Accurate Modelling of AFM Force-Indentation Curves with Blunted Indenters at Small Indentation Depths.

When testing biological samples with atomic force microscopy (AFM) nanoindentation using pyramidal indenters, Sneddon's equation is commonly used for data processing, approximating the indenter as a perfect cone. While more accurate models treat the AFM tip as a blunted cone or pyramid, these are complex and lack a direct relationship between applied force and indentation depth, complicating data analysis. This paper proposes a new equation derived from simple mathematical processes and physics-based criteria. It is accurate for small indentation depths and serves as a viable alternative to complex classical approaches. The proposed equation has been validated for ℎ < 3R (where h is the indentation depth and R is the tip radius) and confirmed through simulations with blunted conical and pyramidal indenters, as well as experiments on prostate cancer cells. It is a reliable method for experiments where the tip radius cannot be ignored, such as in shallow indentations on thin samples to avoid substrate effects.

Assessing sarcoma cell cytoskeleton remodeling in response to varying collagen concentration.

Sarcomas, rare malignant tumors of mesenchymal origin, are often underdiagnosed and have face diagnostic ambiguities and limited treatment options. The main objective of this study was to define the nanomechanical and biophysical properties of sarcoma cells, particularly examining how the cytoskeleton's remodeling and related cellular processes such as cell migration and invasion in response to environmental stimuli due to collagen content. Utilizing one murine fibrosarcoma and one osteosarcoma cell line we employed atomic force microscopy, immunostaining, advanced image processing, in vitro cellular assays, and molecular techniques to investigate cells' cytoskeleton remodeling in response to varying collagen concentration. Our study focused on how alterations in collagen content affects the cytoskeletal dynamics and correlate with changes in gene expression profiles relevant to metastasis and an aggressive cancer phenotypes. Our findings indicate that despite their shared classification, fibrosarcoma and osteosarcoma cells display distinct biophysical properties and respond differently to mechanical forces. Notably, this difference in cellular behavior renders mechanical properties a potent novel biomarkers. Furthermore, the metastasis-related identified genes related to metastatic capability, could be potential therapeutic targets. This study highlights the significance of understanding the unique traits of sarcoma cells to improve diagnostic precision and expand therapeutic strategies, for this rare type of cancer.

Stabilizing Tumor-Resident Mast Cells Restores T-Cell Infiltration and Sensitizes Sarcomas to PD-L1 Inhibition.

PURPOSE: To explore the cellular cross-talk of tumor-resident mast cells (MC) in controlling the activity of cancer-associated fibroblasts (CAF) to overcome tumor microenvironment (TME) abnormalities, enhancing the efficacy of immune-checkpoint inhibitors in sarcoma. EXPERIMENTAL DESIGN: We used a coculture system followed by further validation in mouse models of fibrosarcoma and osteosarcoma with or without administration of the MC stabilizer and antihistamine ketotifen. To evaluate the contribution of ketotifen in sensitizing tumors to therapy, we performed combination studies with doxorubicin chemotherapy and anti-PD-L1 (B7-H1, clone 10F.9G2) treatment. We investigated the ability of ketotifen to modulate the TME in human sarcomas in the context of a repurposed phase II clinical trial. RESULTS: Inhibition of MC activation with ketotifen successfully suppressed CAF proliferation and stiffness of the extracellular matrix accompanied by an increase in vessel perfusion in fibrosarcoma and osteosarcoma as indicated by ultrasound shear wave elastography imaging. The improved tissue oxygenation increased the efficacy of chemoimmunotherapy, supported by enhanced T-cell infiltration and acquisition of tumor antigen-specific memory. Importantly, the effect of ketotifen in reducing tumor stiffness was further validated in sarcoma patients, highlighting its translational potential. CONCLUSIONS: Our study suggests the targeting of MCs with clinically administered drugs, such as antihistamines, as a promising approach to overcome resistance to immunotherapy in sarcomas.

Short-Term l-arginine Treatment Mitigates Early Damage of Dermal Collagen Induced by Diabetes.

