Spatial Dependence and Heterogeneity in Molecular Imaging: Imaging Mass Spectrometry Datasets

You will find the following datasets in our Zenodo repository: 

• Demo_rat_brain_data.pickle is a subset of 100 ion images of MALDI-IMS dataset no1 (rat brain). It was used to demonstrate exporatory spatial data analysis of molecular imaging data with local and global spatial autocorrelation statistics. 
• Zebra_fish_8_clusters_dataset.pickle is a subset of 174 ion images of MALDI-IMS dataset no2 (zebra fish). It was used to demonstrate the Moran-HOG clustering workflow, which which uses the Moran quadrant map to perform colocalization-based ion image clustering. 
• Zebra_fish_UMAP_dataset.pickle is a UMAP embedding of MALDI-IMS dataset no2 (zebra fish). It was used to demonstrate the Moran-Felsenszwalb segmentation workflow, which uses the Moran quadrant map to perform multivariate tissue domain segmentation. 

These datasets relate to "Spatial Dependence and Heterogeneity in Molecular Imaging: Moran Quadrant Maps Enable Advanced Spatial-Statistical Analysis" by Tideman et al. (October 2025). The preprint is available on bioRxiv: https://www.biorxiv.org/content/10.1101/2025.10.27.684518v1. 

About MALDI-IMS dataset no1: 

• Materials: The MALDI matrix 2,5-dihydroxyacetphenone (DHA, Sigma-Aldrich Chemical CO., St. Louis, MO, USA) was applied using a TM Sprayer (HTX Technologies, Carrboro, NC, USA) and rehydrated as described in "Next-generation technologies for spatial proteomics: Integrating ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR imaging mass spectrometry for protein analysis" by Spraggins et al. (Proteomics, 2016) and "Matrix Sublimation/Recrystallization for Imaging Proteins by Mass Spectrometry at High Spatial Resolution" by Yang et al. (Analytical Chemistry, 2011). 
• Sample Preparation: The brain tissue sections of the case study were collected from a rat Parkinson's disease model. Parkinson’s disease is characterized by a degeneration of dopaminergic neurons (i.e., neurons that synthesize dopamine) and the formation of Lewy bodies (i.e., protein aggregates) in the substantia nigra. The substantia nigra is a region of the basal ganglia, which are a group of nuclei (i.e., clusters of neurons) located beneath the cerebral cortex. Dopamine is the primary neurotransmitter (i.e., chemical messenger) of the basal ganglia. The loss of dopaminergic neurons severely impairs the brain’s motor function, leading to movement disorders. The dopaminergic neurons of a rat’s left brain hemisphere were destroyed to make it resemble a diseased brain. The right hemisphere was used as a control. The rat model was obtained as follows: the nigrostriatal dopaminergic neurons of the left hemisphere were destroyed to make it resemble a diseased brain, whereas the right hemisphere was used as a control. An adult male Sprague Dawley rat was deeply anesthetized with isoflurane, pretreated with desmethylimipramine (12.5 mg/kg, ip) and placed in a stereotaxic frame. After an incision of the dorsal surface of the skull and placement of a burr hole, the animal was injected with despramine. 10 min later 1.5:L of 6-hydroxydopamine HBr (6-OHDA; 4.0:g/μL, free base) was unilaterally injected into the substantia nigra (AP:-5.4; L:2.3; DV:-8.4) to selectively destroy nigrostriatal dopaminergic neurons. Although there is a crossed-nigrostriatal pathway, it is quite small, contributing well under 5% of the dopamine content to the contralateral striatum, and thus the contralateral striatum is usually referred to as the intact (control) side. All in-house animal experiments were performed with approval by the Vanderbilt Institutional Animal Care and Use Committee. Brain tissue was harvested, snap frozen using liquid nitrogen, and stored at -80°C until use. Frozen brain tissue was sectioned in the coronal plane at 10μm using a cryostat (-20°C, Leica CM3050S; Buffalo Grove, IL, USA) and thaw mounted onto conductive Indium-tin-oxide coated glass slides (Delta Technologies, Loveland, CO, USA). Samples were washed to remove interfering lipids and salts in sequential washes of 70% ethanol (30s), 100% ethanol (30s), Carnoy's fluid (6:3:1 ethanol:chloroform: acetic acid) (2min), 100% ethanol (30s), water with 0.2% TFA (30s), and 100% ethanol (30s).
• Imaging Mass Spectrometry: MALDI IMS images were collected using a 15T FT-ICR mass spectrometer (Bruker Daltonics, Billerica, MA, USA) with a spatial sampling resolution of 75μm(laser spot size 50 μm) and a mass resolving power of 50,000 (m/FWHM) at m/z 5000. The molecular images focused on a m/z range of 1300 to 23,000 with a total of 20,000 pixels. The instrument was tuned for protein imaging as described in "Next-generation technologies for spatial proteomics: Integrating ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR imaging mass spectrometry for protein analysis" by Spraggins et al. (Proteomics, April 2016).
• Data Processing: Data were imported into MATLAB 2015b (The Mathworks Inc., Natick, MA) for further data analysis, where they were normalized to TIC and peak picked, resulting in a total of 2611 peaks. Refer to "Connecting imaging mass spectrometry and magnetic resonance imaging-based anatomical atlases for automated anatomical interpretation and differential analysis" by Verbeeck et al. (Biochimica et Biophysica Acta - Proteins and Proteomics, 2017) for additional information. 

