Spectrometry Imaging Research Articles

High resolution imaging spectrometry using dispersion shearing interferometer with Amici prisms

High resolution imaging spectrometry using dispersion shearing interferometer with Amici prisms

UTHP spectrometer: an ultra-thin and high-performance imaging spectrometer for ocean color remote sensing in broadband

The principle of ocean color detection involves using satellite sensors to capture variations in received signals, which are subsequently analyzed to infer the concentrations of various components responsible for ocean color changes. In recent years, significant research advancements have been achieved, centered on the development of ocean color detectors and the analysis of the resulting data. We have developed an innovative ultra-thin high-performance (UTHP) scanning imaging spectrometer for ocean color remote sensing across the near ultraviolet-visible-near infrared (NUV-VIS-NIR) spectrum. This spectrometer overcomes the bulkiness of conventional equipment by integrating a waveguide platform that replaces traditional optical elements with a single flat glass panel, featuring curved surfaces and high-reflectivity coatings to perform essential optical functions. We believe this to be a novel design that greatly reduces the size and weight of the device, making it highly suitable for deployment on various platforms, including aircraft and satellites. The optical system underwent extensive testing, including simulations using Zemax software and physical prototyping, achieving a spectral resolution of less than 3 nm across the 400-1000 nm waveband while maintaining low distortion. The UTHP spectrometer’s compactness, lightweight design, and exceptional performance mark a significant advancement in imaging spectrometry, with the potential to transform remote sensing by providing more efficient, accessible methods for studying expansive oceanic regions.

Dual-Modality Accurate Visualization of Drug Synergy Based on Mass Spectrometry and Fluorescence Imaging.

There is a potential synergistic effect between nonsteroidal anti-inflammatory drugs and hydrogen sulfide (H2S), but direct evidence for the study is lacking. With a single fluorescence detection method, it is difficult to accurately confirm the effectiveness of the synergistic effect. In this study, the fluorescent probe and the nonsteroidal anti-inflammatory drug naproxen were combined via different self-immolative spacer groups to obtain a diagnostic and therapeutic integrated fluorescent probe Nap-NP-NSB, which can release H2S. The quantitative release of H2S by Nap-NP-NSB was evaluated in vitro and in cells, and the synergistic effect of H2S and naproxen was confirmed by monitoring the treatment process of cellular inflammation and oxidative damage of gastric mucosa cells. Finally, in vivo fluorescence imaging and mass spectrometry imaging of the liver and stomach tissues and their sections were performed in the mouse model of acute hepatitis. The dual-modal detection method not only confirmed that Nap-NP-NSB had better anti-inflammatory activity and less gastric mucosal damage, but also enabled a more accurate visualization of the drug synergistic effect of naproxen and H2S. This work provides a dual visualization imaging method combining fluorescence and mass spectrometry imaging and develops a new idea for studying drug synergies based on self-immolative structures.

Estimating plant β-diversity using airborne and spaceborne imaging spectroscopy

ABSTRACT The increasing threats to grassland ecosystems from land-use/land-cover change, disturbances, and invasive species underscore the importance of monitoring grassland plant diversity. While most remote sensing studies have mainly focused on quantifying α-diversity (diversity within communities), less attention has been given to remotely estimating β-diversity (diversity between communities) in naturally-assembled grasslands. In this study, we used remote sensing to map plant β-diversity in a naturally-assembled grassland managed using prescribed fire and grazing in a North American tallgrass prairie ecosystem within the US Southern Great Plains. We aimed to assess the impact of time since fire on the relationship between in situ plant β-diversity and remotely–sensed β-diversity, also known as spectral β-diversity. We collected in situ observations at 60 m × 60 m, 120 m × 120 m, 180 m × 180 m, and 240 m × 240 m plots, alongside airborne and spaceborne DESIS imaging spectroscopy data with spatial resolutions of 1 m and 30 m, respectively. To assess how management practices influenced β-diversity, we grouped our in situ observations into three categories based on time since fire, including recently-burned (burn age <1 year), transitional (burn age of 1–2 years), and unburned (burn age >2 years). Our findings showed that the association between in situ and spectral β-diversity was strongly influenced by time since fire. Excluding plots with high soil cover (≥50%) improved our remote estimation of β-diversity, despite soil exposure effects. Our study demonstrated that remote sensing of β-diversity in grassland ecosystems is influenced by spatial scale, time since fire, and soil exposure, all of which can be addressed with scale-appropriate imaging spectrometry. This study also highlighted the capability of spaceborne imaging spectrometers to estimate β-diversity of grassland ecosystems across large spatial domains.

