Integrating Histology and Attenuated Total Reflectance Fourier Transformed Infrared Spectroscopy to Estimate the Postmortem Interval in Rat Liver

Background The precise determination of the postmortem interval (PMI) remains a significant challenge within the field of forensic science. Traditional methods, including algor mortis, rigor mortis, and livor mortis, are often constrained by variations in environmental conditions. Other methods have considerably involved pH, ions and metabolic byproduct measurements with inherent limitations and inaccuracies. Advanced techniques like flow cytometry, single-cell gel electrophoresis, polymerase chain reaction, some of which have been found expensive and cumbersome, have also largely been reported to investigate PMI in different tissues with almost similar consensus. This research aimed to explore the time-dependent postmortem alterations in rat liver tissue through histological analysis and Attenuated Total Reflectance–Fourier Transform Infrared (ATR-FTIR) spectroscopy, focusing on effect sizes and their practical relevance in forensic applications. Results Histological examination revealed the presence of intact hepatocytes at the 1-hour mark, followed by autolytic changes at 12 hours, and necrotic processes accompanied by liquefaction by 48 hours. The ATR-FTIR spectral analysis paralleled these identified phases, demonstrating a consistent decrease in protein absorbance (Cohen’s d = 2.4–2.7), while glycogen levels exhibited the most pronounced decline (d = 14.4). Additionally, there was an observed increase in nucleic acids and the cytoplasmic to nuclear ratio (d = − 7.7; R = 0.913, η² = 0.834; d = − 6.9; R = 0.822, η² = 0.676), indicating substantial and reproducible time-dependent changes. Conclusions The liver shows discernible histological and biochemical transformations postmortem within a 48-hour timeframe. ATR-FTIR spectroscopy effectively detected these molecular modifications, yielding objective and quantifiable metrics such as the cytoplasm to nucleus (C:N) ratio. The integration of histological findings with spectral biomarkers enhances the accuracy, reproducibility, and forensic reliability of PMI estimations.