Stiffness analysis of 3D spheroids using microtweezers - Fig 3

<p>(A) The experimental set up with a well containing a spheroid. B. The cantilever tips were immersed into the media to access the spheroid (arrow) to apply step compression.</p

Stiffness analysis of 3D spheroids using microtweezers - Fig 1

<p>A. Microtweezer system showing the three main components: tweezer arms with a flexible plate spring, a bimorph piezo actuator and two cantilevers as the force sensing tips. The moving arm was actuated (direction indicated by the arrow) to move the cantilever closer to the fixed arm. B. The cantilever holder along with SU8 micro-cantilevers. C. Micro-cantilevers were made from SU8 and brass to obtain a wide measureable force range of sub hundred nN to more than 1 mN.</p

Two materials were used to make replaceable cantilever tips.

<p>The stiffness and force range for each cantilever is indicated.</p

Young’s modulus of agarose measured using two techniques: Microindentation and microtweezer.

<p>Young’s modulus of agarose measured using two techniques: Microindentation and microtweezer.</p

Microindentation set up for mechanical characterization of agarose using stainless steel sphere and 100g load cell.

<p>Microindentation set up for mechanical characterization of agarose using stainless steel sphere and 100g load cell.</p

Spheroid stiffness analysis was done using SU8 cantilevers.

<p>Three cell lines BT474, T47D and MCF 10A spheroids were tested. The protocol similar to agarose pillar was applied for each cell type. Red square indicates reference and green square indicates the position of the cantilever tip with sample. The deviation in the green and red square in A4, B4, and C4 shows cantilever bending due to the spheroid. A1, B1 and C1 being the initial position for each cell line. Panel 1–4 represents step 0, 6, 12 and 18, respectively, for cantilevers. Supplementary movie clips of BT474 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188346#pone.0188346.s002" target="_blank">S2 Movie</a>), T47D (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188346#pone.0188346.s003" target="_blank">S3 Movie</a>) and MCF10A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188346#pone.0188346.s004" target="_blank">S4 Movie</a>) compression are available.</p

Spheroid viability test (A) before, (B) during and (C) 10 mins after microtweezer compression was conducted to show the effect of microtweezers on cell.

<p>Green shows live cells in a spheroid and red shows dead cells. There were no dead cells produced during compression (C) and after release at the point of contact with the microcantilevers (B, shown in dashed white lines) when compared to spheroid before compression (A). Thereby showing no physical damage to the sample during compression.</p

Schematic showing the principle of force and stiffness measurement.

<p>The sample was placed between the cantilevers (A) and stepwise motion of the moving arm produced bending of the flexible cantilevers (<i>d</i>_c) and sample indentation (<i>d</i>_s). The reference cantilever deflection (<i>d</i>_ref) is found in the reference measurement where there was no sample between the cantilevers.</p

The microtweezer system was verified through measurement with agarose pillars (250 μm diameter) with different concentrations.

<p>Panels A1-A4 shows the reference measurement where there is no sample between the cantilevers. Panels B1-B4 are the corresponding images with a sample. Panel 1–4 represents step 0, 6, 12 and 18, respectively. The red image tiles (red square) were selected and tracked from the reference images while the green image tiles were tracked from sample images. The shift in the position of the red and green tile with step compression of the agarose pillar shows the bending of cantilevers and sample indentation. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188346#pone.0188346.s001" target="_blank">S1 Movie</a> is available.</p

The pattern matching algorithm written in MATLAB marked a target area (black square, 50×50 pixels) in image 1 and searched a larger scan area (dashed black square, 110×110 pixels) for a match of the target area in image 2.

<p>The best matched area (2, white square) will be used as the new target in the next step.</p