Changes in the structural properties of the skin due to collagen alterations are an important factor in diabetic skin complications. Using a combination of photonic methods as an optic diagnostic tool, we investigated the structural alteration in rat dermal collagen I in diabetes, and after short-term l-arginine treatment. The multiplex approach shows that in the early phase of diabetes, collagen fibers are partially damaged, resulting in the heterogeneity of fibers, e.g., "patchy patterns" of highly ordered/disordered fibers, while l-arginine treatment counteracts to some extent the conformational changes in collagen-induced by diabetes and mitigates the damage. Raman spectroscopy shows intense collagen conformational changes via amides I and II in diabetes, suggesting that diabetes-induced structural changes in collagen originate predominantly from individual collagen molecules rather than supramolecular structures. There is a clear increase in the amounts of newly synthesized proline and hydroxyproline after treatment with l-arginine, reflecting the changed collagen content. This suggests that it might be useful for treating and stopping collagen damage early on in diabetic skin. Our results demonstrate that l-arginine attenuates the early collagen I alteration caused by diabetes and that it could be used to treat and prevent collagen damage in diabetic skin at a very early stage.

Midkine (MDK) in Hepatocellular Carcinoma: More than a Biomarker.

Midkine (MDK) is a multifunctional secreted protein that can act as a cytokine or growth factor regulating multiple signaling pathways and being implicated in fundamental cellular processes, such as survival, proliferation, and migration. Although its expression in normal adult tissues is barely detectable, MDK serum levels are found to be elevated in several types of cancer, including hepatocellular carcinoma (HCC). In this review, we summarize the findings of recent studies on the role of MDK in HCC diagnosis and progression. Overall, studies show that MDK is a powerful biomarker for HCC early diagnosis, as it can differentiate not only between HCC patients and normal individuals but also between HCC patients and patients with other liver pathologies. It is correlated with high recurrence rates and was shown to be valuable for the diagnosis of early-stage HCC, even in patients negative for α-fetoprotein (AFP), the most commonly used biomarker for HCC diagnosis. A comparison with AFP reveals that MDK is inferior to AFP with regard to specificity but significantly superior with regard to sensitivity, which further indicates the need for using both biomarkers for more effective HCC diagnosis.

A New Elementary Method for Determining the Tip Radius and Young's Modulus in AFM Spherical Indentations.

Atomic force microscopy (AFM) is a powerful tool for characterizing biological materials at the nanoscale utilizing the AFM nanoindentation method. When testing biological materials, spherical indenters are typically employed to reduce the possibility of damaging the sample. The accuracy of determining Young's modulus depends, among other factors, on the calibration of the indenter, i.e., the determination of the tip radius. This paper demonstrates that the tip radius can be approximately calculated using a single force-indentation curve on an unknown, soft sample without performing any additional experimental calibration process. The proposed method is based on plotting a tangent line on the force indentation curve at the maximum indentation depth. Subsequently, using equations that relate the applied force, maximum indentation depth, and the tip radius, the calculation of the tip radius becomes trivial. It is significant to note that the method requires only a single force-indentation curve and does not necessitate knowledge of the sample's Young's modulus. Consequently, the determination of both the sample's Young's modulus and the tip radius can be performed simultaneously. Thus, the experimental effort is significantly reduced. The method was tested on 80 force-indentation curves obtained on an agarose gel, and the results were accurate.

Fascin-1 in Cancer Cell Metastasis: Old Target-New Insights.

As metastasis is responsible for most cancer-related deaths, understanding the cellular and molecular events that lead to cancer cell migration and invasion will certainly provide insights into novel anti-metastatic therapeutic targets. Fascin-1 is an actin-bundling protein fundamental to all physiological or pathological processes that require cell migration. It is responsible for cross-linking actin microfilaments during the formation of actin-rich cellular structures at the leading edge of migrating cells such as filopodia, lamellipodia and invadopodia. While most epithelial tissues express low levels of Fascin-1, it is dramatically elevated in the majority of cancers and its expression has been associated with more aggressive disease and decreased overall survival. Hence, it has been proposed as a potential anti-cancer target. In the present review, we studied recent literature with regard to Fascin-1 expression in different cancers, its role in altering the mechanical properties of cancer cells, promoting cancer cell migration, invasion and metastasis and the effect of its inhibition, via various pharmacological inhibitors, in eliminating metastasis in vitro and/or in vivo. Recent studies corroborate the notion that Fascin-1 is critically involved in metastasis and prove that it is a valuable anti-metastatic target that is worth investigating further.

Multilevel Assessment of Stent-Induced Inflammation in the Adjacent Vascular Tissue.