Tissue derivatization for visualizing lactate and pyruvate in mouse testis tissues using matrix-assisted laser desorption/ionization-mass spectrometry imaging

Pyruvate and lactate are the final metabolites of the glycolytic system that are formed under oxygen-rich and anaerobic conditions, respectively. They play an important role in energy metabolism. Obtaining a tissue distribution image of pyruvate and lactate holds great significance in molecular biology because the glycolytic system plays an essential role in diseases, such as tumors and diabetes; microbial activities, such as alcohol production and lactic acid fermentation; and maintaining homeostasis in the gut environment. However, it is difficult to obtain images of the distribution of in vivo metabolites because of the low detection sensitivities of current methods. In this study, a novel derivatization method for pyruvate and lactate was developed using matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to detect pyruvate and lactate in vivo and obtain biodistribution images. We investigated derivatization methods using readily available 3-nitrophenylhydrazine (3NPH), the addition of which improves the sensitivity of pyruvate detection, and the distribution of pyruvate in mouse testes was successfully visualized. Furthermore, the distribution of lactate in the mouse testes could be visualized, and improved detection sensitivity for the main metabolites of the tricarboxylic acid cycle was demonstrated. This derivatization method can be used to detect carboxyl-containing metabolites, including pyruvate, via MALDI-MSI. Furthermore, 3NPH forms amide bonds with carbonyl, phosphate, and carboxyl groups, suggesting the possibility of visualizing its distribution in many metabolites.Graphical

Metabolic imaging in living plants: A promising field for chemical exchange saturation transfer (CEST) MRI.

Magnetic resonance imaging (MRI) is a versatile technique in the biomedical field, but its application to the study of plant metabolism in vivo remains challenging because of magnetic susceptibility problems. In this study, we report the establishment of chemical exchange saturation transfer (CEST) for plant MRI. This method enables noninvasive access to the metabolism of sugars and amino acids in complex sink organs (seeds, fruits, taproots, and tubers) of major crops (maize, barley, pea, potato, sugar beet, and sugarcane). Because of its high signal detection sensitivity and low susceptibility to magnetic field inhomogeneities, CEST analyzes heterogeneous botanical samples inaccessible to conventional magnetic resonance spectroscopy. The approach provides unprecedented insight into the dynamics and distribution of sugars and amino acids in intact, living plant tissue. The method is validated by chemical shift imaging, infrared microscopy, chromatography, and mass spectrometry. CEST is a versatile and promising tool for studying plant metabolism in vivo, with many applications in plant science and crop improvement.

Evolutionary dynamics in gut-colonizing Candida glabrata during caspofungin therapy: Emergence of clinically important mutations in sphingolipid biosynthesis.

Invasive fungal infections are associated with high mortality, which is exacerbated by the limited antifungal drug armamentarium and increasing antifungal drug resistance. Echinocandins are a frontline antifungal drug class targeting β-glucan synthase (GS), a fungal cell wall biosynthetic enzyme. Echinocandin resistance is generally low but increasing in species like Candida glabrata, an opportunistic yeast pathogen colonizing human mucosal surfaces. Mutations in GS-encoding genes (FKS1 and FKS2 in C. glabrata) are strongly associated with clinical echinocandin failure, but epidemiological studies show that other, as yet unidentified factors also influence echinocandin susceptibility. Furthermore, although the gut is known to be an important reservoir for emergence of drug-resistant strains, the evolution of resistance is not well understood. Here, we studied the evolutionary dynamics of C. glabrata colonizing the gut of immunocompetent mice during treatment with caspofungin, a widely-used echinocandin. Whole genome and amplicon sequencing revealed rapid genetic diversification of this C. glabrata population during treatment and the emergence of both drug target (FKS2) and non-drug target mutations, the latter predominantly in the FEN1 gene encoding a fatty acid elongase functioning in sphingolipid biosynthesis. The fen1 mutants displayed high fitness in the gut specifically during caspofungin treatment and contained high levels of phytosphingosine, whereas genetic depletion of phytosphingosine by deletion of YPC1 gene hypersensitized the wild type strain to caspofungin and was epistatic to fen1Δ. Furthermore, high resolution imaging and mass spectrometry showed that reduced caspofungin susceptibility in fen1Δ cells was associated with reduced caspofungin binding to the plasma membrane. Finally, we identified several different fen1 mutations in clinical C. glabrata isolates, which phenocopied the fen1Δ mutant, causing reduced caspofungin susceptibility. These studies reveal new genetic and molecular determinants of clinical caspofungin susceptibility and illuminate the dynamic evolution of drug target and non-drug target mutations reducing echinocandin efficacy in patients colonized with C. glabrata.

