MALDI Imaging Research Articles

Multiple ion isolation and accumulation events for selective chemical noise reduction and dynamic range enhancement in MALDI imaging mass spectrometry.

Abundant chemical noise in MALDI imaging mass spectrometry experiments can impede the detection of less abundant compounds of interest. This chemical noise commonly originates from the MALDI matrix as well as other endogenous compounds present in high concentrations and/or with high ionization efficiencies. MALDI imaging mass spectrometry of biological tissues measures numerous biomolecular compounds that exist in a wide range of concentrations in vivo. When ion trapping instruments are used, highly abundant ions can dominate the charge capacity and lead to space charge effects that hinder the dynamic range and detection of lowly abundant compounds of interest. Gas-phase fractionation has been previously utilized in mass spectrometry to isolate and enrich target analytes. Herein, we have characterized the use of multiple continuous accumulations of selected ions (Multi CASI) to reduce the abundance of chemical noise and diminish the effects of space charge in MALDI imaging mass spectrometry experiments. Multi CASI utilizes the mass-resolving capability of a quadrupole mass filter to perform multiple sequential ion isolation events prior to a single mass analysis of the combined ion population. Multi CASI was used to improve metabolite and lipid detection in the MALDI imaging mass spectrometry analysis of rat brain tissue.

The glycosylation landscape of prostate cancer tissues and biofluids.

An overview of the role of glycosylation in prostate cancer (PCa) development and progression is presented, focusing on recent advancements in defining the N-glycome through glycomic profiling and glycoproteomic methodologies. Glycosylation is a common post-translational modification typified by oligosaccharides attached N-linked to asparagine or O-linked to serine or threonine on carrier proteins. These attached sugars have crucial roles in protein folding and cellular recognition processes, such that altered glycosylation is a hallmark of cancer pathogenesis and progression. In the past decade, advancements in N-glycan profiling workflows using Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) technology have been applied to define the spatial distribution of glycans in PCa tissues. Multiple studies applying N-glycan MALDI-MSI to pathology-defined PCa tissues have identified significant alterations in N-glycan profiles associated with PCa progression. N-glycan compositions progressively increase in number, and structural complexity due to increased fucosylation and sialylation. Additionally, significant progress has been made in defining the glycan and glycopeptide compositions of prostatic-derived glycoproteins like prostate-specific antigen in tissues and biofluids. The glycosyltransferases involved in these changes are potential drug targets for PCa, and new approaches in this area are summarized. These advancements will be discussed in the context of the further development of clinical diagnostics and therapeutics targeting glycans and glycoproteins associated with PCa progression. Integration of large scale spatial glycomic data for PCa with other spatial-omic methodologies is now feasible at the tissue and single-cell levels.

Preclinical Activity of Two Paclitaxel Nanoparticle Formulations After Intraperitoneal Administration in Ovarian Cancer Murine Xenografts.

Epithelial ovarian cancer is associated with high mortality due to diagnosis at later stages associated with peritoneal involvement. Several trials have evaluated the effect of intraperitoneal treatment. In this preclinical study, we report the efficacy, pharmacokinetics and pharmacodynamics of intraperitoneal treatment with two approved nanomolecular formulations of paclitaxel (nab-PTX and mic-PTX) in a murine ovarian cancer xenograft model. IC50 was determined in vitro on three ovarian cancer cell lines (OVCAR-3, SK-OV-3 and SK-OV-3-Luc IP1). EOC xenografts were achieved using a modified subperitoneal implantation technique. Drug treatment was initiated 2 weeks after engraftment, and tumor volume and survival were assessed. Pharmacokinetics and drug distribution effects were assessed using UHPLC-MS/MS and MALDI imaging mass spectrometry, respectively. Pharmacodynamic effects were analyzed using immunohistochemistry and transmission electron microscopy using standard protocols. We demonstrated sub-micromolar IC50 concentrations for both formulations on three EOC cancer cell lines in vitro. Furthermore, IP administration of nab-PTX or mic-PTX lead to more than 2-fold longer survival compared to a control treatment of IP saline administration (30 days in controls, 66 days in nab-PTX treated animals, and 76 days in mic-PTX animals, respectively). We observed higher tissue uptake of drug following nab-PTX administration when compared to mic-PTX, with highest uptake after 4 hours post-treatment, and confirmed this lower uptake of mic-PTX using HPLC on digested tumor samples. Furthermore, apoptosis was not increased in tumor implants up to 24h post-treatment. Intraperitoneal administration of both nab-PTX and mic-PTX results in a significant anticancer efficacy and survival benefit in a mouse OC xenograft model.

