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

Postmortem computed tomography, or PMCT, has become a mainstay of modern forensic investigation, offering a non-invasive window into projectile trajectories, fracture patterns, and the extensive osseous destruction typical of cranial gunshot injuries. Established PMCT findings include inward and outward bevelling of bone, keyhole lesions, radiating fracture lines, and the distribution of bone fragments, air, and blood along the bullet path. Yet comparatively little systematic attention has been paid to the hyperdense—radiographically bright—findings that appear at the entry wound itself. These bright foci are frequently attributed to gunshot residue, the particulate and combustion-related material expelled from a firearm upon discharge, but their morphology and underlying mechanisms have remained poorly differentiated, despite their potential forensic weight.

The new study set out to determine whether the various hyperdense patterns seen at entry sites represent a single phenomenon or a heterogeneous spectrum. Led by Radiya Sri Rajendran, with Stephan Andreas Bolliger, Michael Josef Thali, and Dominic Gascho as co-authors, the team analyzed fifty cases of gunshot wounds to the head and neck, all considered consistent with short-range shots based on forensic documentation. Thirty-five cases, seventy percent, were through-and-through injuries with an exit wound, while fifteen cases, thirty percent, involved retained projectiles. Pistols accounted for sixty-six percent of the weapons, revolvers for twenty-six percent, and rifles for eight percent, with 9 mm Para and .22 long rifle ammunition the most frequently represented calibers.

Imaging was performed on a 128-slice SOMATOM Definition Flash scanner using standardized parameters: a tube voltage of 120 kV, a tube current-time product of 400 mAs, a pitch of 0.35, and a rotation time of 1.0 second. Datasets were reconstructed at a slice thickness of 0.6 millimeters with a bone kernel to optimize visualization of radiopaque structures. Two readers with very different levels of experience independently assessed gunshot residue-related hyperdensities: Reader 1 was a physician without specific training in forensic radiology or wound ballistics, while Reader 2 was a forensic imaging specialist with thirteen years of experience. Other findings—bone fragments, fracture lines, projectile fragments, and the radiopaque ring sign—were evaluated by consensus. Agreement on residue detection was moderate, with a Cohen’s kappa of 0.55, a figure that itself underscores how difficult these distinctions can be.

The results paint a picture of considerable morphological complexity. Gunshot residue-related hyperdensities were identified in eighty-two percent of cases by the less experienced reader and seventy-two percent by the specialist, with discrepancies concentrated in cases where residue patterns were limited or diffuse. Bone fragments were the most consistent radiopaque finding at the entry site, present in the vast majority of cases, and their radiodensity frequently overlapped with that of residue, complicating differentiation. Projectile fragments, by contrast, could be reliably separated from both because of their markedly higher radiodensity—often reaching the upper measurable limit of the Hounsfield unit scale at 3071 HU—and the characteristic streak artifacts they produce. Residue itself exhibited a wide range of maximum attenuation values, from 550 to 3022 HU with a median of 1705.5, reflecting focal high-density components rather than the overall composition of the deposit.

Against this backdrop of ambiguity, the radiopaque ring sign stood out. In the three cases where it appeared, it manifested as a compact, continuous circumferential structure embedded within the subcutaneous tissue at the wound margin, clearly distinguishable from both diffuse hyperdensities and focal metallic fragments. Two of the three cases involved a revolver firing .22 long rifle ammunition, and the third involved a revolver chambered in .38 S&W. Critically, in cases with through-and-through injuries, no comparable ring-like structure was observed at the exit sites, even though those sites also contained a mixture of bone and projectile fragments. This asymmetry supports the interpretation that the ring sign is a specific phenomenon of the entry site rather than a general accumulation of radiopaque material, and all three ring-sign cases showed external findings clearly attributable to contact gunshot wounds.

The distinction matters because the forensic literature has not always separated these patterns. Early work by Stein and colleagues in 2000 illustrated gunshot residue findings on PMCT, including a ring-like accumulation reproduced experimentally in a porcine model, but did not consistently treat it as a separate entity. Later studies described residue as spontaneously dense material around entrance wounds in intermediate-range shots, implying more diffuse or surface-related patterns. Gascho and colleagues explicitly reported a subdermal ring of radiopaque material at the entry wound of neck injuries from a contact shot, and porcine cadaver experiments by Junno and colleagues in 2022 reinforced the reproducibility of a ring-shaped structure particularly in contact shots, pointing to a distance-dependent mechanism. More recently, Jupin-Delevaux and colleagues introduced the hyperdense rim sign as a marker for entry wound identification, but their definition rests largely on geometric criteria and may encompass both compact rings and ill-defined perilesional hyperdensities under a single term.

The Zurich team argues that this broader usage risks overgeneralization. Their data indicate that superficial hyperdensities at the wound margin, diffuse perilesional deposits within surrounding tissue, and the compact radiopaque ring sign likely reflect distinct underlying mechanisms. The diffuse patterns probably correspond to surface contamination and combustion-related effects, whereas the ring may arise from a more localized deposition process under specific ballistic conditions—possibly related to gases and particulates forced into the subcutaneous tissue when the muzzle is pressed against the skin. The authors also note that Hounsfield unit measurements alone cannot resolve the question, since maximum attenuation values are dominated by metallic fragments and partial volume effects, and they found that three-dimensional visualization techniques such as cinematic rendering helped in selected cases by clarifying the shape and spatial distribution of ambiguous structures.

The study’s implications extend to an open question in classical forensic pathology: whether the radiopaque ring on PMCT relates to the contusion ring seen on external examination of the skin. The authors suggest that prospective or experimental studies with standardized documentation of both external and radiological findings, ideally combined with histopathological analysis, could clarify whether the two phenomena are connected or independent. They also acknowledge limitations: the retrospective design and restriction to head and neck wounds limit generalizability to other anatomical regions; reliable discrimination among contact, near-contact, and close-range shots was not possible in every case due to tissue destruction, decomposition, or inconclusive external findings; and the subgroup of ring-sign cases is small. The imaging data themselves, containing identifiable anatomical features, are not publicly available, though they may be accessible from the corresponding author under institutional and ethical approval.

Even with those caveats, the message for forensic practice is clear. Hyperdense findings at the wound margin are common on PMCT and remain valuable for identifying entry wounds, but they should not be read as a single entity. A true radiopaque ring sign is rare, morphologically distinct, confined to the entry site, and in this cohort appeared only in contact shots, whereas other residue-related hyperdensities occur across a broader range of firing distances and demand cautious, context-informed interpretation. As virtual autopsy techniques continue to spread through forensic institutes worldwide, refining the vocabulary and diagnostic criteria for what appears on the scan could mean the difference between an ambiguous finding and decisive evidence.

Subject of Research: Radiopaque ring sign at gunshot wound entry sites on postmortem CT

Article Title: Radiopaque ring sign as a distinct finding at the entry site of gunshot wounds on postmortem CT

Article References: Rajendran, R. S., Bolliger, S. A., Thali, M. J., & Gascho, D. (2026). Radiopaque ring sign as a distinct finding at the entry site of gunshot wounds on postmortem CT. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-04004-8

Image Credits: AI Generated

DOI: 10.1007/s00414-026-04004-8

Keywords: gunshot residue, postmortem CT, radiopaque ring sign, forensic imaging, virtual autopsy, wound ballistics, entry wound, hyperdense rim sign, contact shot, forensic radiology, Radiopaque, ring