The review’s methodology tells its own story. De-Giorgio and Benedetti searched PubMed, Scopus, and Web of Science without date restrictions, combining AFM-related terms with post-mortem and autopsy keywords, and screened references manually to catch anything the databases missed. Despite the breadth of the search, only five original research articles met their strict criteria of focusing on human post-mortem materials with forensic relevance. That number is striking. AFM has been embraced enthusiastically in materials science, nanotechnology, biology, and clinical medicine for decades, yet its application to the tissues of the deceased remains in its infancy. The five qualifying studies concentrated on just four substrates: bone, skeletal muscle, tendinous tissue, and hair. This scarcity, the authors argue, reflects a combination of high instrumentation costs, small sample sizes, and substantial methodological heterogeneity that has kept the field fragmented.

Bone emerges as the most scientifically mature substrate in the review. Building on earlier animal studies that mapped elastic and inelastic deformation behavior and visualized the lacunar-canalicular network with exceptional precision, researchers turned to human autopsy samples to probe age-related changes in bone composition. In one key investigation, Milovanovic and colleagues characterized mineral grain size in bone harvested from the lateral femoral neck of two groups of women: young women aged 20 to 40 and elderly women aged 73 to 94. The older cohort exhibited larger and more heterogeneous mineral grains, a structural condition associated with reduced mechanical resistance. Crucially, chemical analysis of calcium and phosphorus levels revealed no significant differences between the groups, ruling out a major confounder and reinforcing the validity of the purely structural observations.

A follow-up study translated those structural differences into mechanical terms using AFM-based nanoindentation. The technique works by applying a controlled force to the sample surface and recording the corresponding force-distance curves, from which local mechanical properties can be computed with precision. The results showed that the differences in mineral grain size between young and elderly women translated into distinct elastic behaviors, with significantly reduced elasticity in the elderly group, an effect closely linked to higher fracture susceptibility. Although limited by sample size, the work demonstrated a reproducible methodological framework that the review’s authors believe could be extended to contexts far more directly relevant to forensic investigation, including the characterization of fracture margins in cases of suspected trauma.

It is in forensic traumatology that the review’s most provocative proposals appear. AFM topography and force mapping can quantify nanoscale roughness, microcrack patterns, and local stiffness gradients along fracture edges, capabilities that could support the differentiation between perimortem trauma, injuries inflicted around the time of death, and post-mortem damage that occurred afterward. One high-impact translational scenario involves cardiopulmonary resuscitation. CPR is well recognized to generate rib fracture patterns that can complicate post-mortem interpretation, sometimes mimicking injuries inflicted by an assailant. AFM could add objective surface and mechanical descriptors to complement macroscopic evaluation, radiology, and conventional microscopy, helping pathologists distinguish resuscitation artifacts from thoracic fractures unrelated to resuscitative maneuvers. In an era when CPR-related rib injuries are common and medico-legal disputes over their origin are frequent, such a tool could prove decisive.

Bone is also highly informative in cases of thermal exposure, including fires and explosions. Experimental work has shown that heating produces progressive physicochemical and structural modifications of bone mineral: crystallite parameters begin to change at temperatures of 500 degrees Celsius and above, while marked crystallite growth and recrystallization become prominent at higher temperatures, commonly reported around 700 degrees Celsius and beyond, with downstream effects on the stability and fragility of calcined remains. Post-mortem computed tomography, or PMCT, has already proven valuable in assessing burned victims, including the recognition of typical heat-related changes and the differentiation of heat-related fractures from traumatic ones. The review proposes a correlative framework in which PMCT provides non-destructive, case-level documentation of fracture patterns, ATR-FTIR spectroscopy and X-ray diffraction characterize temperature-related mineral and matrix transformations, and AFM targets selected regions for nanoscale topographic and nanomechanical mapping. The open question is whether thermal overprinting modifies or preserves the nanoscale features relevant to forensic interpretation, and AFM is positioned to answer it.

