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Ehlers-Danlos syndromes (EDS) represent a group of rare genetic disorders affecting connective tissues. Globally, approximately 1.5 million individuals suffer from EDS, with 10,000 reported cases in Canada alone. Understanding the histological properties of collagen in EDS has been challenging, but advanced techniques like atomic force microscopy (AFM) have opened up new possibilities for label-free skin imaging. This approach, which explores Type I collagen fibrils at the nanoscale, could potentially enhance EDS diagnosis and our knowledge of collagen type I-related connective tissue disorders. In the current study, we have employed AFM to examine ex-vivo skin biopsies from four individuals: one with classical EDS (cEDS), one with hypermobile EDS (hEDS), one with hEDS and Scleroderma (hEDS-Scleroderma), and one healthy control. Picrosirius red (PS) staining was used to highlight collagen differences in the samples. For each case, 14 images and 1400 force curves were obtained, with seven images and 700 force curves representing healthy collagen (PS-induced red staining) and the rest showcasing disrupted collagen (yellow staining). The results showed that PS staining was uniform throughout the control section, while cEDS and hEDS displayed localized areas of yellow staining. In the case of hEDS-Scleroderma, the yellow staining was widespread throughout the section. AFM images revealed irregular collagen fibrils in the disrupted, yellow-stained areas, contrasting with aligned and well-registered collagen fibrils in healthy, red-stained regions. Additionally, the study assessed the ability of non-AFM specialists to differentiate between healthy and disrupted collagen in AFM images, yielding substantial agreement among raters according to Fleiss's and Cohen's kappa scores (0.96 and 0.79±0.1, respectively). Biomechanical analysis revealed that normal healthy collagen exhibited a predominant population at 2.5 GPa. In contrast, EDS-affected collagen displayed subpopulations with lower compressive elastic modulus, indicating weaker collagen fibrils in EDS patients. Although these findings pertain to a limited number of cases, they offer valuable insights into the nanoscale collagen structure and biomechanics in individuals with EDS. Over time, these insights could be developed into specific biomarkers for the condition, improving diagnosis and treatment for EDS and related connective tissue disorders., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Neshatian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The aim of the current work is twofold: to demonstrate the application of in situ non-invasive imaging by portable atomic force microscopy (AFM) on the surfaces of a violin and to integrate compositional and mechanical analysis at the nano scale level on model samples of varnished wood. These samples were prepared according to traditional recipes by an Italian lute-maker family well practised in the art. Samples of oil and spirit-based varnishes on maple wood, naturally and accelerated light aged, were studied. AFM was used to measure the nanomechanical properties of the model samples and established that the spirit-based varnish was stiffer than the oil-based. Synchrotron radiation micro- Fourier Transform Infra-red analysis of the layer structure revealed that stiffer spirit-based varnish showed less penetration into the wood than the oil-based. Further PeakForce Quantitative Nanomechanical Mapping (QNM) demonstrated a difference in adhesion values between the oil- and spirit-based samples.

Growing interest in exploring mechanically mediated biological phenomena has resulted in cell culture substrates and 3D matrices with variable stiffnesses becoming standard tools in biology labs. However, correlating stiffness with biological outcomes and comparing results between research groups is hampered by variability in the methods used to determine Young's (elastic) modulus, E, and by the inaccessibility of relevant mechanical engineering protocols to most biology labs. Here, we describe a protocol for measuring E of soft 2D surfaces and 3D hydrogels using atomic force microscopy (AFM) force spectroscopy. We provide instructions for preparing hydrogels with and without encapsulated live cells, and provide a method for mounting samples within the AFM. We also provide details on how to calibrate the instrument, and give step-by-step instructions for collecting force-displacement curves in both manual and automatic modes (stiffness mapping). We then provide details on how to apply either the Hertz or the Oliver-Pharr model to calculate E, and give additional instructions to aid the user in plotting data distributions and carrying out statistical analyses. We also provide instructions for inferring differential matrix remodeling activity in hydrogels containing encapsulated single cells or organoids. Our protocol is suitable for probing a range of synthetic and naturally derived polymeric hydrogels such as polyethylene glycol, polyacrylamide, hyaluronic acid, collagen, or Matrigel. Although sample preparation timings will vary, a user with introductory training to AFM will be able to use this protocol to characterize the mechanical properties of two to six soft surfaces or 3D hydrogels in a single day.