Electrically tunable planar liquid-crystal singlets for simultaneous spectrometry and imaging

Conventional hyperspectral cameras cascade lenses and spectrometers to acquire the spectral datacube, which forms the fundamental framework for hyperspectral imaging. However, this cascading framework involves tradeoffs among spectral and imaging performances when the system is driven toward miniaturization. Here, we propose a spectral singlet lens that unifies optical imaging and computational spectrometry functions, enabling the creation of minimalist, miniaturized and high-performance hyperspectral cameras. As a paradigm, we capitalize on planar liquid crystal optics to implement the proposed framework, with each liquid-crystal unit cell acting as both phase modulator and electrically tunable spectral filter. Experiments with various targets show that the resulting millimeter-scale hyperspectral camera exhibits both high spectral fidelity ( > 95%) and high spatial resolutions ( ~1.7 times the diffraction limit). The proposed “two-in-one” framework can resolve the conflicts between spectral and imaging resolutions, which paves a practical pathway for advancing hyperspectral imaging systems toward miniaturization and portable applications.

Soil fertility prediction using combined USB-microscope based soil image, auxiliary variables, and portable X-ray fluorescence spectrometry

This study explored the application of portable X-ray fluorescence (PXRF) spectrometry and soil image analysis to rapidly assess soil fertility, focusing on critical parameters such as available B, organic carbon (OC), available Mn, available S, and the sulfur availability index (SAI). Analyzing 1133 soil samples from various agro-climatic zones in Eastern India, the research combined color and texture features from microscopic soil images, PXRF data, and auxiliary soil variables (AVs) using a Random Forest model. Results indicated that integrating image features (IFs) with auxiliary variables (AVs) significantly enhanced prediction accuracy for available B (R² = 0.80) and OC (R² = 0.88). A data fusion approach, incorporating IFs, AVs, and PXRF data, further improved predictions for available Mn and SAI with R² values of 0.72 and 0.70, respectively. The study demonstrated how these integrated technologies have the potential to provide quick and affordable options for soil testing, opening up access to more sophisticated prediction models and a better comprehension of the fertility and health of the soil. Future research should focus on the application of deep learning models on a larger dataset of soil images, developed using soils from a broader range of agro-climatic zones under field condition.

Combination of gas chromatography-mass spectrometry and hyperspectral imaging for identification of adulterated Safflower seed oil

Safflower seed oil (SSO), a crucial edible oil, occupies a significant position in human diet due to its unique nutritional components. However, because of its high economic value, SSO has become a primary target for food adulteration. To rapidly and effectively detect the concentration of adulterants in SSO, a research approach using hyperspectral imaging and gas chromatography-mass spectrometry (GC-MS) was proposed as technical means, employing machine learning as the methodology. Specifically, different preprocessing methods were compared, with median filtering (MF) selected for spectral data to significantly reduce noise and improve the robustness and generalization ability of the model. Regarding the selection of feature bands, bands around 440 nm, 530 nm, and 880 nm – 950 nm were identified as more favorable for establishing a predictive model for the concentration of adulterants in SSO, while also shortening the modeling time. By constructing an ensemble learning model with ridge regression (Ridge) and partial least squares regression (PLSR) as base models and LightGBM as the meta-model, achieving high-precision prediction of the concentration of adulterants in SSO. Furthermore, by jointly modeling the linoleic acid, oleic acid and palmitic acid measured by GC-MS with hyperspectral data, the model's R2 was improved to 0.976, highlighting its outstanding performance. Therefore, this study identifies MF-Ridge-Stacking as the optimal model for predicting the concentration of adulterants in SSO. The research not only provides theoretical and technical support for adulteration identification but also presents a new method for predicting the concentration of adulterants in SSO, with potential practical application value.