Soft tissue tells a different story, one in which water plays an unexpectedly large role. Studies on human iliotibial band samples investigated how variations in water content influence the mechanical properties of post-mortem soft tissues, and the findings were clear: lower water content increases the elastic modulus of the tissue. This has direct implications for anyone designing experiments on post-mortem soft tissues, since hydration status must be controlled for or at least reported. With specific regard to estimating the post-mortem interval, the elapsed time since death and one of the central questions in forensic practice, De-Giorgio and colleagues performed a preliminary investigation on skeletal muscle samples collected at different post-mortem intervals, applying AFM-based measurements of the Young’s modulus and hysteresis, the latter reflecting energy dissipation during loading cycles. Their results indicated that hysteresis is less influenced by inter-individual variability than the elastic modulus, making it a parameter worth prioritizing in future PMI-focused studies. This matters because traditional PMI methods, which rely on qualitative assessment of livor mortis, rigor mortis, and algor mortis, are fundamentally constrained by the examiner’s subjective evaluation and by intrinsic factors including age, sex, muscle mass, and ambient temperature.

Hair, remarkably durable and easy to sample, offers a third window into the post-mortem nanoworld. Mansilla and colleagues compared hair samples from contemporary autopsies with those from archaeological mummies to examine nanostructural cellular features. Compared with modern hair, the mummy samples exhibited a depletion of fatty acids and a reduction in the regularity of the cuticular scale pattern, although the scales themselves remained remarkably preserved even after centuries. The review’s authors suggest extending this analytical approach by comparing mummy hair across different historical periods and geographic regions, potentially revealing correlations between long-term environmental exposure and nanostructural hair alterations. Such a comparative framework could eventually help investigators assess whether remains are consistent with a claimed historical context or with a more recent death.

Perhaps the most forward-looking sections of the review concern substrates that have barely been touched in forensic settings. Skin, which has been extensively investigated with AFM in dermatological research on the epidermis and dermis, remains underexplored in forensic pathology, despite the fact that establishing wound vitality and age is often critical for medico-legal reconstruction and remains methodologically challenging. AFM-derived parameters such as surface topography, local stiffness mapping, and viscoelastic signatures could be investigated for their association with the post-trauma interval, provided that major confounders including hydration status, sampling site, and fixation protocol are explicitly controlled. The authors also point to dried bloodstains, where experimental studies have used AFM to estimate stain age by tracking changes in the mechanical properties of red blood cells over time, with subsequent work showing that substrate type, whether glass, metal, or ceramic, significantly affects the mechanical signature. Gunshot residue analysis represents another avenue, since AFM has already been applied to high-resolution characterization of GSR particle morphology and size distribution, with evidence that particle size distributions may vary as a function of shooting distance. Extending such analyses to peri-wound skin at entry and exit sites could reveal reproducible patterns related to muzzle-to-target distance and ammunition type. Even the subtle electrical current mark seen in electrocution victims, currently assessed mainly through histopathology and scanning electron microscopy with energy-dispersive X-ray analysis, could yield additional microfeatures to AFM’s nanomechanical mapping.

The review is candid about the obstacles standing between these visions and courtroom reality. The evidence base is constrained by small sample sizes, high instrumentation costs, and methodological heterogeneity spanning differences in sampling, preparation, AFM operational modes, and reporting standards. To support AFM as a reliable forensic tool, and ultimately strengthen its potential admissibility in court, the authors call for robust statistical reporting including appropriate summary measures, dispersion indices, and explicit assessment of reproducibility and measurement uncertainty. They also advocate multicentric research designs to increase sample representativeness and distribute financial and logistical burdens, along with standardized post-mortem imaging biobanks built on harmonized acquisition protocols to facilitate cross-study comparison and accelerate the integration of AFM-based metrics into forensic science. Bone, skeletal muscle, and hair remain the most practical starting points, being generally accessible at autopsy and collectable with limited manipulation. If those foundations are laid, the nanoscale may soon become a routine crime scene, one measured not in meters but in nanometers.

Subject of Research: Applications of atomic force microscopy to human post-mortem tissues in forensic medicine

Article Title: Atomic force microscopy in human post-mortem samples: current applications and future directions

Article References: De-Giorgio, F., & Benedetti, B. (2026). Atomic force microscopy in human post-mortem samples: current applications and future directions. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-03976-x

Image Credits: AI Generated

DOI: 10.1007/s00414-026-03976-x

Keywords: atomic force microscopy, forensic science, post-mortem interval, forensic pathology, bone nanomechanics, nanoindentation, skeletal muscle, mummified hair, thermal bone alteration, wound age estimation, gunshot residue, legal medicine