Multimodal MALDI imaging mass spectrometry for improved diagnosis of melanoma.

Imaging mass spectrometry (IMS) provides promising avenues to augment histopathological investigation with rich spatio-molecular information. We have previously developed a classification model to differentiate melanoma from nevi lesions based on IMS protein data, a task that is challenging solely by histopathologic evaluation. Most IMS-focused studies collect microscopy in tandem with IMS data, but this microscopy data is generally omitted in downstream data analysis. Microscopy, nevertheless, forms the basis for traditional histopathology and thus contains invaluable morphological information. In this work, we developed a multimodal classification pipeline that uses deep learning, in the form of a pre-trained artificial neural network, to extract the meaningful morphological features from histopathological images, and combine it with the IMS data. To test whether this deep learning-based classification strategy can improve on our previous results in classification of melanocytic neoplasia, we utilized MALDI IMS data with collected serial H&E stained sections for 331 patients, and compared this multimodal classification pipeline to classifiers using either exclusively microscopy or IMS data. The multimodal pipeline achieved the best performance, with ROC-AUCs of 0.968 vs. 0.938 vs. 0.931 for the multimodal, unimodal microscopy and unimodal IMS pipelines respectively. Due to the use of a pre-trained network to perform the morphological feature extraction, this pipeline does not require any training on large amounts of microscopy data. As such, this framework can be readily applied to improve classification performance in other experimental settings where microscopy data is acquired in tandem with IMS experiments.

Mobilized Electrospray Device for On-Tissue Chemical Derivatization in MALDI-MS Imaging.

Applying solutions of a matrix or derivatization agent via microdroplets is a common sample preparation technique for matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) experiments. Mobilized nebulizer sprayers are commonly used to create a homogeneous matrix or reagent layer across large surfaces. Electrospray devices have also been used to produce microdroplets for the same purpose but are rarely used for large tissues due to their immobility. Herein, we present a movable electrospray device that can be used for large tissue sample preparation through a simple modification to an automatic commercial nebulizer device. As demonstrated for on-tissue chemical derivatization (OTCD) with Girard's reagent T using a mimetic tissue model, the sprayer has the additional benefit of being able to investigate reaction acceleration in OTCD when comparing electrostatically charged spray to electrostatically neutral spray. Finally, MALDI-MSI of fatty aldehydes is successfully demonstrated in rat brain tissues using this device for both OTCD and matrix application.

Spatialized Metabolomic Annotation Combining MALDI Imaging and Molecular Networks.

MALDI mass spectrometry imaging has gained major interest in the field of chemical imaging. This technique makes it possible to locate tens to hundreds of ionic signals on the sample surface without any a priori. One of the current challenges is still the limited ability to annotate signals in order to convert m/z values into probable chemical structures. At the same time, data obtained by LC-MS/MS have benefited from the development of numerous chemoinformatics tools, in particular molecular networks, for their efficient annotation. For the first time, we present here the combination of MALDI-FT-ICR imaging with molecular networks from MALDI-MS/MS data directly acquired on plant tissue sections. Annotation improvements are demonstrated, paving the way for new annotation pipelines for MALDI imaging.