A recent U.S. Food and Drug Administration report presented the currently available scientific information related to biological response to metal implants. In this work, a multilevel approach was employed to assess the implant-induced and biocorrosion-related inflammation in the adjacent vascular tissue using a mouse stent implantation model. The implications of biocorrosion on peri-implant tissue were assessed at the macroscopic level via in vivo imaging and histomorphology. Elevated matrix metalloproteinase activity, colocalized with the site of implantation, and histological staining indicated that stent surface condition and implantation time affect the inflammatory response and subsequent formation and extent of neointima. Hematological measurements also demonstrated that accumulated metal particle contamination in blood samples from corroded-stetted mice causes a stronger immune response. At the cellular level, the stent-induced alterations in the nanostructure, cytoskeleton, and mechanical properties of circulating lymphocytes were investigated. It was found that cells from corroded-stented samples exhibited higher stiffness, in terms of Young's modulus values, compared to noncorroded and sham-stented samples. Nanomechanical modifications were also accompanied by cellular remodeling, through alterations in cell morphology and stress (F-actin) fiber characteristics. Our analysis indicates that surface wear and elevated metal particle contamination, prompted by corroded stents, may contribute to the inflammatory response and the multifactorial process of in-stent restenosis. The results also suggest that circulating lymphocytes could be a novel nanomechanical biomarker for peri-implant tissue inflammation and possibly the early stage of in-stent restenosis. Large-scale studies are warranted to further investigate these findings.

An Ethnobotanical Investigation into the Traditional Uses of Mediterranean Medicinal and Aromatic Plants: The Case of Troodos Mountains in Cyprus.

The Troodos mountains in Cyprus are a hotspot of plant diversity and cultural heritage. However, the traditional uses of medicinal and aromatic plants (MAPs), a significant aspect of local culture, have not been thoroughly studied. The aim of this research was to document and analyze the traditional uses of MAPs in Troodos. Data on MAPs and their traditional uses were collected through interviews. A database was constructed with categorized information on the uses of 160 taxa belonging to 63 families. The quantitative analysis included the calculation and comparison of six indices of ethnobotanical importance. The cultural value index was selected to reveal the most culturally significant MAPs taxa, while the informant consensus index was utilized to quantify the consensus in information obtained related to uses of MAPs. Furthermore, the 30 most popular MAPs taxa, exceptional and fading uses, and the plant parts used for different purposes are described and reported. The results reveal a deep connection between the people of Troodos and the plants of the area. Overall, the study provides the first ethnobotanical assessment for the Troodos mountains in Cyprus, contributing to a better understanding of the diverse uses of MAPs in mountain regions of the Mediterranean.

Pancreatic Cancer Presents Distinct Nanomechanical Properties During Progression.

Cancer progression is closely related to changes in the structure and mechanical properties of the tumor microenvironment (TME). In many solid tumors, including pancreatic cancer, the interplay among the different components of the TME leads to a desmoplastic reaction mainly due to collagen overproduction. Desmoplasia is responsible for the stiffening of the tumor, poses a major barrier to effective drug delivery and has been associated with poor prognosis. The understanding of the involved mechanisms in desmoplasia and the identification of nanomechanical and collagen-based properties that characterize the state of a particular tumor can lead to the development of novel diagnostic and prognostic biomarkers. In this study, in vitro experiments were conducted using two human pancreatic cell lines. Morphological and cytoskeleton characteristics, cells' stiffness and invasive properties were assessed using optical and atomic force microscopy techniques and cell spheroid invasion assay. Subsequently, the two cell lines were used to develop orthotopic pancreatic tumor models. Tissue biopsies were collected at different times of tumor growth for the study of the nanomechanical and collagen-based optical properties of the tissue using Atomic Force Microscopy (AFM) and picrosirius red polarization microscopy, respectively. The results from the in vitro experiments demonstrated that the more invasive cells are softer and present a more elongated shape with more oriented F-actin stress fibers. Furthermore, ex vivo studies of orthotopic tumor biopsies on MIAPaCa-2 and BxPC-3 murine tumor models highlighted that pancreatic cancer presents distinct nanomechanical and collagen-based optical properties relevant to cancer progression. The stiffness spectrums (in terms of Young's modulus values) showed that the higher elasticity distributions were increasing during cancer progression mainly due desmoplasia (collagen overproduction), while a lower elasticity peak was evident - due to cancer cells softening - on both tumor models. Optical microscopy studies highlighted that collagen content increases while collagen fibers tend to form align patterns. Consequently, during cancer progression nanomechanical and collagen-based optical properties alter in relation to changes in collagen content. Therefore, they have the potential to be used as novel biomarkers for assessing and monitoring tumor progression and treatment outcomes.

