Raman Spectroscopy for Forensic Pathology Applications: A Case Series and Narrative Literature Review

Introduction: Forensic pathology benefits from analytical methods that can add molecular information while preserving tissue morphology and evidentiary material. Confocal Raman microspectroscopy provides non-destructive, spatially resolved chemical information with limited sample preparation. This study evaluated its complementary value in three representative forensic scenarios and contextualized the findings through a structured narrative review of Raman applications in forensic science. materials and methods: The aim of our study was to validate this technique on three specific sample types—fat embolism, gunshot residue, and cocaine abuse. Additionally, we conducted a literature review to provide an updated overview of the potential forensic applications of RS. In cases of fat embolism, Raman analysis confirmed the presence of lipid emboli within pulmonary tissues, revealing characteristic bands corresponding to saturated and unsaturated fatty acids, as well as protein and carbohydrate components. These findings were consistent with histological ex-amination and Sudan III staining. In deaths due to cocaine intoxication, spectroscopy revealed structural and biochemical alterations in brain tissue, including increased levels of lipids, nucleic acids, and aromatic amino acids, suggesting substance-induced neurodegeneration. Regarding gunshot wounds, RS identified lead and barium residues, thereby validating its efficacy as a non-destructive method for detecting gunshot residues. Methods: Confocal Raman microspectroscopy was applied to formalin-fixed, paraffin-embedded tissue sections from 73 judicial autopsy cases, comprising pulmonary fat embolism (n = 15), acute cocaine intoxication (n = 27), and firearm-related fatalities (n = 31), together with seven sudden cardiac death controls. Spectra were acquired in the fingerprint (660–1800 cm⁸¹) and highwavenumber (2500–3200 cm⁸¹) regions for biological tissue analysis, with additional lowwavenumber acquisition for inorganic gunshot-residue characterization. The spectroscopic analysis was exploratory and descriptive rather than a validated classification study. In parallel, a structured narrative review was conducted using PubMed/MEDLINE, Scopus, and Web of Science to summarize major forensic applications of Raman spectroscopy. Results: In pulmonary fat embolism, Raman spectra showed characteristic lipid-associated bands, including C–H stretching at 2851, 2928, and 3007 cm⁸¹ and an ester carbonyl band at 1746 cm⁸¹. In cocaine-related fatalities, MCR-ALS mapping demonstrated spatial differences in lipid- and protein-associated spectral contributions relative to controls, including bands around 700, 1000, 1450, and 1650–1690 cm⁸¹. In firearm fatalities, Raman microspectroscopy detected inorganic gunshot-residue particles at wound margins, including a mendipite-associated signature at approximately 148–152 cm⁸¹ and barium-containing spectral features. The narrative review further identified applications in biological fluid discrimination, bloodstain pattern analysis, skeletal and postmortem interval assessment, trace evidence, and chemometric classification. Discussion: The present findings support its role as an adjunct to established histopathological, toxicological, and trace-evidence methods rather than as a stand-alone diagnostic technique. Confocal Raman microspectroscopy provided complementary, spatially resolved molecular information across the three forensic case categories while preserving the analyzed tissue sections. Conclusion: Standardized acquisition protocols, case-level quantitative validation, multicenter reproducibility studies, and shared forensic spectral libraries remain necessary before routine implementation.