Ultrahigh-Mass Resolution Mass Spectrometry Imaging with an Orbitrap Externally Coupled to a High-Performance Data Acquisition System.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool that enables molecular sample analysis while simultaneously providing the spatial context of hundreds or even thousands of analytes. However, because of the lack of a separation step prior to ionization and the immense diversity of biomolecules, such as lipids, including numerous isobaric species, the coupling of ultrahigh mass resolution (UHR) with MSI presents one way in which this complexity can be resolved at the spectrum level. Until now, UHR MSI platforms have been restricted to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Here, we demonstrate the capabilities of an Orbitrap-based UHR MSI platform to reach over 1,000,000 mass resolution in a lipid mass range (600-950 Da). Externally coupling the Orbitrap Q Exactive HF with the high-performance data acquisition system FTMS Booster X2 provided access to the unreduced data in the form of full-profile absorption-mode FT mass spectra. In addition, it allowed us to increase the time-domain transient length from 0.5 to 10 s, providing improvement in the mass resolution, signal-to-noise ratio, and mass accuracy. The resulting UHR performance generates high-quality MALDI MSI images and simplifies the identification of lipids. Collectively, these improvements resulted in a 1.5-fold increase in annotations, demonstrating the advantages of this UHR imaging platform for spatial lipidomics using MALDI-MSI.

Improving the Signal Intensity of Cryosections Using a Conductive Adhesive Film in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique was used to obtain the molecular images of cryosections without labeling. Although MALDI-MSI has been widely used to detect small molecules from biological tissues, issues remain due to the technical process of cryosectioning and limited mass spectrometry parameters. The use of a conductive adhesive film is a unique method to obtain high-quality sections from cutting tissue, such as bone, muscle, adipose tissue, and whole body of mice or fish, and we have reported the utilization of the film for MALDI-MSI in previous. However, some signal of the small molecules using the conductive adhesive films was still lower than on the indium tin oxide (ITO) glass slide. Here, the sample preparation and analytical conditions for MALDI-MSI using an advanced conductive adhesive film were optimized to obtain strong signals from whole mice heads. The effects of tissue thickness and laser ionization power on signal intensity were verified using MALDI-MSI. The phospholipid signal intensity was measured for samples with three tissue thicknesses (5, 10, and 20 μm); compared to the signals from the samples on the ITO glass slides, the signals with conductive adhesive films exhibited significantly higher intensities when a laser with a higher range of power was used to ionize the small molecules. Thus, the technique using the advanced conductive adhesive film showed an improvement in MALDI-MSI analysis.

Mass spectrometry imaging of natural carbonyl products directly from agar-based microbial interactions using 4-APEBA derivatization.

Aliphatic carboxylic acids, aldehydes, and ketones play diverse roles in microbial adaptation to their microenvironment, from excretion as toxins to adaptive metabolites for membrane fluidity. However, the spatial distribution of these molecules throughout biofilms and how microbes in these environments exchange these molecules remain elusive for many of these bioactive species due to inefficient molecular imaging strategies. Herein, we apply on-tissue chemical derivatization (OTCD) using 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) on a co-culture of a soil bacterium (Bacillus subtilis NCIB 3610) and fungus (Fusarium sp. DS 682) grown on agar as our model system. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we spatially resolved more than 300 different metabolites containing carbonyl groups within this model system. Various spatial patterns are observable in these species, which indicate possible extracellular or intercellular processes of the metabolites and their up- or downregulation during microbial interaction. The unique chemistry of our approach allowed us to bring additional confidence in accurate carbonyl identification, especially when multiple isomeric candidates were possible, and this provided the ability to generate hypotheses about the potential role of some aliphatic carbonyls in this B. subtilis/Fusarium sp. interaction. The results shown here demonstrate the utility of 4-ABEBA-based OTCD MALDI-MSI in probing interkingdom interactions directly from microbial co-cultures, and these methods will enable future microbial interaction studies with expanded metabolic coverage.IMPORTANCEThe metabolic profiles within microbial biofilms and interkingdom interactions are extremely complex and serve a variety of functions, which include promoting colonization, growth, and survival within competitive and symbiotic environments. However, measuring and differentiating many of these molecules, especially in an in situ fashion, remains a significant analytical challenge. We demonstrate a chemical derivatization strategy that enabled highly sensitive, multiplexed mass spectrometry imaging of over 300 metabolites from a model microbial co-culture. Notably, this approach afforded us to visualize over two dozen classes of ketone-, aldehyde-, and carboxyl-containing molecules, which were previously undetectable from colonies grown on agar. We also demonstrate that this chemical derivatization strategy can enable the discrimination of isobaric and isomeric metabolites without the need for orthogonal separation (e.g., online chromatography or ion mobility). We anticipate that this approach will further enhance our knowledge of metabolic regulation within microbiomes and microbial systems used in bioengineering applications.