Correlative Raman, Backscattered Electron and X-ray Imaging and Energy Dispersive X-ray Spectrometry Uncovers Unique Chemical Signatures Surrounding Nanoparticles and Wear Debris in Periprosthetic Tissue

Correlative Raman, Backscattered Electron and X-ray Imaging and Energy Dispersive X-ray Spectrometry Uncovers Unique Chemical Signatures Surrounding Nanoparticles and Wear Debris in Periprosthetic Tissue

Second harmonic generation of focused beams on the LFEX laser facility.

There is a strong demand for efficient second harmonic generation (SHG) in ultra-intense short-pulse lasers. This paper demonstrates the generation of an unconverted fundamental (1ω)+second harmonics (2ω) mixed laser on the LFEX laser system. The experimental setup utilizes 0.5 mm-thick LBO crystal plates in a focusing beams implemented after an off-axis parabola, the design reduces the size and cost of the SHG system. The LFEX laser beams with four-beams combined energy of 222 J and a pulse duration of 1.5 ps, is successfully converted to 102 J of 2ω light and 100 J of unconverted 1ω light, 20 J is lost through surface reflections, and they are mixed at the focal point. Verification of successful SHG is confirmed through X-ray pinhole imaging and electron spectrometry. This novel technique is not limited to LFEX lasers and holds applicability for various ultra-intense lasers. Consequently, this accomplishment significantly contributes to expanding the capability for high-energy density laser-plasma experiments.

Combined matrix-assisted laser desorption/ionisation-mass spectrometry imaging with liquid chromatography-tandem mass spectrometry for observing spatial distribution of lipids in whole Caenorhabditis elegans.

Matrix-assisted laser desorption/ionisation-mass spectrometry imaging (MALDI-MSI) is a powerful label-free technique for biomolecule detection (e.g., lipids), within tissue sections across various biological species. However, despite its utility in many applications, the nematode Caenorhabditis elegans is not routinely used in combination with MALDI-MSI. The lack of studies exploring spatial distribution of biomolecules in nematodes is likely due to challenges with sample preparation. This study developed a sample preparation method for whole intact nematodes, evaluated using cryosectioning of nematodes embedded in a 10% gelatine solution to obtain longitudinal cross sections. The slices were then subjected to MALDI-MSI, using a RapifleX Tissuetyper in positive and negative polarities. Samples were also prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using an Exploris 480 coupled to a HPLC Vanquish system to confirm the MALDI-MSI results. An optimised embedding method was developed for longitudinal cross-sectioning of individual worms. To obtain longitudinal cross sections, nematodes were frozen at -80°C so that all worms were rod shaped. Then, the samples were defrosted and transferred to a 10% gelatine matrix in a cryomold; the worms aligned, and the whole cryomold submerged in liquid nitrogen. Using MALDI-MSI, we were able to observe the distribution of lipids within C. elegans, with clear differences in their spatial distribution at a resolution of 5μm. To confirm the lipids from MALDI-MSI, age-matched nematodes were subjected to LC-MS/MS. Here, 520 lipids were identified using LC-MS/MS, indicating overlap with MALDI-MSI data. This optimised sample preparation technique enabled (un)targeted analysis of spatially distributed lipids within individual nematodes. The possibility to detect other biomolecules using this method thus laid the basis for prospective preclinical and toxicological studies on C. elegans.

Multitemporal airborne imaging spectrometry and fluorometry reveal contrasting photoprotective responses of trees

The Photochemical Reflectance Index (PRI) and solar induced fluorescence (SIF) provide information on plant photosynthetic activity. PRI and SIF are both strongly influenced by irradiance, but uncertainties related to the interpretation of these light responses at large spatial scales remain, partly due to a shortage of suitable data from aircraft or satellite platforms. The goal of this study was to explore interpretations of the PRI- and SIF-light responses of trees owing to species, functional types (evergreen and deciduous) and season. Using airborne hyperspectral and ultraspectral imagery in a North American urban forest, we derived PRI, SIF, and albedo (an indicator of illumination) at the 1-m pixel level. We then quantified crown-level PRI and SIF light responses of ten different tree species at three time points from late-summer to autumnal senescence using hierarchical models. Our results confirmed that both PRI and SIF were strongly influenced by illumination with PRI decreasing and SIF increasing with illumination. Both slope and intercept of the PRI-albedo relationship changed with season, but the pattern varied among species and functional types. SIF values decreased during autumnal senescence for all species, but evergreen species exhibited less seasonal decline in the slope of SIF-albedo relationship compared to deciduous species. The PRI and SIF light responses derived from the airborne imagery offer complementary information on dynamic photosynthesis responses presumably due to varying canopy structure, pigmentation and photoprotection among species and functional types. From airborne platforms, PRI- and SIF-light responses can be used to explore the contrasting physiological responses of individual tree crowns, providing a spatially and temporally explicit view of dynamic plant traits related to photoregulation and a novel view of functional diversity for entire landscapes.