3D AFM Nanomechanical Characterization of Biological Materials.

Atomic Force Microscopy (AFM) is a powerful tool enabling the mechanical characterization of biological materials at the nanoscale. Since biological materials are highly heterogeneous, their mechanical characterization is still considered to be a challenging procedure. In this paper, a new approach that leads to a 3-dimensional (3D) nanomechanical characterization is presented based on the average Young's modulus and the AFM indentation method. The proposed method can contribute to the clarification of the variability of the mechanical properties of biological samples in the 3-dimensional space (variability at the x-y plane and depth-dependent behavior). The method was applied to agarose gels, fibroblasts, and breast cancer cells. Moreover, new mathematical methods towards a quantitative mechanical characterization are also proposed. The presented approach is a step forward to a more accurate and complete characterization of biological materials and could contribute to an accurate user-independent diagnosis of various diseases such as cancer in the future.

Determining Spatial Variability of Elastic Properties for Biological Samples Using AFM.

Measuring the mechanical properties (i.e., elasticity in terms of Young's modulus) of biological samples using Atomic Force Microscopy (AFM) indentation at the nanoscale has opened new horizons in studying and detecting various pathological conditions at early stages, including cancer and osteoarthritis. It is expected that AFM techniques will play a key role in the future in disease diagnosis and modeling using rigorous mathematical criteria (i.e., automated user-independent diagnosis). In this review, AFM techniques and mathematical models for determining the spatial variability of elastic properties of biological materials at the nanoscale are presented and discussed. Significant issues concerning the rationality of the elastic half-space assumption, the possibility of monitoring the depth-dependent mechanical properties, and the construction of 3D Young's modulus maps are also presented.

Polymeric micelles effectively reprogram the tumor microenvironment to potentiate nano-immunotherapy in mouse breast cancer models.

Nano-immunotherapy improves breast cancer outcomes but not all patients respond and none are cured. To improve efficacy, research focuses on drugs that reprogram cancer-associated fibroblasts (CAFs) to improve therapeutic delivery and immunostimulation. These drugs, however, have a narrow therapeutic window and cause adverse effects. Developing strategies that increase CAF-reprogramming while limiting adverse effects is urgent. Here, taking advantage of the CAF-reprogramming capabilities of tranilast, we developed tranilast-loaded micelles. Strikingly, a 100-fold reduced dose of tranilast-micelles induces superior reprogramming compared to free drug owing to enhanced intratumoral accumulation and cancer-associated fibroblast uptake. Combination of tranilast-micelles and epirubicin-micelles or Doxil with immunotherapy increases T-cell infiltration, resulting in cures and immunological memory in mice bearing immunotherapy-resistant breast cancer. Furthermore, shear wave elastography (SWE) is able to monitor reduced tumor stiffness caused by tranilast-micelles and predict response to nano-immunotherapy. Micellar encapsulation is a promising strategy for TME-reprogramming and SWE is a potential biomarker of response.

Nanomechanical properties of solid tumors as treatment monitoring biomarkers.

Many tumors, such as types of sarcoma and breast cancer, stiffen as they grow in a host healthy tissue, while individual cancer cells are becoming softer. Tumor stiffening poses major pathophysiological barriers to the effective delivery of drugs and compromises treatment efficacy. It has been established that normalization of the mechanical properties of a tumor by targeting components of the tumor microenvironment (TME) enhances the delivery of anti-cancer agents and consequently the therapeutic outcome. Consequently, there is an urgent need for the development of biomarkers, which characterize the mechanical state of a particular tumor for the development of personalized treatments or for monitoring therapeutic strategies that target the TME. In this work, Atomic Force Microscopy (AFM) was used to assess human and murine nanomechanical properties from tumor biopsies. In the case of murine tumor models, the nanomechanical properties during tumor progression were measured and a TME normalization drug (tranilast) along with chemotherapy doxorubicin were employed in order to investigate whether AFM has the ability to capture changes in the nanomechanical properties of a tumor during treatment. The nanomechanical data were further correlated with ex vivo characterization of structural components of the TME. The results highlighted that nanomechanical properties alter during cancer progression and AFM measurements are sensitive enough to capture even small alterations during different types of treatments, namely normalization and chemotherapy. The identification of unique AFM-based nanomechanical properties can lead to the development of biomarkers for treatment prediction and monitoring. STATEMENT OF SIGNIFICANCE: Cancer progression is associated with vast remodeling of the tumor microenvironment resulting in changes in the mechanical properties of the tissue. Indeed, many tumors stiffen as they grow and this stiffening compromises treatment efficacy. As a result, a number of treatments target tumor microenvironment in order to normalize its mechanical properties. Consequently, there is an urgent need for the development of innovative tools that can assess the mechanical properties of a particular tumor and monitor tumor progression and treatment outcomes. This work highlights the use of atomic force microscopy (AFM) for assessing the elasticity spectrum of solid tumors at different stages and during treatment. This knowledge is essential for the development of AFM-based nanomechanical biomarkers for treatment prediction and monitoring.