Multimodal analytical tools for the molecular and elemental characterisation of lesions in brain tissue of multiple sclerosis patients

Multiple sclerosis (MS) is a prevalent immune-mediated inflammatory disease of the central nervous system inducing a widespread degradation of myelin and resulting in neurological deficits. Recent advances in molecular and atomic imaging provide the means to probe the microenvironment in affected brain tissues at an unprecedented level of detail and may provide new insights. This study showcases state-of-the-art spectroscopic and mass spectrometric techniques to compare distributions of molecular and atomic entities in MS lesions and surrounding brain tissues.MS brains underwent post-mortem magnetic resonance imaging (MRI) to locate and subsequently dissect MS lesions and surrounding white matter. Digests of lesions and unaffected white matter were analysed via ICP-MS/MS revealing significant differences in concentrations of Li, Mg, P, K, Mn, V, Rb, Ag, Gd and Bi. Micro x-ray fluorescence spectroscopy (μXRF) and laser ablation – inductively coupled plasma – time of flight – mass spectrometry (LA-ICP-ToF-MS) were used as micro-analytical imaging techniques to study distributions of both endogenous and xenobiotic elements. The essential trace elements Fe, Cu and Zn were subsequently calibrated using in-house manufactured gelatine standards. Lipid distributions were studied using IR-micro spectroscopy and matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI). MALDI-MSI was complemented with high-resolution tandem mass spectrometry and trapped ion mobility spectroscopy for the annotation of specified phospho- and sphingolipids, revealing specific lipid species decreased in MS lesions compared to surrounding white matter.This explorative study demonstrated that modern molecular and atomic mapping techniques provide high-resolution imaging for relevant bio-indicative entities which may complement our current understanding of the underlying pathophysiological processes.

Lipidomic Phenotyping Of Human Small Intestinal Organoids Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

In the past decade, interest in organoids for biomedical research has surged, resulting in a higher demand for advanced imaging techniques. Traditional specimen embedding methods pose challenges, such as analyte delocalization and histological assessment. Here, we present an optimized sample preparation approach utilizing an Epredia M-1 cellulose-based embedding matrix, which preserves the structural integrity of fragile small intestinal organoids (SIOs). Additionally, background interference (delocalization of analytes, nonspecific (histological) staining, matrix ion clusters) was minimized, and we demonstrate the compatibility with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). With our approach, we can conduct label-free lipid imaging at the single-cell level, thereby yielding insights into the spatial distribution of lipids in both positive and negative ion modes. Moreover, M-1 embedding allows for an improved coregistration with histological and immunohistochemical (IHC) stainings, including MALDI-IHC, facilitating combined untargeted and targeted spatial information. Applying this approach, we successfully phenotyped crypt-like (CL) and villus-like (VL) SIOs, revealing that PE 36:2 [M - H]- (m/z 742.5) and PI 38:4 [M - H]- (m/z 885.5) display higher abundance in CL organoids, whereas PI 36:1 [M - H]- (m/z 863.6) was more prevalent in VL organoids. Our findings demonstrate the utility of M-1 embedding for advancing organoid research and unraveling intricate biological processes within these in vitro models.

Unraveling spatial metabolome of the aerial and underground parts of Scutellaria baicalensis by matrix-assisted laser desorption/ionization mass spectrometry imaging

