Figure 2.

Graphic representation illustrating the main methods to estimate PMI and the time frame within which can be employed (adapted from [133]). Algor mortis, the cooling of the body after death, is probably the most widely used method for estimating PMI and is based on the decrease in rectal temperature [68]. Livor mortis corresponds to the change in skin color due to the deposition of stagnant blood in the lower parts of the body after death and is assessed by the color and fixation of lividity through the bleaching test [68,134]. Rigor mortis, also called postmortem rigidity, represents the third stage of death characterized by the stiffening of muscles due to the binding of actin and myosin filaments (starts around 2–3 h after death) [68]. The vitreous humor, a clear and colorless fluid that fills the space between the lens and the retina of the eye, offers an alternative biological matrix for estimating the PMI through a valuable biochemical method [12,135]. Entomology, the study of insects and their interactions with other organisms, humans, and the environment, has been used for forensic purposes since the 13th century, providing insights into PMI through the analysis of insect colonization and life cycle evolution on decomposing cadavers [136]. The process of decomposition after death involves the degradation of soft tissues through autolysis (cellular breakdown) and putrefaction (bacterial consumption), with various factors influencing its progression; there is a somewhat predictable sequence of stages, including fresh, bloat, decay, and dry [134]. Desiccation, the process of drying of the skin and mucous membranes after death, leads to changes in color and texture, with notable effects observed in the eyes as well as in the skin where the lips and genitalia are particularly affected [134]. Microscopic changes, including alterations in tissue histopathology, immunohistochemical, and protein densities, can provide valuable information for estimating the PMI based on the decrease or increase in specific proteins and compounds [134]. Molecular methods, including RNA and DNA analysis, offer valuable tools for PMI estimations by assessing nucleic acid integrity, measuring degradation rates, and quantitatively amplifying target genes, although DNA degradation poses limitations for longer PMIs [134]. Microbiology, the main focus of this review is discussed in detail in the next section. Ocular postmortem changes include variations in corneal opacity, pupil diameter, blood vessel striation, retinal color, and intraocular pressure [134]. Forensic radiology is an emerging area of forensic sciences that deals with the utilization of imaging techniques such as radiography, computed tomography, and magnetic resonance imaging for the examination and analysis of deceased individuals [134]. Radionuclide-based methods, commonly employed in forensic anthropology for skeletonized remains, utilize radionuclides such as 210Pb and 210Po, 14C radiocarbon, as well as measurements of citrate and nitrogen content [134,137,138]. Forensic odontology uses dental evidence to provide expert analysis and answers to forensic inquiries, serving both the field of justice and anthropology [139]. Forensic anthropology analyzes bone evidence, whether fragmented or intact, to respond to the law in several aspects, including identification, gender or age estimation, and PMI calculation [137]. Through botanical classification and traditional knowledge of plant species, the relationships between their development, specific habits, and geographical origins can be established, adding valuable information about the environment surrounding a corpse and possibly in the PMI estimation [140].