Computational protocol: Microsurgery-aided in-situ force probing reveals extensibility and viscoelastic properties of individual stress fibers

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Protocol publication

[…] Experiments were performed on an inverted ZEISS Axiovert 200M system with a 40× Plan-Neofluar, air objective (N.A. 0.75). Two three-axis micromanipulators (NMN-25 mounted on top of MX-1, Narishige Scientific Instruments Lab.) were used to position the cantilever. The cantilever holder was kept above the sample and the beam hanged vertically in the field of view of the microscope, free to move. A motorized 3-axis sample stage (MS-2000, Applied Scientific Instrumentation, with NanoDrive controller, Mad City Labs Inc.) was used to move the cells towards the cantilever. Stage motion was controlled by a custom script written in MATLAB® (The MathWorks Inc., MA, USA). Images were acquired using a CoolSnap HQ2 camera (Photometrics, AZ, USA) through the ultra-fast acquisition streaming mode in Metamorph (Molecular Devices, CA, USA).Cantilever was placed into the hole immediately after surgery, as soon as the hole was sufficiently large to accommodate it (~few minutes, see ). That was done to block the beginning of the process of resealing, which eventually occurred in some hours. Stage displacement was triggered as soon as the cantilever position was adjusted to the fiber midpoint and the setup stabilized. We controlled that the cantilever motion was perpendicular to the axis of the fiber; if we observed a significant lateral motion during fiber stretching, indicating a major imbalance between both sides of the fiber, the results were discarded. After each successful trial, we removed the cantilever and waited for the fiber to return to its straight position (~a few seconds) before the fiber was stretched again. Total time of force application for one fiber typically did not exceed ~1 min.Stage displacement (δs) and cantilever displacement (δclv) were separately determined using a slice alignment plugin in ImageJ (NIH, Bethesda, Maryland, USA,, as previously described. Acquisition frequencies needed to be of at least 10 Hz to have enough sampling frequency for the deformation velocity of 5 μm/s. Isolated stress fiber half-length d was manually measured on image after isolation. The average length of the fiber probed was 28.4 μm +/−3.6 μm (s.d.) with minimal length 22 μm and maximum length 35 μm.For non-isolated stress fibers, the experimental and analysis procedures follow. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Diseases Tension-Type Headache