Scleroderma (or systemic sclerosis, SSc) is a disease caused by excess crosslinking of collagen. The skin stiffens and becomes painful, while internally, organ function can be compromised by the less elastic collagen. Diagnosis of SSc is often only possible in advanced cases by which treatment time is limited. A more detailed analysis of SSc may provide better future treatment options and information of disease progression. Recently, the histological stain picrosirius red showing collagen register has been combined with atomic force microscopy (AFM) to study SSc. Skin from healthy individuals and SSc patients was biopsied, stained and studied using AFM. By investigating the crosslinking of collagen at a smaller hierarchical stage, the effects of SSc were more pronounced. Changes in morphology and Young's elastic modulus were observed and quantified; giving rise to a novel technique, we have termed "quantitative nanohistology". An increase in nanoscale stiffness in the collagen for SSc compared with healthy individuals was seen by a significant increase in the Young's modulus profile for the collagen. These markers of stiffer collagen in SSc are similar to the symptoms experienced by patients, giving additional hope that in the future, nanohistology using AFM can be readily applied as a clinical tool, providing detailed information of the state of collagen., Competing Interests: The authors report no conflicts of interest in this work.

As titanium (Ti) continues to be utilized in great extent for the fabrication of artificial implants, it is important to understand the crucial bacterium-Ti interaction occurring during the initial phases of biofilm formation. By employing a single-cell force spectroscopy technique, the nanoadhesive interactions between the early-colonizing Streptococcus sanguinis and a clinically analogous smooth Ti substrate were explored. Mean adhesion forces between S. sanguinis and Ti were found to be 0.32±0.00, 1.07±0.06, and 4.85±0.56 nN for 0, 1, and 60 seconds contact times, respectively; while adhesion work values were reported at 19.28±2.38, 104.60±7.02, and 1,317.26±197.69 aJ for 0, 1, and 60 seconds, respectively. At 60 seconds surface delays, minor-rupture events were modeled with the worm-like chain model yielding an average contour length of 668±12 nm. The mean force for S. sanguinis minor-detachment events was 1.84±0.64 nN, and Poisson analysis decoupled this value into a short-range force component of -1.60±0.34 nN and a long-range force component of -0.55±0.47 nN. Furthermore, a solution of 2 mg/mL chlorhexidine was found to increase adhesion between the bacterial probe and substrate. Overall, single-cell force spectroscopy of living S. sanguinis cells proved to be a reliable way to characterize early-bacterial adhesion onto machined Ti implant surfaces at the nanoscale.

The mechanical properties of bacterial cells play an important role in crucial bacterial processes such as cell growth, colonisation and biofilm formation. Recent developments in the field of nanotechnology and atomic force microscopy (AFM) have made it possible to observe, characterise and understand the nanomechanic behaviour of live bacterial cells as never before. Unlike traditional techniques, AFM makes it possible to employ living bacteria in their physiological environment with minimal or no sample preparation. The technique of AFM nanoindentation opens new possibilities to study bacterial cell wall stiffness under different mechanical and buffer conditions. Also, by attaching bacterial cells to functionalised AFM cantilevers, single-cell force spectroscopy (SCFS) can be used to measure the adhesion of bacteria to biological and non-biological substrates at the nano-newton and pico-newton scale, and provide specific information on receptor-ligand interactions. By studying the biophysics of the bacterial-surface interaction with the abovementioned techniques, it has been possible to gain new insight on the early stages of bacterial colonisation and biofilm formation.

