Time-resolved local strain tracking microscopy for cell mechanics

Abstract

A uniaxial cell stretching technique to measure time-resolved local substrate strain while simultaneously imaging adherent cells is presented. The experimental setup comprises a uniaxial stretcher platform compatible with inverted microscopy and transparent elastomer samples with embedded fluorescent beads. This integration enables the acquisition of real-time spatiotemporal data, which is then processed using a single-particle tracking algorithm to track the positions of fluorescent beads for the subsequent computation of local strain. The present local strain tracking method is demonstrated using polydimethylsiloxane (PDMS) samples of rectangular and dogbone geometries. The comparison of experimental results and finite element simulations for the two sample geometries illustrates the capability of the present system to accurately quantify local deformation even when the strain distribution is non-uniform over the sample. For a regular dogbone sample, the experimentally obtained value of local strain at the center of the sample is 77%, while the average strain calculated using the applied cross-head displacement is 48%. This observation indicates that considerable errors may arise when cross-head measurement is utilized to estimate strain in the case of non-uniform sample geometry. Finally, the compatibility of the proposed platform with biological samples is tested using a unibody PDMS sample with a well to contain cells and culture media. HeLa S3 cells are plated on collagen-coated samples and cell adhesion and proliferation are observed. Samples with adherent cells are then stretched to demonstrate simultaneous cell imaging and tracking of embedded fluorescent beads.

Centrosomal and ciliary targeting of CCDC66 requires cooperative action of centriolar satellites, microtubules and molecular motors

“…This trafficking model predicts directional movement to or from centrosomes at a rate similar to that of motor-mediated transport along microtubules. To determine whether satellite dynamics is consistent with this model, we quantitatively assayed their dynamic behavior by combining time-lapse confocal imaging of interphase RPE1::GFP-CCDC66 cells with single particle tracking algorithms that we developed based on previous studies (described in Materials and Methods) 60,61 . We tracked a total of 144 satellites (n = 4) and a representative cell indicating the satellites with their associated trajectories is shown in Fig.…”

“…The trajectories of GFP labelled satellites were determined by using a custom script written in MATLAB (R2017b, Mathworks, Natick, MA). Particle tracking code to determine the time-dependent positions of satellites was partially adapted from previous studies 60,61 . To remove point defects in images, all frames were initially convoluted by using a gaussian filter with a lower bound of 3 pixels and an upper bound of 40 pixels.…”

Identification of SERPINE1 as a Regulator of Glioblastoma Cell Dispersal with Transcriptome Profiling

“…The trajectories of cells on uncoated and vitronectin-coated surfaces were determined by using a custom script written in MATLAB (R2017b, Mathworks, Natick, MA, USA). Single-cell tracking code to determine time-dependent positions of cells was partially adapted from previous studies [58,59]. Briefly, point defects were removed by using a Gaussian filter with a lower bound of 3 pixels.…”

Membrane-Bound Vimentin Filaments Reorganize and Elongate under Strain

“…From the trajectories of the tracked bead positions, a displacement field relative to a reference bead was derived (Figure B), which is a characteristic of a uniaxial stretching device based on PDMS. − Beads located on the crosshairs ( x = 0, y or y = 0, x ) move along the axes, either away from the reference bead along the y -axis (longitudinal) or toward the reference bead along the x -axis (lateral), while beads in between the crosshairs move in both the x - and y -directions. For the characterization of the uniaxial stretcher, the longitudinal strain ( y -component) and the lateral strain ( x -component) were calculated using elastix (Figure C).…”