Integrated Opto–Biomechatronics For Single Muscle Fibre Structure‐Function Assessment: The MyoRobot 3.0

Figures

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  FIGURE 1. Drawbacks of a limited and static FOV in current biomechatronics systems. (A) a typical FOV with sufficient magnification to detect sarcomeres allows to image a ∼200 μm segment of a single muscle fibre (often less). This area is coarsely highlighted in an image of an entire dystrophic mdx single fibre taken at lower magnification. The loss of structural information from a limited FOV is imminent. (B) particularly in stretch protocols, such as stress–strain recordings sketched here, the muscle fibre shifts out of the FOV. This is highlighted by the red arrows indicating a shift of some marked structures on the fibre over 10% strain.

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  FIGURE 2. Overview of the Integrated Optics System. (A1) Technical drawing showing a cross‐section of the entire beam path. A light source (10) provides Köhler illumination to the sample plane which is picked up by the objective (5) below, deflected by a mirror (6) and focused onto the camera (8) by a tube lens (7). Measures given in mm. (A2) Displays the imaging system below the rack (3) in which the muscle fibre will be immersed. (A3) Shows the entire CAD drawing of the MyoRobot 3.0, including the VC (2) and FT (1) between which the sample is mounted. The entire imaging unit is attached to two cross‐stages for motion control. (B) Photograph of the MyoRobot 3.0 including all key‐components.

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  FIGURE 3. Quantitative comparison of average SL values obtained from stitched brightfield images. Top: Brightfield image stitching of a single EDL muscle fibre at 0% (L0) and 40% strain. Insets highlight preserved sarcomere detail under stretch. Bottom right: Comparison of SL values obtained using three analytical pipelines: SHG‐based FFT algorithm (SHG‐FFT), Myo‐FFT, and Myo‐SIGN (algorithms described in Ref. [31]). Data from n = 20 fibres show large agreement across methods, confirming the accuracy of the MyoRobot 3.0 imaging and analysis pipeline.

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  FIGURE 4. Fibre thinning during stretch causes stress underestimation unless dynamically accounted for. (A) Representative stress–strain recording comparing restoration stress based on either a static (gray) or dynamic (black) fibre diameter. At 40% strain, ignoring diameter thinning leads to ∼27% underestimation (Δ S∼8 kPa). (B) Boxplots showing the average fibre diameter of seven fibres at 0% and 40% strain, highlighting a significant reduction of ∼13%. (C1,C2) Corresponding diameter histograms across the full fibre length confirm diameter thinning under stretch. (D) Approximation using a cylinder model predicts a 15% diameter reduction at 40% strain, leading to a 15% diameter and thus, 27.75% area loss–mirroring the empirically observed stress deviation in (A).

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  FIGURE 5. Comparison of stress–strain vs. stepwise‐stretch recordings for assessing axial elasticity in single muscle fibres. (A) Schematic of the stretch‐jump protocol. The fibre was stretched in four discrete steps of 10% L0, each followed by a ∼1‐min relaxation phase to allow for visco–elastic stress decay. (B) Representative comparison between a continuous stress–strain recording (black) and a stepwise stretch‐jump protocol (teal). The stress–strain curve is plotted as a function of continuously increasing strain. In contrast, the original stretch‐jump recording is time‐based and consists of discrete stretch events followed by relaxation phases. For visual comparison, the stepwise recording was segmented and aligned to corresponding strain levels (10%–40%), such that the steady‐state plateau stress after each relaxation phase is mapped to the respective strain (highlighted by teal labels). The apparent discontinuities and non‐continuous progression of the teal trace arise from this transformation of time‐based data into strain space and do not reflect interruptions in the original recording. Red dashed lines indicate the strain levels at which plateau stresses from the stretch‐jump…

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  FIGURE 6. Calcium‐dependent modulation of passive restoration stress and Young's modulus in chemically skinned fibres. (A) Representative stress–strain trace used to derive restoration stress and Young's modulus. (B) Maximum restoration stress at 40% strain across the four conditions. (C) Young's modulus comparison across the same four conditions. Data were obtained from the same fibres across all conditions. Asterisks indicate statistical significance (p <0.05). Note that the highest stiffness was observed in control fibres (no BTS), and significant calcium‐dependent stiffening was seen under cross‐bridge block at pCa 4.92.

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  FIGURE 7. Calcium‐dependent changes in passive stress characteristics differ depending on stretch protocol. (A,) Representative trace of a single fibre subjected to stepwise “stretch‐jumps” under calcium‐free conditions (pCa 9.0, ‐BTS). Vertical lines mark the visco–elastic peak stress (green) and the elastic plateau stress (blue), the latter measured after stress relaxation. (B) Exemplary calculation of Young's modulus from a linear fit to elastic stress values plotted against strain. (C) Grouped data of visco–elastic peak stress; all p >0.05 within each strain level. (D) Elastic stress values after stress relaxation (1 min); all p >0.05 within each strain level. (E) Comparison of Young's moduli obtained from stress–strain curves (grey) and stepwise “stretch‐jumps” (blue). No group‐level differences were significant across conditions. A significantly higher Young's modulus was detected in the control group (pCa 9.0 ‐BTS) when derived from stress–strain recordings.

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  FIGURE A1. Handling artifacts in real‐time diameter tracking using a dedicated smoothing algorithm. (A–C) Simulated fibre diameter traces (grey) were overlaid with artificial artifacts of different profiles: (A) rectangular; (B) triangular; (C) spike‐shaped. In all cases, the processed traces (black) show accurate reconstruction of the underlying trend. (D) Application of the same algorithm to an actual muscle fibre recording. The raw data (grey) exhibit multiple high‐amplitude artifacts, likely due to debris or sedimentation in the open MyoRobot 3.0 well. After filtering (black), the trace reflects the physiologically expected gradual decrease in diameter during stretch. Insets 1‐3 exemplarily display the occurrence of 'spikes' in the diameter trace. At some stage (1), a particle may sediment into the detection area (green rectangle). For the short time in focus (2), this particle is interpreted as obstacle, instantaneously but transiently increasing the computed diameter. Once past the focal plane (3) the actual fibre diameter continues to be recorded.