The use of the atomic force microscope (AFM) in microbiology has progressed significantly throughout the years since its first application as a high-resolution imaging instrument. Modern AFM setups are capable of characterizing the nanomechanical behaviour of bacterial cells at both the cellular and molecular levels, where elastic properties and adhesion forces of single bacterium cells can be examined under different experimental conditions. Considering that bacterial and biofilm-mediated infections continue to challenge the biomedical field, it is important to understand the biophysical events leading towards bacterial adhesion and colonization on both biological and non-biological substrates. The purpose of this review is to present the latest findings concerning the field of single-bacterium nanomechanics, and discuss future trends and applications of nanoindentation and single-cell force spectroscopy techniques in biomedicine.

The structural properties of collagen have been the subject of numerous studies over past decades, but with the arrival of new technologies, such as the atomic force microscope and related techniques, a new era of research has emerged. Using microthermal analysis, it is now possible to image samples as well as performing localized thermal measurements without damaging or destroying the sample itself. This technique was successfully applied to characterize the thermal response between native collagen fibrils and their denatured form, gelatin. Thermal transitions identified at (150 ± 10)°C and (220 ± 10)°C can be related to the process of gelatinization of the collagen fibrils, whereas at higher temperatures, both the gelatin and collagen samples underwent two-stage transitions with a common initial degradation temperature at (300 ± 10)°C and a secondary degradation temperature of (340 ± 10)°C for the collagen and of (420 ± 10)°C for the gelatin, respectively. The broadening and shift in the secondary degradation temperature was linked to the spread of thermal degradation within the gelatin and collagen fibrils matrix further away from the point of contact between probe and sample. Finally, similar measurements were performed inside a bone resorption lacuna, suggesting that microthermal analysis is a viable technique for investigating the thermomechanical response of collagen for in situ samples that would be, otherwise, too challenging or not possible using bulk techniques., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Mineralised tissues such as bone consist of two material phases: collagen protein fibrils, secreted by osteoblasts, form model structures for subsequent deposition of mineral, calcium hydroxyapatite. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair. Bone active drugs have recently been developed for skeletal diseases, and there is revived interest in changes in the structure of mineralised tissues seen in disease and upon treatment. The resolution of atomic force microscopy and use of unmodified samples has enabled us to image bone and dentine collagen exposed by the natural process of cellular dissolution of mineralised matrix. The morphology of bone and dentine has been analysed when fully mineralised and after osteoclast-mediated bone resorption, and compared with results from other microscopy techniques. Banded type I collagen, with 66.5+/-1.4 nm axial D-periodicity and 62.2+/-7.0 nm diameter, has been identified within resorption lacunae in bone and 69.4+/-4.3 nm axial D-periodicity and 140.6+/-12.4 nm diameter in dentine substrates formed by human and rabbit osteoclasts, respectively. This observation suggests a route by which the material and morphological properties of bone collagen can be analysed in situ, compared with collagen from non-skeletal sites, and contrasted in diseases of medical importance, such as osteoporosis, where skeletal tissue is mechanically weakened.

Mineralised tissues, such as bone, consist of two material phases: collagen protein fibrils that form the structural models upon which the mineral, calcium hydroxyapatite, is subsequently deposited. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair, and matrix proteins are replaced by the synthetic activity of osteoblasts and then calcify. The resolution of atomic force microscopy and use of unmodified, fully calcified samples has enabled the imaging of the overall bone and dentine structure, including collagen and mineral phases. Mineral crystals, in the diameter size range of 225 nm up to 1.4 microm, were found in unmodified bone and dentine respectively. D-banded collagen is observed in dentine after acid treatment and in bone after osteoclast-mediated matrix resorption; axial periodicity values of approximately 67 and 69 nm are observed, respectively. These experimental approaches have enabled the structure of mineralised tissues to be examined in native samples and will facilitate the study of bone structure in important clinical disorders of the skeleton, such as osteoporosis.