Next Generation MALDI Imaging: FluoMALDI, RaMALDI & QMALDI

Kristine Glunde (1, 2, 3)
(1) Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA, (2) Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA, (3) Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Kristine Glunde, PhD (Presenter) The Johns Hopkins University School of Medicine

Presenter Bio: Dr. Glunde is Professor of Radiology, Oncology, and Pathology, and her research is focused on developing and sharing innovative matrix-assisted laser desorption/ionization (MALDI) imaging applications that enable biomedical discovery. Since joining the Faculty of the Johns Hopkins School of Medicine in 2003, Dr. Glunde's research lab has been focusing on cancer metabolism, novel metabolic cancer treatment targets, and molecular and metabolic imaging of cancer. More recently, she is also focusing on developing various new, clinically oriented MALDI imaging applications. She has specific training and expertise in chemistry, biochemistry, cancer biology, fluorescence microscopy, optical imaging, mass spectrometry, and mass spectrometric imaging. She has been involved in numerous research studies on molecular imaging of cancer, brain metabolism, and MALDI imaging methods development as Principal Investigator and Co-Investigator. Throughout this time, she has mentored more than 65 students, postdoctoral fellows, and junior faculty. She has published over 130 publications in the field of cancer metabolism, molecular imaging of cancer, and MALDI imaging methods development. Since 2019, she is the founding director of the Applied Imaging Mass Spectrometry (AIMS) Core at the Johns Hopkins Medical Institutions (JHMI), making available this highly multiplexed, high throughput tissue imaging technology to faculty at Johns Hopkins and outside institutions. She has built a quickly expanding mass spectrometry imaging program at Johns Hopkins, where her team members interact with a diverse group of over 80 users, spanning multiple departments at Johns Hopkins and several institutions on the East Coast and nationwide.

Relevant Financial Disclosures (within past 24 months, reported on May 11, 2026) No relevant financial relationship(s) to disclose.

To further advance matrix assisted laser desorption/ionization (MALDI) imaging, we have developed three innovative application driven technologies that expand the analytical capabilities of MALDI imaging across spatial biology, multimodal molecular discovery, and quantitative analysis. These technologies: (1) integrate MALDI imaging with fluorescence microscopy for precise spatial targeting of defined tissue and cellular compartments, (2) combine MALDI imaging with Raman microscopy to enable robust, complementary biomolecular discovery and identification, and (3) establish quantitative MALDI imaging approaches for the detection of drug metabolites and imaging contrast agents.

First, we developed FluoMALDI microscopy, a multimodal imaging technique that seamlessly integrates fluorescence microscopy with MALDI imaging on the same biological sample. A key innovation underlying FluoMALDI is the discovery that co-crystallization of fluorophores with MALDI matrices significantly enhances fluorescence brightness, enabling improved sensitivity and spatial registration. Using representative applications in neuroscience, developmental biology, and cell biology, we demonstrate that FluoMALDI microscopy enables spatially guided molecular profiling driven by enhanced fluorescent dyes, genetic tracers, and immunofluorescence. This approach facilitates comprehensive molecular characterization of targeted regions and cell populations within a single tissue section.

Second, to integrate complementary chemical information, we developed RaMALDI, a streamlined multimodal workflow that combines Raman spectroscopic imaging (RSI) and MALDI mass spectrometry imaging (MSI) on a single tissue section using a unified sample-preparation protocol. By fusing RSI and MALDI MSI data, RaMALDI leverages the strengths of both modalities – label-free chemical specificity and high molecular sensitivity – to generate spatially resolved biomolecular maps. We show that RaMALDI imaging across multiple tissue types effectively integrates molecular information acquired by both techniques, thereby enabling new opportunities for discovery in cell biology, biomedicine, and pathology, as well as advancing tissue-based diagnostics.

Finally, we developed quantitative MALDI (QMALDI) imaging to support the investigation of drug metabolism and molecular imaging agents in vivo. As a proof of concept, we repurposed aspirin as an activatable contrast agent for chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) of its metabolite salicylic acid (SA). For orthogonal validation, QMALDI imaging was applied to map aspirin metabolites, including SA, in mouse models of breast cancer. QMALDI imaging revealed pronounced accumulation of SA in the kidney medulla and within the tumor rim, particularly in vascularized, viable tumor regions following systemic administration. These results highlight the potential of QMALDI imaging as a powerful tool for quantitatively interrogating drug distribution, metabolism, and molecular imaging agents across diverse tissues.

Together, FluoMALDI, RaMALDI, and QMALDI represent a new generation of MALDI-based imaging approaches that expand the scope, sensitivity, and utility of mass spectrometry imaging for spatially resolved biological and biomedical research.