Atomic Force Microscopy Nanoindentation Method on Collagen Fibrils.

Atomic Force Microscopy nanoindentation method is a powerful technique that can be used for the nano-mechanical characterization of bio-samples. Significant scientific efforts have been performed during the last two decades to accurately determine the Young's modulus of collagen fibrils at the nanoscale, as it has been proven that mechanical alterations of collagen are related to various pathological conditions. Different contact mechanics models have been proposed for processing the force-indentation data based on assumptions regarding the shape of the indenter and collagen fibrils and on the elastic or elastic-plastic contact assumption. However, the results reported in the literature do not always agree; for example, the Young's modulus values for dry collagen fibrils expand from 0.9 to 11.5 GPa. The most significant parameters for the broad range of values are related to the heterogeneous structure of the fibrils, the water content within the fibrils, the data processing errors, and the uncertainties in the calibration of the probe. An extensive discussion regarding the models arising from contact mechanics and the results provided in the literature is presented, while new approaches with respect to future research are proposed.

Assessing Collagen D-Band Periodicity with Atomic Force Microscopy.

The collagen superfamily includes more than fifty collagen and/or collagen-like proteins with fibril-forming collagen type I being the most abundant protein within the extracellular matrix. Collagen type I plays a crucial role in a variety of functions, it has been associated with many pathological conditions and it is widely used due to its unique properties. One unique nano-scale characteristic of natural occurring collagen type I fibers is the so-called D-band periodicity, which has been associated with collagen natural structure and properties, while it seems to play a crucial role in the interactions between cells and collagen and in various pathological conditions. An accurate characterization of the surface and structure of collagen fibers, including D-band periodicity, on collagen-based tissues and/or (nano-)biomaterials can be achieved by Atomic Force Microscopy (AFM). AFM is a scanning probe microscope and is among the few techniques that can assess D-band periodicity. This review covers issues related to collagen and collagen D-band periodicity and the use of AFM for studying them. Through a systematic search in databases (PubMed and Scopus) relevant articles were identified. The study of these articles demonstrated that AFM can offer novel information concerning D-band periodicity. This study highlights the importance of studying collagen D-band periodicity and proves that AFM is a powerful tool for investigating a number of different properties related to collagen D-band periodicity.

How did correlative atomic force microscopy and super-resolution microscopy evolve in the quest for unravelling enigmas in biology?

With the invention of the Atomic Force Microscope (AFM) in 1986 and the subsequent developments in liquid imaging and cellular imaging it became possible to study the topography of cellular specimens under nearly physiological conditions with nanometric resolution. The application of AFM to biological research was further expanded with the technological advances in imaging modes where topographical data can be combined with nanomechanical measurements, offering the possibility to retrieve the biophysical properties of tissues, cells, fibrous components and biomolecules. Meanwhile, the quest for breaking the Abbe diffraction limit restricting microscopic resolution led to the development of super-resolution fluorescence microscopy techniques that brought the resolution of the light microscope comparable to the resolution obtained by AFM. The instrumental combination of AFM and optical microscopy techniques has evolved over the last decades from integration of AFM with bright-field and phase-contrast imaging techniques at first to correlative AFM and wide-field fluorescence systems and then further to the combination of AFM and fluorescence based super-resolution microscopy modalities. Motivated by the many developments made over the last decade, we provide here a review on AFM combined with super-resolution fluorescence microscopy techniques and how they can be applied for expanding our understanding of biological processes.

Silencing of Growth Differentiation Factor-15 Promotes Breast Cancer Cell Invasion by Down-regulating Focal Adhesion Genes.