BackgroundScutellaria baicalensis Georgi, a traditional Chinese medicine, is clinically applied mainly as the dried root of Scutellaria baicalensis, and the aerial parts of Scutellaria baicalensis, its stems and leaves, are often consumed as "Scutellaria baicalensis tea" to clear heat, dry dampness, reduce fire and detoxify, while few comparative analyses of the spatial metabolome of the aerial and underground parts of Scutellaria baicalensis have been carried out in current research. MethodsIn this work, Matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) was used to visualize the spatial imaging of the root, stem, and leaf of Scutellaria baicalensis at a high resolution of 10 μm, respectively, investigating the spatial distribution of the different secondary metabolites in the aerial and underground parts of Scutellaria baicalensis. ResultsIn the present results, various metabolites, such as flavonoid glycosides, flavonoid metabolites, and phenolic acids, were systematically characterized in Scutellaria baicalensis root, stem, and leaf. Nine glycosides, 18 flavonoids, one organic acid, and four other metabolites in Scutellaria baicalensis root; nine glycosides, nine flavonoids, one organic acid in Scutellaria baicalensis stem; and seven flavonoids and seven glycosides in Scutellaria baicalensis leaf were visualized by MALDI-MSI. In the underground part of Scutellaria baicalensis, baicalein, wogonin, baicalin, wogonoside, and chrysin were widely distributed, while there was less spatial location in the aerial parts. Moreover, scutellarein, carthamidin/isocarthamidin, scutellarin, carthamidin/isocarthamidin-7-O-glucuronide had a high distribution in the aerial parts of Scutellaria baicalensis. In addition, the biosynthetic pathways involved in the biosynthesis of significant flavonoid metabolites in aerial and underground parts of Scutellaria baicalensis were successfully localized and visualized. ConclusionsMALDI-MSI offers a favorable approach for investigating the spatial distribution and effective utilization of metabolites of Scutellaria baicalensis. The detailed spatial chemical information can not only improve our understanding of the biosynthesis pathways of flavonoid metabolites, but more importantly, suggest that we need to fully exert the overall medicinal value of Scutellaria baicalensis, strengthening the reuse and development of the resources of Scutellaria baicalensis aboveground parts.

Abstract A039: Detection of immune cell glycosylation as an indicator of metabolic activity in the tumor tissue microenvironment using multimodal mass spectrometry imaging

Abstract Glycosylation on the cell surface is a major target and mediator of the immune response to cancers. It is expected that any immune cells present in these tissues will be actively engaged in responding to the presence of the tumor, however, multiple escape mechanisms used by the targets are known to suppress these immune responses. It follows that in this tumor microenvironment, any response, or non-response, to a cancer immunotherapeutic will also be mediated by glycans present in the target tissues and immune cells interacting with them. From a data archive of over 500 FFPE human tumor tissues assessed for N-glycan imaging MS analysis, a subset of tissues (prostate, colon, pancreas, lung) with notable intra- and peri-tumor immune cell clusters were re-evaluated for detection of N-glycans that co-localize to these regions. The metabolic premise for this is that the Warburg metabolites glucose and glutamine are both required for N-glycan biosynthesis and therefore active immune cells would have detectable glycan signatures. In most tumor types evaluated, there was minimal to no detection of N-glycans. This would be consistent with a tumor microenvironment deficient in metabolites due to the presence of the tumor, or other immunosuppressive mechanisms. A subset of the tissues did have glycan signatures associated with immune cell clusters, and the glycan structures present in each cluster were recorded. The same tissues were assessed by multiplexed MALDI-immunohistochemistry (IHC) to identify the immune cell types present in each cluster. Established method workflows were used for the N-glycan MALDI imaging and MALDI-IHC analyses. Previously characterized N-glycans and immune cell clusters (CD4, CD8, CD11b, CD163) in SARS-CoV2 infected autopsy lung tissues were used as positive controls for an active immune microenvironment. Distinct N-glycan species are associated with each immune cell type in these tissues. Detected N-glycans included high mannose structures, and a series of tri- and tetra-antennary structures with one fucose and a bisecting N-acetylglucosamine (GlcNAc). While the high mannose glycans were detected in other areas of the tissues, the bisecting branched N-glycans were distinctly enriched in the immune cell clusters. In these selected tissue subsets, immune cell clusters distal to tumor regions had readily detected high mannose N-glycans and tri-and tetra-antennary bisecting GlcNAc structure. In general, the immune cell clusters adjacent to the tumor region had minimal to no glycan expression relative to the more distal regions. We hypothesize that when N-glycans are detected by imaging mass spectrometry in tissue immune cell clusters that this represents tumors with more active immune functional states, and the lack of detection represents immune-suppressed tumors. Citation Format: Richard R Drake, Kameisha Radford, Caroline Kittrell, Kristin Wallace, Peggi M Angel. Detection of immune cell glycosylation as an indicator of metabolic activity in the tumor tissue microenvironment using multimodal mass spectrometry imaging [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A039.