For more information about MALDI-IMS dataset no1, refer to Section 4.1 of the Supplementary Material of Tideman et al. and/or refer to "Connecting imaging mass spectrometry and magnetic resonance imaging-based anatomical atlases for automated anatomical interpretation and differential analysis" by Verbeeck et al. (Biochimica et Biophysica Acta - Proteins and Proteomics, 2017).

About MALDI-IMS dataset no2: 

• Ethics statement: Zebrafish studies were approved by the Institutional Animal Care and Use Committees (IACUC) of Vanderbilt University Medical Center (protocol number M2200064) in accordance with the Public Health Service Policy on the Human Care and Use of Laboratory Animals under the United States of America National Institutes of Health (NIH) Office of Laboratory Animal Welfare (OLAW).
• Zebrafish husbandry and animal work: The zebrafish line used in this study had an AB background and were maintained on a 14:10 h light:dark cycle in a recirculating aquaculture system. A single Adult (>90 days post fertilization) male zebrafish was euthanized according to IACUC protocol.
• Materials: Acetonitrile, isopentane, and gelatin from cold water fish were purchased from Sigma-Aldrich (St. Louis, MO). The matrix 4-(dimethylamino) cinnamic acid (DMACA) with 99% purity was bought from Thermo Scientific (Waltham, MA). 
• Sample preparation: Whole-body adult male zebrafish were frozen using a dry ice/isopentane freezing method. Samples were then embedded in 10% fish gelatine followed by a second embedding of the swim bladders. Cryosectioning was done at a 10μm thickness using a CM3050 S cryostat (Leica Biosystems, Wetzlar, Germany) and mounted onto indium tin oxide-coated glass slides (Delta Technologies, Loveland, CO, USA). Matrix application of DMACA at a concentration of 2mg/mL was applied using an in-house sublimation apparatus. Autofluorescence image was collected on a Zeiss Axio Scan.Z1 (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) prior to MALDI imaging. 
• Imaging Mass Spectrometry: Experiment was carried out on a prototype MALDI timsTOF Pro mass spectrometer (Bruker Daltonics, Bremen, Germany) in positive ionization mode at a spatial resolution of 10μm (30% laser power at 10kHz, 200 shots per pixel). 

For more information about MALDI-IMS dataset no2, refer to Section 4.3 of the Supplementary Material of Tideman et al. and/or contact Jacquelyn Spathies (data collector) from Vanderbilt University Medical Center.