A novel non-antibody fluorescent dual-staining technique for sperm detection in forensic samples.

In sexual assault investigations, the detection of spermatozoa from victims' clothing or vaginal samples constitutes critical evidence indicative of ejaculation. Staining methods such as Baecchi's staining and fluorescence immunostaining are widely used to enhance microscopic examination for sperm detection. However, these approaches have inherent limitations, including limited specificity (e.g., Baecchi's staining) and reduced performance in aged samples (e.g., immunostaining), underscoring the need for novel staining strategies. In the present study, we developed a dual-staining method targeting the protamine-DNA complex in sperm heads, utilizing a thiol-reactive fluorescent dye in combination with a DNA-binding fluorescent dye. This CPM/AO dual staining method produced strong green fluorescence localized to sperm heads under ultraviolet excitation. Based on the finding that sperm heads exhibited stronger fluorescence than somatic cell nuclei, this phenomenon may involve Förster resonance energy transfer (FRET). Although the nuclei of oral and vaginal epithelial cells, as well as other debris, exhibited similar coloration of the sperm head, spermatozoa could be effectively distinguished based on fluorescence intensity and related features, enabling reliable visual identification. Moreover, the method demonstrated consistent effectiveness in detecting spermatozoa in aged forensic specimens. These findings indicate that the proposed staining technique serves as a useful tool for sperm detection in forensic contexts, particularly when dealing with degraded samples.