Membrane deformation of unfixed erythrocytes in air with time lapse investigated by tapping mode atomic force microscopy

Estimation of the time of death is one of the most important problems for forensic medicine and law. Physical and chemical postmortem changes are evaluated together while estimating the time of death. The pattern analysis of antemortem and postmortem bloodstains is one of the important parameters for forensic science, and cellular changes of blood cells can be useful for the quantitative assessment of the time of death. In this study, by successively investigating erythrocytes exposed in air on mica for 5 days using tapping mode atomic force microscopy (TM-AFM), we observed deformation of whole cell and membrane surface of unfixed erythrocytes with time lapse. We found that the time-dependent cellular changes occurred after exposure of erythrocytes in air for several days. At 0.5 days of exposure, fissures and cell shrinkage were observed. At 2.5 days of exposure, the emergence of nanometer-scale protuberances were observed and these protuberances increased in number with increasing time. The changes of cell shape and cell membrane surface ultrastructure can be used to estimate the time of death. Futhermore, smear-induced abnormal erythrocytes and immunostained erythrocytes were observed here. The need for more precise research is indicated, such as the correlation of membrane changes to intervals of less than 0.5 day of air exposure, and use of various substrates in addition to mica, including glass, metals, fabrics, among others, on which the bloodstains appear in crime scenes. The results of this research demonstrate the efficacy of AFM as a potentially powerful analytical tool in forensic science.