Forensic estimation of semen stains' time since deposition on porous substrates using ATR FTIR and Raman spectroscopy supported by OPLS-DA models

Semen stains play a critical role in the investigation of sexual crimes, where their correct identification and age estimation are essential. In real casework, they are predominantly found on porous substrates, such as the underwear of victims or perpetrators. Currently, no validated method exists for estimating their age. In this study, semen stains deposited on four porous substrates commonly encountered in sexual assault and abuse scenarios were analysed using attenuated total reflectance Fourier-transform infrared (ATR FTIR) and Raman spectroscopy. Spectral data were modelled using orthogonal partial least squares-discriminant analysis (OPLS-DA) and continuous OPLS to classify stains by age and describe their aging patterns. Both spectroscopic techniques successfully discriminated semen stains according to their time since deposition (TSD), independently of the substrate, revealing a gradual degradation process with subtle early spectral changes, followed by more pronounced alterations after approximately one week. ATR FTIR exhibited higher statistical performance than Raman, likely due to fluorescence interference and the more localized sampling inherent to Raman, whereas ATR FTIR provides a more representative bulk signal. This study is the first to examine semen stain aging using Raman spectroscopy and provides a direct comparison of ATR FTIR and Raman for TSD estimation on the same substrates. The respective strengths and limitations of each technique are evaluated, along with their complementary value. Together, these methods demonstrate strong potential as complementary, non-destructive tools for forensic analysis of semen stains in sexual assault and abuse investigations. • ATR FTIR and Raman spectroscopy can be used to estimate the TSD of semen stains. • First study that uses Raman spectroscopy to establish semen stain aging. • First direct comparison of ATR FTIR and Raman spectroscopy for semen TSD estimation. • OPLS-DA discriminates semen on porous substrates by TSD using ATR FTIR and Raman. • OPLS continuous models can be used to track semen stain changes with time.