High-Speed Nanomechanical Mapping of the Early Stages of Collagen Growth by Bimodal Force Microscopy

Abstract

High-speed AFM enabled the imaging of protein interactions with millisecond time resolutions (10 fps). However, the acquisition of nanomechanical maps of proteins is about 100 times slower. Here, we developed a high-speed bimodal AFM that provided high-spatial resolution maps of the elastic modulus, the loss tangent and the topography at imaging rates of 5.7 fps. The new microscope was applied to identify the initial stages of the self-assembly of the collagen structures. By following the changes in the physical properties we identified four stages, nucleation and growth of collagen precursors, formation of tropocollagen molecules, assembly of tropocollagens into microfibrils, and alignment of microfibrils to generate microribbons. Some emerging collagen structures never matured and, after an existence of several seconds, they disappeared into the solution. The elastic modulus of a microfibril (~4 MPa) implied very small stiffness (~3x10 -6 N/m). Those values amplified the amplitude of the collagen thermal fluctuations on the mica plane which facilitated microribbon built-up.

Nano-Mechanical Analyses of Native and Cross-Linked Collagen I Matrices Reveal the Mechanical Complexity of Homogenous Samples

“…Since the density of collagen fibers appears to influence the second peak, it follows that this peak is likely related to the way collagen fibers are assembled within the gels, while the first peak being the same (within the bounds of experimental error), irrelevant of collagen density gels, is likely related to the gaps between fibrils (or areas of very low collagen fibril concentration) filled with liquid. Similar bimodal distribution of moduli was recently observed by Gisbert et al [26], who analyzed the changes in the mechanical properties associated with the early stages of collagen assembly on a mica surface (we note that the authors report much higher modulus values, which are most likely due to the effect of the substrate stiffness as only single fibers rather than a thick collagen gel layer were analyzed). Gisbert et al attributed the lowest moduli values to the gaps (between d-spacing) and the presence of monomers.…”

Accurate Wide-Modulus-Range Nanomechanical Mapping of Ultrathin Interfaces with Bimodal Atomic Force Microscopy

“…It involves the application of the virial theorem to excited modes. , This leads to two independent equations, one for each excited mode where z i is the deflection of the excited modes and z is the total cantilever deflection ( z ≈ z 0 + z 1 + z 2 ). In many situations of interest, T is well approximated by the period of the first eigenmode ( T ≈ T 1 ). , The above equation can be solved by two independent approaches. First, by integrating the equation of motion of the excited modes, it has been shown that for A 01 ≫ A 02 , We have used the subscript m to indicate that the virials are directly determined (measured) from the observables ( A 1 , A 2 , ϕ 1 , Δ f 2 ).…”

“…Bimodal AFM is arguably the fastest and most versatile method for mapping the nanomechanical properties of surfaces and interfaces. , It has been applied to characterize the elastic properties of polymers, , hybrid materials, inorganic and organic crystals, , self-assembled monolayers, lipid membranes, DNA, , proteins, − and cells . It combines high spatial resolution (angstrom or nanoscale) with quantitative accuracy for the determination of the Young’s modulus.…”

“…A variety of theories and simulations have supported or demonstrated the capabilities of bimodal AFM. − Currently, it is the only method that provides nanomechanical maps at 5 frames per second (high speed) . Despite its widespread use, the accuracy of the bimodal nanomechanical measurements performed on ultrathin materials might be jeopardized by the bottom-effect artifact. , …”

High-Speed Atomic Force Microscopy and Nanomechanical Mapping as Tools for Studying Dynamic Membrane Remodeling Processes

“…The different ceramide localization in the membrane and mechanical properties is relevant in several biological contexts as apoptosis or viral infection. Finally, we will discuss possible approaches to evaluate the dynamic changes in viscoelasticity of lipid membranes [8], i.e., using fast mapping with bimodal AFM [9].…”