibiotic levels in the outer biofilm layer over time while maintaining low levels in the inner region. Moreover, the MACS method demonstrated its universality and applicability to other bacterial strains. ConclusionThese results unveil complex cell activities within biofilm colonies, offering insights into microbe communities. The MACS method is universally applicable to loosely packed microorganism colonies, overcoming the limitations of previously reported MSI methods. It has great potential for studying bacterial-infected cancer tissues and artificial organs, making it a valuable tool in microbiological research.

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.

DeepS: Accelerating 3D Mass Spectrometry Imaging via a Deep Neural Network.

Mass spectrometry imaging (MSI) is a powerful methodology that enables the visualization of the spatial distribution of biomolecules, including lipids, peptides, and proteins, from biological tissue sections. While two-dimensional (2D) MSI has been widely reported in various applications, three-dimensional (3D) MSI can enable the mapping of biomolecule distribution in complex biological structures (e.g., organs) with an added dimension. However, traditional 3D MSI techniques are time-consuming since 3D MS images are constructed from 2D MSI analyses of a series of tissue sections. In this study, we propose a 3D MSI workflow, termed DeepS, which uses a 3D sparse sampling network (3D-SSNet) and a sparse sampling strategy to significantly accelerate 3D MSI analyses. Sparsely sampled tissue sections are reconstructed using 3D-SSNet, yielding results comparable to those using full sampling MSI, even at a sampling ratio of 20-30%. The workflow performed well when applied to 3D imaging of a mouse brain with Alzheimer's disease, and combined with transfer learning, it is successfully used for the 3D MSI analyses of more heterogeneous samples, e.g., a mouse brain with glioblastoma and a mouse kidney.

Recent advances in mass spectrometry imaging of single cells.

Mass spectrometry imaging (MSI) is a sensitive, specific, label-free imaging analysis technique that can simultaneously obtain the spatial distribution, relative content, and structural information of hundreds of biomolecules in cells and tissues, such as lipids, small drug molecules, peptides, proteins, and other compounds. The study of molecular mapping of single cells can reveal major scientific issues such as the activity pattern of living organisms, disease pathogenesis, drug-targeted therapy, and cellular heterogeneity. Applying MSI technology to the molecular mapping of single cells can provide new insights and ideas for the study of single-cell metabolomics. This review aims to provide an informative resource for those in the MSI community who are interested in single-cell imaging. Particularly, we discuss advances in imaging schemes and sample preparation, instrumentation improvements, data processing and analysis, and 3D MSI over the past few years that have allowed MSI to emerge as a powerful technique in themolecular imaging of single cells. Also, we highlight some of the most cutting-edge studies in single-cell MSI, demonstrating the future potential of single-cell MSI. Visualizing molecular distribution at the single-cell or even sub-cellular level can provide us with richer cell information, which strongly contributes to advancing research fields such as biomedicine, life sciences, pharmacodynamic testing, and metabolomics. At the end of the review, we summarize the current development of single-cell MSI technology and look into the future of this technology.

Multiplexed neuropeptide mapping in ant brains integrating microtomography and three-dimensional mass spectrometry imaging.

Neuropeptides are important regulators of animal physiology and behavior. Hitherto the gold standard for the localization of neuropeptides have been immunohistochemical methods that require the synthesis of antibody panels, while another limiting factor has been the brain's opacity for subsequent in situ light or fluorescence microscopy. To address these limitations, we explored the integration of high-resolution mass spectrometry imaging (MSI) with microtomography for a multiplexed mapping of neuropeptides in two evolutionary distant ant species, Atta sexdens and Lasius niger. For analyzing the spatial distribution of chemically diverse peptide molecules across the brain in each species, the acquisition of serial mass spectrometry images was essential. As a result, we have comparatively mapped the three-dimensional (3D) distributions of eight conserved neuropeptides throughout the brain microanatomy. We demonstrate that integrating the 3D MSI data into high-resolution anatomy models can be critical for studying organs with high plasticity such as brains of social insects. Several peptides, like the tachykinin-related peptides (TK) 1 and 4, were widely distributed in many brain areas of both ant species, whereas others, for instance myosuppressin, were restricted to specific regions only. Also, we detected differences at the species level; many peptides were identified in the optic lobe of L. niger, but only one peptide (ITG-like) was found in this region in A. sexdens. Building upon MS imaging studies on neuropeptides in invertebrate model systems, our approach leverages correlative MSI and computed microtomography for investigating fundamental neurobiological processes by visualizing the unbiased 3D neurochemistry in its complex anatomic environment.