BACKGROUND/AIM: As metastasis accounts for most breast cancer (BC)-related deaths, identifying key players becomes research priority. Growth differentiation factor-15 (GDF15), a member of the transforming growth factor-ß superfamily, is affected by the actin cytoskeleton and has been associated with cancer. However, its exact role in BC cell invasiveness is vague. MATERIALS AND METHODS: GDF15 short-hairpin (shRNA)-mediated silencing was used to inhibit GDF15 expression in MCF-7 and MDA-MB-231 BC cells and gene expression of relevant focal adhesion (FA) genes, cell migration, invasion and tumor spheroid invasion were subsequently analyzed. RESULTS: GDF15 silencing promoted cell migration, cell invasion as well as tumor spheroid invasion and up-regulated urokinase plasminogen activator (uPA) and FA genes, integrin-linked kinase (ILK), LIM zinc finger domain containing 1 (LIMS1), α-parvin (PARVA), and RAS suppressor-1 (RSU1). Computational analysis of Cancer Genome Atlas BC dataset however, revealed no significant correlation between GDF15 expression and metastasis pointing towards a more complex molecular interplay between GDF15, actin cytoskeleton and FA-related genes which ultimately affects their expression pattern, in vivo. CONCLUSION: GDF15 suppresses BC cell invasion in vitro through down-regulation of FA genes but its role in BC is more complicated in vivo and warrants further investigation.

TGF-ß inhibition combined with cytotoxic nanomedicine normalizes triple negative breast cancer microenvironment towards anti-tumor immunity.

Tumor normalization strategies aim to improve tumor blood vessel functionality (i.e., perfusion) by reducing the hyper-permeability of tumor vessels or restoring compressed vessels. Despite progress in strategies to normalize the tumor microenvironment (TME), their combinatorial antitumor effects with nanomedicine and immunotherapy remain unexplored. Methods: Here, we re-purposed the TGF-ß inhibitor tranilast, an approved anti-fibrotic and antihistamine drug, and combined it with Doxil nanomedicine to normalize the TME, increase perfusion and oxygenation, and enhance anti-tumor immunity. Specifically, we employed two triple-negative breast cancer (TNBC) mouse models to primarily evaluate the therapeutic and normalization effects of tranilast combined with doxorubicin and Doxil. We demonstrated the optimized normalization effects of tranilast combined with Doxil and extended our analysis to investigate the effect of TME normalization to the efficacy of immune checkpoint inhibitors. Results: Combination of tranilast with Doxil caused a pronounced reduction in extracellular matrix components and an increase in the intratumoral vessel diameter and pericyte coverage, indicators of TME normalization. These modifications resulted in a significant increase in tumor perfusion and oxygenation and enhanced treatment efficacy as indicated by the notable reduction in tumor size. Tranilast further normalized the immune TME by restoring the infiltration of T cells and increasing the fraction of T cells that migrate away from immunosuppressive cancer-associated fibroblasts. Furthermore, we found that combining tranilast with Doxil nanomedicine, significantly improved immunostimulatory M1 macrophage content in the tumorigenic tissue and improved the efficacy of the immune checkpoint blocking antibodies anti-PD-1/anti-CTLA-4. Conclusion: Combinatorial treatment of tranilast with Doxil optimizes TME normalization, improves immunostimulation and enhances the efficacy of immunotherapy.

Atomic Force Microscopy on Biological Materials Related to Pathological Conditions.

Atomic force microscopy (AFM) is an easy-to-use, powerful, high-resolution microscope that allows the user to image any surface and under any aqueous condition. AFM has been used in the investigation of the structural and mechanical properties of a wide range of biological matters including biomolecules, biomaterials, cells, and tissues. It provides the capacity to acquire high-resolution images of biosamples at the nanoscale and allows at readily carrying out mechanical characterization. The capacity of AFM to image and interact with surfaces, under physiologically relevant conditions, is of great importance for realistic and accurate medical and pharmaceutical applications. The aim of this paper is to review recent trends of the use of AFM on biological materials related to health and sickness. First, we present AFM components and its different imaging modes and we continue with combined imaging and coupled AFM systems. Then, we discuss the use of AFM to nanocharacterize collagen, the major fibrous protein of the human body, which has been correlated with many pathological conditions. In the next section, AFM nanolevel surface characterization as a tool to detect possible pathological conditions such as osteoarthritis and cancer is presented. Finally, we demonstrate the use of AFM for studying other pathological conditions, such as Alzheimer's disease and human immunodeficiency virus (HIV), through the investigation of amyloid fibrils and viruses, respectively. Consequently, AFM stands out as the ideal research instrument for exploring the detection of pathological conditions even at very early stages, making it very attractive in the area of bio- and nanomedicine.