Estimation of the time of zolpidem intake and differentiation between consumption and external contamination using MALDI-MSI for investigations on single hair samples

Estimation of drug ingestion time (event time) and distinguishing between drug ingestion and external contamination are important for interpreting hair analysis results in forensics practice. Here, we present a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) method for in situ analysis of intact hair. We applied a longitudinal cutting method for a single hair to analysis authentic hair samples from a victim of a drug-facilitated sexual assault (DFSA) case and zolpidem-soaked hair. MALDI-MSI showed that zolpidem-positive segments distributed at 4−6 mm or 6−8 mm from the root in three single hairs of a DFSA victim collected 25 days after the event, at concentrations ranging from 0.1 to 5.7 pg mm−1, in agreement with the results from segmental analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS). The estimation of drug intake time was about 20–30 days before sampling, which was consistent with the known time of drug intake. This MALDI-MS method allows imaging analysis of trace substances in a single hair and can realize the intuitive reflection of drug taking time. In addition, zolpidem applied by soaking was mainly distributed on both sides of the longitudinal hair shaft, whereas ingested zolpidem was found only in the middle of the hair shaft of the DFSA victim. The MALDI-MS images of unwashed and washed hair suggested that the amount of externally applied drug was decreased by washing, it was still present on surface layer (cuticle) sides although. Visualization using MALDI-MSI could therefore distinguish between drug ingestion and contamination by reflecting the distribution and deposition site of the drug in hair.

Multi-omics analysis of antagonistic interactions among free-living Pseudonocardia from diverse ecosystems.

Actinomycetes are a phylogenetically diverse bacterial group which are widely distributed across terrestrial and aquatic ecosystems. Within this order, the genus Pseudonocardia and their specialised metabolites have been the focus of previous ecological studies due to their antagonistic interactions with other microorganisms and their mutualistic interactions with insects. However, the chemical ecology of free-living Pseudonocardia remains understudied. This study applies a multi-omics approach to investigate the chemical ecology of free-living actinomycetes from the genus Pseudonocardia. In a comparative genomics analysis, it was observed that the biosynthetic gene cluster family distribution was influenced mainly by phylogenetic distance rather than the geographic or ecological origin of strains. This finding was also observed in the mass spectrometry-based metabolomic profiles of nine Pseudonocardia species isolated from marine sediments and two terrestrial species. Antagonist interactions between these 11 species were examined, and matrix-assisted laser desorption/ionisation-mass spectrometry imaging was used to examine in situ chemical interactions between the Southern Ocean strains and their phylogenetically close relatives. Overall, it was demonstrated that phylogeny was the main predictor of antagonistic interactions among free-living Pseudonocardia. Moreover, two features at m/z 441.15 and m/z 332.20 were identified as metabolites related to these interspecies interactions.

Nanoparticle-loaded microbubbles for treatment of lung cancer

Lung cancer is one of the most common cancers and a leading cause of death, with poor prognosis and high unmet clinical need. Chemotherapy is a common part of the treatment, either alone or in combination with other treatment modalities, but with limited efficacy and severe side effects. Encapsulation of drugs into nanoparticles can enable a more targeted delivery with reduced off-target toxicity. Delivery to the lungs is however often insufficient due to various biological barriers in the body and in the tumor microenvironment. Here we demonstrate that by incorporating drug-loaded nanoparticles into air-filled microbubbles, a more effective targeting to the lungs can be achieved. Fluorescence imaging and mass spectrometry revealed that the microbubbles could significantly improve accumulation of drug in the lungs of mice, compared to injecting either the free drug by itself or only the drug-loaded nanoparticles. Therapeutic efficacy was verified in a preclinical mouse model with non-small cell lung cancer, monitoring tumor growth by luminescence.