2,5-Dihydroxyterephthalic Acid: A Matrix for Improved Detection and Imaging of Amino Acids.

Amino acids (AAs), which are low-molecular-weight (low-MW) metabolites, serve as essential building blocks not only for protein synthesis but also for maintaining the nitrogen balance in living systems. In situ detection and imaging of AAs are crucial for understanding more complex biological processes. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a label-free mass spectrometric imaging technique that enables the simultaneous detection and imaging of the spatial distribution and relative abundance of different endogenous/exogenous compounds in biological samples. The excellent efficiency of MALDI-MSI is attributed to the choice of the MALDI matrix. However, to the best of our knowledge, no matrix has been specifically developed for AAs. Herein, we report a MALDI matrix, 2,5-dihydroxyterephthalic acid (DHT), which can improve the detection and imaging of AAs in biological samples by MALDI-MS. Our results indicated that DHT exhibited strong ultraviolet-visible (UV-vis) absorption, uniform matrix deposition, and high vacuum stability. Moreover, the matrix-related ion signals produced from DHT were reduced by 50 and 71.8% at m/z < 500 compared to the commonly used matrices of 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA), respectively, in their respective organic solvents. In terms of quantitative performance, arginine, glutamic acid, glutamine, and proline can be detected with limits of detection of 6, 4, 6, and 4 ng/mL, respectively, using the DHT as the matrix. Using DHT as the matrix, all 20 protein AAs were successfully detected in human serum by MALDI-MS, whereas only 7 and 10 AAs were detected when DHB and CHCA matrices were used, respectively. Furthermore, 20 protein AAs and taurine were successfully detected and imaged in a section of edible Crassostrea gigas (oyster) tissue for the first time. Our study demonstrates that using DHT as a matrix can improve the detection and imaging of AAs in biological samples by MALDI-MS.

Mass spectrometry imaging reveals flavor distribution in edible mushrooms

The spatial distribution of molecules and compounds responsible for the flavor profile of edible button mushrooms (Agaricus bisporous) has never been determined. The food industry is interested in knowing the localization of these compounds. Such knowledge would enable extraction of flavor compounds from a particular regions of the mushroom, which is safer for consumption compared to alternatives such as synthetic flavoring agents. The present study utilizes matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI), to determine the spatial distribution of flavor compounds in a mushroom. As MALDI-MSI requires very thin sections, a sample preparation protocol was optimized and sectioning fresh frozen mushrooms at 35 µm thickness was considered the best method to evaluate the distribution of flavor compounds. Further, the effect of heat on the spatial distribution of flavor compounds was investigated by heating whole mushrooms to 140 ℃ prior to sectioning. Heating reduced the water content of the mushroom and thus enabled the generation of even-thinner 17 µm thick sections. MALDI-MSI measurements performed on underivatized and on-tissue derivatized fresh frozen and heat-treated mushroom sections elucidated the spatial distribution of several flavor-related compounds.

Advances in cellular and tissue-based imaging techniques for sarcoid granulomas.

Sarcoidosis embodies a complex inflammatory disorder spanning multiple systems, with its origin remaining elusive. It manifests as the infiltration of inflammatory cells that coalesce into distinctive noncaseous granulomas within afflicted organs. Unraveling this disease necessitates the utilization of cellular or tissue-based imaging methods to both visualize and characterize the biochemistry of these sarcoid granulomas. Although hematoxylin and eosin stain, standard in routine use alongside cytological stains have found utility in diagnosis within clinical contexts, special stains such as Masson's trichrome, reticulin, methenamine silver, and Ziehl-Neelsen provide additional varied perspectives of sarcoid granuloma imaging. Immunohistochemistry aids in pinpointing specific proteins and gene expressions further characterizing these granulomas. Finally, recent advances in spatial transcriptomics promise to divulge profound insights into their spatial orientation and three-dimensional (3-D) molecular mapping. This review focuses on a range of preexisting imaging methods employed for visualizing sarcoid granulomas at the cellular level while also exploring the potential of the latest cutting-edge approaches like spatial transcriptomics and matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), with the overarching goal of shedding light on the trajectory of sarcoidosis research.