Three-Dimensional Mass Spectrometry Imaging Reveals Distributions of Lipids and the Drug Metabolite Associated with the Enhanced Growth of Colon Cancer Cell Spheroids Treated with Triclosan.

The application of mass spectrometry imaging (MSI) to explore the responses of cancer cell spheroids (CCS) after treatment of exogenous molecules has attracted growing attention. Increasing studies have utilized MSI to image the two-dimensional distributions of exogenous and endogenous molecules in planar CCS sections. However, because CCS are volumetric and heterogenous, maintaining their three-dimensional (3D) information is essential for acquiring a better understanding of the tumor microenvironment and mechanisms of action of exogenous molecules. Here, an established method of 3D MSI was applied to distinguish the distributions of triclosan sulfate and endogenous lipids in three microregions of colon CCS with an enhanced growth induced by the treatment of triclosan, a common antimicrobial agent. The results of 3D MSI showed that triclosan sulfate gradually accumulated from the periphery to the entire structure of CCS and finally localized in the core region. Spatial lipidomics analysis revealed that the upregulated phosphatidylethanolamine (fold change (FD) = 1.26, p = 0.0021), phosphatidylinositol (FD = 1.17, p = 0.0180), and phosphatidylcholine (FD = 1.22, p = 0.0178) species mainly distributed in the outer proliferative region, while the upregulated sphingomyelin (FD = 1.18, p = 0.024) species tended to distribute in the inner necrotic region. Our results suggest that a competitive mechanism between inhibiting and promoting CCS growth might be responsible for the proliferation of CCS treated with triclosan.

Three-dimensional (3D) imaging of lipids in skin tissues with infrared matrix-assisted laser desorption electrospray ionization (MALDESI) mass spectrometry.

Three-dimensional (3D) mass spectrometry imaging (MSI) has become a growing frontier as it has the potential to provide a 3D representation of analytes in a label-free, untargeted, and chemically specific manner. The most common 3D MSI is accomplished by the reconstruction of 2D MSI from serial cryosections; however, this presents significant challenges in image alignment and registration. An alternative method would be to sequentially image a sample by consecutive ablation events to create a 3D image. In this study, we describe the use of infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) in ablation-based 3D MSI for analyses of lipids within fresh frozen skin tissue. Depth resolution using different laser energy levels was explored with a confocal laser scanning microscope to establish the imaging parameters for skin. The lowest and highest laser energy level resulted in a depth resolution of 7μm and 18μm, respectively. A total of 594 lipids were putatively detected and detailed lipid profiles across different skin layers were revealed in a 56-layer 3D imaging experiment. Correlated with histological information, the skin structure was characterized with differential lipid distributions with a lateral resolution of 50μm and a z resolution of 7μm.

Three-dimensional quantitative mass spectrometry imaging in complex system: From subcellular to whole organism.

Mass spectrometry imaging (MSI) has been applied for label-free three-dimensional (3D) imaging from position array across the whole organism, which provides high-dimensional quantitative data of inorganic or organic compounds that may play an important role in the regulation of cellular signaling, including metals, metabolites, lipids, drugs, peptides, and proteins. While MSI is suitable for investigation of the spatial distribution of molecules, it has a limitation with visualization and quantification of multiple molecules. 3D-MSI, however, can be applied toward exploring metabolic pathway as well as the interactions of lipid-protein, protein-protein, and metal-protein in complex systems from subcellular to the whole organism through an untargeted methodology. In this review, we highlight the methods and applications of MS-based 3D imaging to address the complexity of molecular interaction from nano- to micrometer lateral resolution, with particular focus on: (a) common and hybrid 3D-MSI techniques; (b) quantitative MSI methodology, including the methods using a stable isotope labeling internal standard (SILIS) and SILIS-free approaches with tissue extinction coefficient or virtual calibration; (c) reconstruction of the 3D organ; (d) application of 3D-MSI for biomarker screening and environmental toxicological research. 3D-MSI quantitative analysis provides accurate spatial information and quantitative variation of biomolecules, which may be valuable for the exploration of the molecular mechanism of the disease progresses and toxicological assessment of environmental pollutants in the whole organism. Additionally, we also discuss the challenges and perspectives on the future of 3D quantitative MSI.

Localized Metabolomic Gradients in Patient-Derived Xenograft Models of Glioblastoma.

