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

Abstract

High-speed atomic force microscopy (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 fps. The 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 (∼3 × 10^-6 N/m). Those values amplified the amplitude of the collagen thermal fluctuations on the mica plane, which facilitated microribbon build-up.

Keywords: bimodal AFM; collagen; high-speed AFM; nanomechanics; viscoelasticity.

Figure 1

High-speed bimodal AFM maps of topography, elastic modulus, and loss tangent of collagen microribbons. (a) Sequence of topography, elastic modulus, and loss tangent maps showing collagen microribbon growth on mica (see Movie S1 in SI). The sequence captures the merging of and the alignment of the D-bands of several microribbons. The numbers in parentheses indicate the frame order in the video. (b) Cross sections along the lines marked in the frames recorded at t = 512 s. All of the properties oscillate with the periodicity of the D-band structure. Some of the cross section points were generated using a cubic interpolation regression obtained from 10 contiguous experimental pixels. The maps were obtained in buffer by applying a peak force on the microribbons of 1.2 nN (18 nN on mica). Imaging rate, 0.2 fps (512 × 512 pixels, scanline of 100 Hz). Additional bimodal AFM data: f₁ = 132 kHz, k₁ = 0.18 nN/nm, Q₁ = 1.7; f₂ = 1045 kHz, k₂ = 12 nN/nm, Q₂ = 5.3; A₀₁ = 10 nm, A₀₂ = 1.5 nm, and A₁ = 9 nm.

Figure 2

Growth dynamics of a single microfibril. (a) Kymograph of the height, the elastic modulus, and the loss tangent of a growing collagen microfibril. The images show the transition from the accretion of collagen precursors from the solution to the formation of a collagen microfibril (five tropocollagen molecules) with the D-band structure. (b) Cross sections along the dashed lines marked in the bimodal AFM images. At t = 0 s, there is no evidence of collagen precursors on the mica; at t = 150 s, the data showed the changes associated with the self-assembly of an emerging collagen structure (procollagen molecule); at t = 225 s, the collagen structure reached the diameter of a tropocollagen molecule, and the growth kinetics entered a stand still phase. The maps were obtained in buffer by applying a peak force of 1.2 nN on the collagen (18 nN on mica). Imaging rate, 1.12 fps (256 × 256 pixels, scanline of 300 Hz, see also Movie S2). Additional bimodal AFM data: f₁ = 158 kHz, k₁ = 0.35 nN/nm, Q₁ = 1.5; f₂ = 1159 kHz, k₂ = 21 nN/nm, Q₂ = 5.1; A₀₁ = 7.1 nm, A₀₂ = 0.2 nm, and A₁ = 6.3 nm.

Figure 3

Physical properties in the first stages of the self-assembly of collagen. First stage: adsorption, nucleation, and growth of collagen precursors. Second stage: formation of tropocollagen. Third stage: assembly of tropocollagen molecules to form microfibrils. Fourth stage: merging and alignment of microfibrils to form microribbons. Those stages are characterized by different values in the physical properties.

Figure 4

Pathways and growth dynamics of collagen self-assembly. Sequence of HS bimodal AFM images showing different collagen growth pathways marked as 1, 2, and 3 at t = 0 s. The tip (1) evolves to merge with a large microribbon at t = 97 s; (2) after a fast growth in length (t = 31 s), a disintegration process started that led to its disappearance (t = 51 s). The trajectory of (3) underlined the role of thermal fluctuations in the growth dynamics. Images were obtained in buffer by applying a peak force of 1 nN to the collagen microribbons (18 nN on mica). The images were extracted from images of larger area (1 μm × 1 μm). Imaging rate, 0.2 fps (512 × 512 pixels, scanline of 100 Hz). See Movie S3 in SI. Bimodal AFM data as in Figure 1.

Figure 5

High-speed bimodal iso-time maps of collagen growth on mica. (a) Iso-time map shows the contour of a microribbon as a function of time. The trajectory of the growing collagen tip is marked by a dashed line. (b) Longitudinal and (c) lateral profiles as a function of time. The images were obtained in buffer by applying a peak force of 1.2 nN on the collagen (18 nN on mica). Imaging rate, 0.2 fps (512 × 512 pixels, scanline of 100 Hz, Figure S1 in SI). Bimodal AFM data as in Figure 1.