Porous silicon microneedle patches for delivery of polymyxin‐based antimicrobials

AbstractThere is a global trend of increasing antibiotic resistance to Gram‐negative bacteria and the medical research community is looking for alternative antibiotics. Antibiotics such as polymyxin B (PMB) show excellent efficacy and low resistance rates against Gram‐negative bacteria. The topical administration of PMB, however, has clinical limitations such as minimal residence time, making it difficult for its use to treat skin infections. Here, we present porous silicon microneedle (pSi MN) patches to deliver PMB to the skin. pSi MN patches are fabricated by, first, generating the projections by dry Si etching techniques, followed by an electrochemical etching to create porous layers around the MNs. After loading PMB into the porous layer, the antibacterial activity of pSi MNs is determined by measuring the inhibition zone of Gram‐negative Escherichia coli. Next, PMB is efficiently delivered into the epidermal layer of ex vivo skin models, as confirmed by chemical mapping using matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI). The results demonstrate that pSi MN patches are an efficient and versatile tool for the transdermal delivery of PMB with maintained bioactivity and antimicrobial efficacy, thus expanding the possibilities of current transdermal drug delivery systems.

B4galt1 Regulates the WNT-β-Catenin Axis to Control Hematopoietic Stem and Progenitor Cells (HSPCs) Fitness

In achieving the optimal numbers of fully functional blood cells across different lineages, intrinsic developmental programs within the cells and external guidance mechanisms are crucial. Glycans on the cell surface and extracellular environment are pivotal in regulating cell maintenance, differentiation, and function. We recently discovered that the glycosyltransferase β-1,4-galactosyltransferase 1 (B4galt1) leads to dysplastic megakaryocytes, impaired thrombopoiesis, and increased hematopoietic stem cells (HSC). To investigate the relationship between B4galt-dependent glycosylation and HSC function, we analyzed the glycan composition in control and B4galt1 null (B4 -/-) femurs by spatial mass spectrometry using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI). In control femurs, we observed a distinct gradient of complex N-glycans, with higher expression at the distal ends and decreasing complexity towards the shaft, showcasing diverse glycan expression in the hematopoietic environment. However, this gradient was absent in B4 -/-femurs, where we detected an increase of immature N-glycans and a concurrent decrease in complex N-glycan structures (p&amp;lt;0.05). Additionally, we observed aberrant O-glycosylation in B4 -/- LT-HSCs, as determined using lectin arrays. The glycan composition in the absence of B4galt1-dependent glycosylation mirrors cancer-associated glycan patterns. To further understand the role of B4galt1 in steady-state hematopoiesis, we next examined the bone marrow (BM) immunophenotypic composition of B4 -/- mice. Flow cytometry analysis revealed a significant expansion of immunophenotypically defined long term (LT)-HSCs (p=0.0061) and multipotent progenitors (MPPs) (p&amp;lt;0.05) in B4 -/-BM. Additionally, B4 -/- LT-HSCs showed higher expression of the platelet marker CD41 (p=0.0171), suggesting the presence of an expanded megakaryocyte (MK) biased LT-HSCs population (LT-HSC CD41+ cells). This expansion occurred without pro-inflammatory cues, as determined through cytokine arrays. To investigate if the global transcriptional landscape in B4 -/- cells could explain the observed expansion phenotype, we conducted droplet-based single-cell RNA-seq. Transfer learning classification allowed us to identify an expansion of the transcriptional output associated with LT-HSC and MPP2 signatures in B4 -/- cells (p&amp;lt;0.05). Further transcriptional analysis revealed an increased number of B4 -/- cells expressing MKs specific markers (CD41, Pf4, GP9, Vwf, GP1ba), providing additional support for the expansion of B4 -/- MK-biased LT-HSCs. Gene Set Enrichment Analysis (GSEA) of B4 -/- LT-HSCs demonstrated enrichment in metabolism-associated pathways and cell cycle regulation (FDR&amp;lt;0.05). Conversely, critical differentiation processes at the stem cell level, such as cellular adhesion and lineage commitment, were downregulated (FDR&amp;lt;0.05). To investigate the role of B4galt1 in HSC expansion, we transcriptionally classified LT-HSCs based on their cell cycle status (G1, G2M, and S). Analysis of LT-HSC CD41+ cell cycle distribution revealed an increased in S phase in B4 -/- LT-HSC CD41+ cells. Additionally, B4 -/- LT-HSC CD41+ cells exhibited enrichment in cell cycle regulation pathways, including DNA replication, DNA strand elongation, and G1 to S cell cycle control (FDR&amp;lt;0.05). Conversely, critical pathways associated with HSC homeostasis and quiescence were depleted in the absence of B4galt1 (FDR&amp;lt;0.05). GSEA analysis unveiled upregulation of Wnt and Myc pathways in B4 -/- LT-HSC CD41+ cells. Immunofluorescence and immunoblot analysis demonstrated elevated total β-catenin levels in B4 -/- LSK cells and enhanced nuclear translocation of non-phosphorylated Ser33/37/Thr41 β-catenin. To confirm the functional significance, we treated B4 -/- LSK cells with the Wnt inhibitor XAV939 which normalized cell numbers to control levels and cause a reduction in the observed expansion at the stem and progenitor compartments. These findings suggest that B4galt1 is vital in regulating HSC expansion, likely through the Wnt/β-catenin signaling pathway. Our data provide compelling evidence that B4galt1 regulates steady-state hematopoiesis, through its influence on glycosylation in the bone marrow niche, highlighting the critical role of the B4galt1-Wnt-β-catenin axis in regulating LT-HSC fitness and expansion.