Glioblastoma (GBM) is increasingly recognized as a disease involving dysfunctional cellular metabolism. GBMs are known to be complex heterogeneous systems containing multiple distinct cell populations and are supported by an aberrant network of blood vessels. A better understanding of GBM metabolism, its variation with respect to the tumor microenvironment, and resulting regional changes in chemical composition is required. This may shed light on the observed heterogeneous drug distribution, which cannot be fully described by limited or uneven disruption of the blood-brain barrier. In this work, we used mass spectrometry imaging (MSI) to map metabolites and lipids in patient-derived xenograft models of GBM. A data analysis workflow revealed that distinctive spectral signatures were detected from different regions of the intracranial tumor model. A series of long-chain acylcarnitines were identified and detected with increased intensity at the tumor edge. A 3D MSI dataset demonstrated that these molecules were observed throughout the entire tumor/normal interface and were not confined to a single plane. mRNA sequencing demonstrated that hallmark genes related to fatty acid metabolism were highly expressed in samples with higher acylcarnitine content. These data suggest that cells in the core and the edge of the tumor undergo different fatty acid metabolism, resulting in different chemical environments within the tumor. This may influence drug distribution through changes in tissue drug affinity or transport and constitute an important consideration for therapeutic strategies in the treatment of GBM. SIGNIFICANCE: GBM tumors exhibit a metabolic gradient that should be taken into consideration when designing therapeutic strategies for treatment.See related commentary by Tan and Weljie, p. 1231.

Maintenance of Deep Lung Architecture and Automated Airway Segmentation for 3D Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) is a technique for mapping the spatial distributions of molecules in sectioned tissue. Histology-preserving tissue preparation methods are central to successful MSI studies. Common fixation methods, used to preserve tissue morphology, can result in artifacts in the resulting MSI experiment including delocalization of analytes, altered adduct profiles, and loss of key analytes due to irreversible cross-linking and diffusion. This is especially troublesome in lung and airway samples, in which histology and morphology is best interpreted from 3D reconstruction, requiring the large and small airways to remain inflated during analysis. Here, we developed an MSI-compatible inflation containing as few exogenous components as possible, forgoing perfusion, fixation, and addition of salt solutions upon inflation that resulted in an ungapped 3D molecular reconstruction through more than 300 microns. We characterized a series of polyunsaturated phospholipids (PUFA-PLs), specifically phosphatidylinositol (-PI) lipids linked to lethal inflammation in bacterial infection and mapped them in serial sections of inflated mouse lung. PUFA-PIs were identified using spatial lipidomics and determined to be determinant markers of major airway features using unsupervised hierarchical clustering. Deep lung architecture was preserved using this inflation approach and the resulting sections are compatible with multiple MSI modalities, automated interpretation software, and serial 3D reconstruction.

Automatic 3D Nonlinear Registration of Mass Spectrometry Imaging and Magnetic Resonance Imaging Data.

Multimodal integration between mass spectrometry imaging (MSI) and radiology-established modalities such as magnetic resonance imaging (MRI) would allow the investigations of key questions in complex biological systems such as the central nervous system. Such integration would provide complementary multiscale data to bridge the gap between molecular and anatomical phenotypes, potentially revealing new insights into molecular mechanisms underlying anatomical pathologies presented on MRI. Automatic coregistration between 3D MSI/MRI is a computationally challenging process due to dimensional complexity, MSI data sparsity, lack of direct spatial-correspondences, and nonlinear tissue deformation. Here, we present a new computational approach based on stochastic neighbor embedding to nonlinearly align 3D MSI to MRI data, identify and reconstruct biologically relevant molecular patterns in 3D, and fuse the MSI datacube to the MRI space. We demonstrate our method using multimodal high-spectral resolution matrix-assisted laser desorption ionization (MALDI) 9.4 T MSI and 7 T in vivo MRI data, acquired from a patient-derived, xenograft mouse brain model of glioblastoma following administration of the EGFR inhibitor drug of Erlotinib. Results show the distribution of some identified molecular ions of the EGFR inhibitor erlotinib, a phosphatidylcholine lipid, and cholesterol, which were reconstructed in 3D and mapped to the MRI space. The registration quality was evaluated on two normal mouse brains using the Dice coefficient for the regions of brainstem, hippocampus, and cortex. The method is generic and can therefore be applied to hyperspectral images from different mass spectrometers and integrated with other established in vivo imaging modalities such as computed tomography (CT) and positron emission tomography (PET).