Comparative microplastic analysis in urban waters using μ-FTIR and Py-GC-MS: A case study in Amsterdam

The contamination of freshwater with microplastics (MPs) has been established globally. While the analysis of MPs has predominantly involved spectroscopic methods for revealing particle numbers, the potential of employing spectroscopy for mass estimation has been underutilized. Consequently, there is a need to enhance our understanding of the mass loads of MPs and ensure the complementarity and comparability of various techniques for accurate quantification. This study presents the first comparative results on urban water samples using micro Fourier-transform infrared (μ-FTIR) imaging and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) to identify and quantify MPs in both particle numbers and mass concentration. Two sampling campaigns in summer and winter were conducted at 11 locations within the Amsterdam canal network. An advanced in-situ volume-reducing sampling pump was employed to collect MPs from the surface water within the size fraction of 10–300 μm. The analysis revealed MP concentrations within the range of 16–107 MP/m3, estimated to be 2.0–789 μg/m3 by μ-FTIR imaging and 8.5–754 μg/m3 by Py-GC-MS. The results of the two analysis techniques showed good comparability in terms of the general trends of MP abundances, with variations in polymer compositions due to the inherent inter-methodological differences. Elevated MP concentrations were observed in the city center compared to the suburban areas. In addition, seasonal differences in MP abundances were noted at the locations with high human activity.

Human Airway Organoids and Multimodal Imaging-Based Toxicity Evaluation of 1-Nitropyrene.

Despite significant advances in understanding the general health impacts of air pollution, the toxic effects of air pollution on cells in the human respiratory tract are still elusive. A robust, biologically relevant in vitro model for recapitulating the physiological response of the human airway is needed to obtain a thorough understanding of the molecular mechanisms of air pollutants. In this study, by using 1-nitropyrene (1-NP) as a proof-of-concept, we demonstrate the effectiveness and reliability of evaluating environmental pollutants in physiologically active human airway organoids. Multimodal imaging tools, including live cell imaging, fluorescence microscopy, and MALDI-mass spectrometry imaging (MSI), were implemented to evaluate the cytotoxicity of 1-NP for airway organoids. In addition, lipidomic alterations upon 1-NP treatment were quantitatively analyzed by nontargeted lipidomics. 1-NP exposure was found to be associated with the overproduction of reactive oxygen species (ROS), and dysregulation of lipid pathways, including the SM-Cer conversion, as well as cardiolipin in our organoids. Compared with that of cell lines, a higher tolerance of 1-NP toxicity was observed in the human airway organoids, which might reflect a more physiologically relevant response in the native airway epithelium. Collectively, we have established a novel system for evaluating and investigating molecular mechanisms of environmental pollutants in the human airways via the combinatory use of human airway organoids, multimodal imaging analysis, and MS-based analyses.

Spatiotemporal metabolic responses to water deficit stress in distinct leaf cell-types of poplar.

The impact of water-deficit (WD) stress on plant metabolism has been predominantly studied at the whole tissue level. However, plant tissues are made of several distinct cell types with unique and differentiated functions, which limits whole tissue 'omics'-based studies to determine only an averaged molecular signature arising from multiple cell types. Advancements in spatial omics technologies provide an opportunity to understand the molecular mechanisms underlying plant responses to WD stress at distinct cell-type levels. Here, we studied the spatiotemporal metabolic responses of two poplar (Populus tremula× P. alba) leaf cell types -palisade and vascular cells- to WD stress using matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). We identified unique WD stress-mediated metabolic shifts in each leaf cell type when exposed to early and prolonged WD stresses and recovery from stress. During water-limited conditions, flavonoids and phenolic metabolites were exclusively accumulated in leaf palisade cells. However, vascular cells mainly accumulated sugars and fatty acids during stress and recovery conditions, respectively, highlighting the functional divergence of leaf cell types in response to WD stress. By comparing our MALDI-MSI metabolic data with whole leaf tissue gas chromatography-mass spectrometry (GC-MS)-based metabolic profile, we identified only a few metabolites including monosaccharides, hexose phosphates, and palmitic acid that showed a similar accumulation trend at both cell-type and whole leaf tissue levels. Overall, this work highlights the potential of the MSI approach to complement the whole tissue-based metabolomics techniques and provides a novel spatiotemporal understanding of plant metabolic responses to WD stress. This will help engineer specific metabolic pathways at a cellular level in strategic perennial trees like poplars to help withstand future aberrations in environmental conditions and to increase bioenergy sustainability.