Low-melting point agarose as embedding medium for MALDI mass spectrometry imaging and laser-capture microdissection-based proteomics

The combination of MALDI mass spectrometry imaging, laser-capture microdissection, and quantitative proteomics allows the identification and characterization of molecularly distinct tissue compartments. Such workflows are typically performed using consecutive tissue sections, and so reliable sectioning and mounting of high-quality tissue sections is a prerequisite of such investigations. Embedding media facilitate the sectioning process but can introduce contaminants which may adversely affect either the mass spectrometry imaging or proteomics analyses. Seven low-temperature embedding media were tested in terms of embedding temperature and cutting performance. The two media that provided the best results (5% gelatin and 2% low-melting point agarose) were compared with non-embedded tissue by both MALDI mass spectrometry imaging of lipids and laser-capture microdissection followed by bottom-up proteomics. Two out of the seven tested media (5% gelatin and 2% low-melting point agarose) provided the best performances on terms of mechanical properties. These media allowed for low-temperature embedding and for the collection of high-quality consecutive sections. Comparisons with non-embedded tissues revealed that both embedding media had no discernable effect on proteomics analysis; 5% gelatin showed a light ion suppression effect in the MALDI mass spectrometry imaging experiments, 2% agarose performed similarly to the non-embedded tissue. 2% low-melting point agarose is proposed for tissue embedding in experiments involving MALDI mass spectrometry imaging of lipids and laser-capture microdissection, proteomics of consecutive tissue sections.

Sample Preparation Method for MALDI Mass Spectrometry Imaging of Fresh-Frozen Spines.

Technologies assessing the lipidomics, genomics, epigenomics, transcriptomics, and proteomics of tissue samples at single-cell resolution have deepened our understanding of physiology and pathophysiology at an unprecedented level of detail. However, the study of single-cell spatial metabolomics in undecalcified bones faces several significant challenges, such as the fragility of bone, which often requires decalcification or fixation leading to the degradation or removal of lipids and other molecules. As such, we describe a method for performing mass spectrometry imaging on undecalcified spine that is compatible with other spatial omics measurements. In brief, we use fresh-frozen rat spines and a system of carboxyl methylcellulose embedding, cryofilm, and polytetrafluoroethylene rollers to maintain tissue integrity while avoiding signal loss from variations in laser focus and artifacts from traditional tissue processing. This reveals various tissue types and lipidomic profiles of spinal regions at 10 μm spatial resolutions using matrix-assisted laser desorption/ionization mass spectrometry imaging. We expect this method to be adapted and applied to the analysis of the spinal cord, shedding light on the mechanistic aspects of cellular heterogeneity, development, and disease pathogenesis underlying different bone-related conditions and diseases. This study furthers the methodology for high spatial metabolomics of spines and adds to the collective efforts to achieve a holistic understanding of diseases via single-cell spatial multiomics.