3D MALDI mass spectrometry imaging reveals specific localization of long-chain acylcarnitines within a 10-day time window of spinal cord injury

We report, for the first time, the detection and specific localization of long-chain acylcarnitines (LC ACs) along the lesion margins in an experimental model of spinal cord injury (SCI) using 3D mass spectrometry imaging (MSI). Acylcarnitines palmitoylcarnitine (AC(16:0)), palmitoleoylcarnitine (AC(16:1)), elaidic carnitine (AC(18:1)) and tetradecanoylcarnitine (AC(14:1)) were detected as early as 3 days post injury, and were present along the lesion margins 7 and 10 days after SCI induced by balloon compression technique in the rat. 3D MSI revealed the heterogeneous distribution of these lipids across the injured spinal cord, appearing well-defined at the lesion margins rostral to the lesion center, and becoming widespread and less confined to the margins at the region located caudally. The assigned acylcarnitines co-localize with resident microglia/macrophages detected along the lesion margins by immunofluorescence. Given the reported pro-inflammatory role of these acylcarnitines, their specific spatial localization along the lesion margin could hint at their potential pathophysiological roles in the progression of SCI.

Interactive Visual Exploration of 3D Mass Spectrometry Imaging Data Using Hierarchical Stochastic Neighbor Embedding Reveals Spatiomolecular Structures at Full Data Resolution.

Technological advances in mass spectrometry imaging (MSI) have contributed to growing interest in 3D MSI. However, the large size of 3D MSI data sets has made their efficient analysis and visualization and the identification of informative molecular patterns computationally challenging. Hierarchical stochastic neighbor embedding (HSNE), a nonlinear dimensionality reduction technique that aims at finding hierarchical and multiscale representations of large data sets, is a recent development that enables the analysis of millions of data points, with manageable time and memory complexities. We demonstrate that HSNE can be used to analyze large 3D MSI data sets at full mass spectral and spatial resolution. To benchmark the technique as well as demonstrate its broad applicability, we have analyzed a number of publicly available 3D MSI data sets, recorded from various biological systems and spanning different mass-spectrometry ionization techniques. We demonstrate that HSNE is able to rapidly identify regions of interest within these large high-dimensionality data sets as well as aid the identification of molecular ions that characterize these regions of interest; furthermore, through clearly separating measurement artifacts, the HSNE analysis exhibits a degree of robustness to measurement batch effects, spatially correlated noise, and mass spectral misalignment.

Mass spectrometry imaging goes three dimensional.

Two complementary approaches improve 3D mass spectrometry imaging of biomolecules on 3D surfaces and within tissue sections and single cells.

Application of 3D Mass Spectrometry Imaging to TKIs

Mass spectrometry imaging (MSI) allows visualization of endogenous and exogenous compound in tissue sections based on its molecular mass. The 3D reconstruction by MSI provides a more informative description of the tumor drug distribution compared to the high-performance liquid chromatography method, highlighting the heterogeneity of intratumor drug concentration. This additional information can be important in understanding chemoresistance to target agents. Here, we present the 3D visualization of the tyrosine kinase inhibitor (TKI), imatinib, in a xenograft model of resistant malignant pleural mesothelioma.

3D-Plant2cells project: Spatial metabolomic, spatial metagenomic, and 3D mass spectrometry imaging, to explore the impact of pesticides on plant metabolome and microbiota

The 3D­Plant2Cells project investigates the impact of the agricultural mode on crops grown for human consumption. We are developing an innovative interdisciplinary approach to study the metabolome and the microbiota of plants in three­ dimension (3D) at three different scales: the whole plant, the fruit, and the cellular scales. The objective of this study is to get a new insight on the metabolome and microbiota response to pesticide treatment. The hypothesis is that the chemistry and microbiome could change upon the use of conventional cultivation managements. To explore this hypothesis, two groups of plants are cultivated in growth chamber and one receives pesticide treatments. The metabolome and the plant­ associated microbiota are explored on the whole plant surface (plant scale) by using an innovative interdisciplinary approach [1,2]. Samples are collected on the whole plant and their metabolite content profiled by high­resolution tandem­ mass spectrometry (HR­MS/MS) and plant­associated microorganism communities by amplicon sequence analysis (metagenomics) [1]. Results of these both inputs are compiled into a bioinformatic model representing a 3D topological map of the metabolome/microbiome over the entire organism surface [1]. Mass spectrometry data will be further interpreted using MS/MS molecular networking [1]. In the meantime, we are studying fruits of the cultivated specimens by 3D mass spectrometry imaging to study the metabolome/microbiota response to pesticide treatments along with the pesticide distribution in 3D. The fruit and cell scale are explored by analyzing serial sections using MALDI, nanoDESI, 3D­ TOF­SIMS coupled to HR­MS/MS. The presentation will aim at introducing the project, and to highlight some methodological approaches used to study the microbiota­chemistry relationships in species or ecosystem.

