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Here, we present the first use of the newly developed LARAPPI\/CI-MSI 3D (laser ablation remote atmospheric pressure photoionization\/chemical ionization mass spectrometry imaging) method for direct three-dimensional (3D) mass spectrometry imaging of bacterial and fungal metabolites in solid culture media. Two-dimensional (2D) MSI was also performed, and it indicated the presence of metabolites belonging to, and including, amino acids and their derivatives, dipeptides, organic acids, fatty acids, sugars and sugar derivatives, benzene derivatives, and indoles. Distribution at a selected depth within the culture medium with the estimation of concentration across all dimensions of 16 metabolites was visualized using LARAPPI\/CI-MSI 3D. The imaging results were correlated with the results of ultra-high-performance liquid chromatography\u2013ultra-high-resolution mass spectrometry (UHPLC\u2013UHRMS). A total of 351\u2013393 chemical compounds, depending on the tested microorganism, were identified, while 242\u2013262 were recognized in the HMDB database in MetaboAnalyst (v 6.0). The LARAPPI\/CI-MSI 3D method enables the rapid screening of the biotechnological potential of environmental strains, facilitating the discovery of industrially valuable biomolecules.<\/jats:p>","DOI":"10.3390\/molecules30061317","type":"journal-article","created":{"date-parts":[[2025,3,14]],"date-time":"2025-03-14T13:07:51Z","timestamp":1741957671000},"page":"1317","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Direct 3D Mass Spectrometry Imaging Analysis of Environmental Microorganisms"],"prefix":"10.3390","volume":"30","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6601-2376","authenticated-orcid":false,"given":"Justyna","family":"Szulc","sequence":"first","affiliation":[{"name":"Department of Environmental Biotechnology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, 90-530 Lodz, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8108-3933","authenticated-orcid":false,"given":"Tomasz","family":"Grzyb","sequence":"additional","affiliation":[{"name":"Department of Environmental Biotechnology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, 90-530 Lodz, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4783-8615","authenticated-orcid":false,"given":"Joanna","family":"Nizio\u0142","sequence":"additional","affiliation":[{"name":"Department of Inorganic and Analytical Chemistry, Faculty of Chemistry, Rzeszow University of Technology, 35-959 Rzeszow, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-8916-0946","authenticated-orcid":false,"given":"Sumi","family":"Krupa","sequence":"additional","affiliation":[{"name":"Department of Inorganic and Analytical Chemistry, Faculty of Chemistry, Rzeszow University of Technology, 35-959 Rzeszow, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wiktoria","family":"Szuberla","sequence":"additional","affiliation":[{"name":"Department of Inorganic and Analytical Chemistry, Faculty of Chemistry, Rzeszow University of Technology, 35-959 Rzeszow, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9899-8627","authenticated-orcid":false,"given":"Tomasz","family":"Ruman","sequence":"additional","affiliation":[{"name":"Department of Inorganic and Analytical Chemistry, Faculty of Chemistry, Rzeszow University of Technology, 35-959 Rzeszow, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Fouillaud, M., and Dufoss\u00e9, L. 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