Identification of invisible biological traces in forensic evidences by hyperspectral NIR imaging combined with chemometrics

The importance of detecting minute biological traces in forensic evidences feeds the continuous interest towards the development of new dedicated technologies both sensitive and reliable. The present study describes the opportunity to combine chemical properties derived from NIR signals with spatial features typical of RGB images by means of hyperspectral imaging (HSI). An analytical procedure based on HSI data collection and their multivariate processing followed by normalized difference images (NDI) is proposed as a screening method to highlight otherwise invisible traces of biological fluids on different supports in view of their collection for DNA analysis. The pattern features identified inside the NDI provided insight into the nature of the biological trace, on the basis of the wavelength at which the stain is highlighted and irrespective of the support on which the stain is deposited. In particular, the procedure allowed to detect and distinguish traces (i.e., 10 and 20 μL volumes) of dehydrated blood, urine, and semen on glass, paper, cotton, denim and polyblend fabric. Beside the simulated specimens used to develop and test the protocol, its robustness was demonstrated also on real and unknown validation samples, confirming its feasibility in some real case studies. An interesting evolution of the proposed strategy is to lay the scientific foundations for the development of a handheld device directly applicable in field