3D Mass Spectrometry Imaging Reveals a Very Heterogeneous Drug Distribution in Tumors.

Mass Spectrometry Imaging (MSI) is a widespread technique used to qualitatively describe in two dimensions the distribution of endogenous or exogenous compounds within tissue sections. Absolute quantification of drugs using MSI is a recent challenge that just in the last years has started to be addressed. Starting from a two dimensional MSI protocol, we developed a three-dimensional pipeline to study drug penetration in tumors and to develop a new drug quantification method by MALDI MSI. Paclitaxel distribution and concentration in different tumors were measured in a 3D model of Malignant Pleural Mesothelioma (MPM), which is known to be a very heterogeneous neoplasm, highly resistant to different drugs. The 3D computational reconstruction allows an accurate description of tumor PTX penetration, adding information about the heterogeneity of tumor drug distribution due to the complex microenvironment. The use of an internal standard, homogenously sprayed on tissue slices, ensures quantitative results that are similar to those obtained using HPLC. The 3D model gives important information about the drug concentration in different tumor sub-volumes and shows that the great part of each tumor is not reached by the drug, suggesting the concept of pseudo-resistance as a further explanation for ineffective therapies and tumors relapse.

Single-Cell Analysis: Visualizing Pharmaceutical and Metabolite Uptake in Cells with Label-Free 3D Mass Spectrometry Imaging.

Detecting metabolites and parent compound within a cell type is now a priority for pharmaceutical development. In this context, three-dimensional secondary ion mass spectrometry (SIMS) imaging was used to investigate the cellular uptake of the antiarrhythmic agent amiodarone, a phospholipidosis-inducing pharmaceutical compound. The high lateral resolution and 3D imaging capabilities of SIMS combined with the multiplex capabilities of ToF mass spectrometric detection allows for the visualization of pharmaceutical compound and metabolites in single cells. The intact, unlabeled drug compound was successfully detected at therapeutic dosages in macrophages (cell line: NR8383). Chemical information from endogenous biomolecules was used to correlate drug distributions with morphological features. From this spatial analysis, amiodarone was detected throughout the cell, with the majority of the compound found in the membrane and subsurface regions and absent in the nuclear regions. Similar results were obtained when the macrophages were doped with amiodarone metabolite, desethylamiodarone. The fwhm lateral resolution measured across an intracellular interface in high lateral resolution ion images was approximately 550 nm. Overall, this approach provides the basis for studying cellular uptake of pharmaceutical compounds and their metabolites on the single cell level.

Serial 3D imaging mass spectrometry at its tipping point.

Since biology is by and large a 3-dimensional phenomenon, it is hardly surprising that 3D imaging has had a significant impact on many challenges in the life sciences. Imaging mass spectrometry (MS) is a spatially resolved label-free analytical technique that recently maturated into a powerful tool for in situ localization of hundreds of molecular species. Serial 3D imaging MS reconstructs 3D molecular images from serial sections imaged with mass spectrometry. As such, it provides a novel 3D imaging modality inheriting the advantages of imaging MS. Serial 3D imaging MS has been steadily developing over the past decade, and many of the technical challenges have been met. Essential tools and protocols were developed, in particular to improve the reproducibility of sample preparation, speed up data acquisition, and enable computationally intensive analysis of the big data generated. As a result, experimental data is starting to emerge that takes advantage of the extra spatial dimension that 3D imaging MS offers. Most studies still focus on method development rather than on exploring specific biological problems. The future success of 3D imaging MS requires it to find its own niche alongside existing 3D imaging modalities through finding applications that benefit from 3D imaging and at the same time utilize the unique chemical sensitivity of imaging mass spectrometry. This perspective critically reviews the challenges encountered during the development of serial-sectioning 3D imaging MS and discusses the steps needed to tip it from being an academic curiosity into a tool of choice for answering biological and medical questions.