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PeakForce QNM Investigation of Cancer Cells
It’s currently accepted that cancer cells are usually softer and more deformable than their normal counterparts.20–22 Glioblastoma is the most aggressive and common form of malignant brain cancer, and is one of the most difficult to treat forms of cancer because the tumor cells are resistant to available therapies and because few drugs can cross the blood-brain barrier to act on the tumor. U-251 is an isogenic cell line of glioblastoma that is extremely invasive.
This tendency to invade can be suppressed by overexpressing some tumor suppressive factors (TSFs). This modification is also expected to induce changes in mechanical properties. Those changes have been investigated in PeakForce QNM mode and the results are reported in Figure 7.
PeakForce QNM investigation of live U-251 Glioblastoma cells
Figure 7. PeakForce QNM investigation of live U-251 Glioblastoma cells. (A and B) 20x20µm 512x512 3D-rendered height and peak force error images respectively of control cells. The loading force has been increased on purpose to reveal high-resolution details of the cell cytoskeleton (typical capture time = 35 minutes). (C, D and E) Peak force error, Young’s modulus, and deformation maps (80x80µm 128x128 pixels) of cells transfected to overexpress TSF (typical capture time = 5-6 minutes). The right graphics summarize the measurements performed on control and transfected cells using traditional force volume and PeakForce QNM, showing good agreement between the techniques, but highlighting the significantly better statistics obtained with PeakForce QNM. (click image to enlarge)
PeakForce QNM can either be used to image challenging cells at a high resolution (Figure 7A and 7B: 20x20µm images showing sharp details of the cell cytoskeleton) or to sense the mechanical properties (Figure 7C, 7D and 7E show peak force error, Young’s modulus and deformation in 80x80µm images of U-251 live cells over-expressing a TSF). Those images have a 128x128 resolution. Using Adaptive Scanning (a feed-forward control allowing dramatic improvement in XY positioning for large scan sizes), allows a capture time of 5 to 6 minutes. Force volume and PeakForce QNM have been tested on a high number of wildtype and TSF-transfected cells. The results can be summarized as follows:
  • 1. Both force volume and PeakForce QNM clearly demonstrate that the transfection causes the cells to become much stiffer (and less deformable), as expected.
  • 2. The standard deviations on the Young’s modulus for these measurements are significantly higher in force volume than in PeakForce QNM measurements. Additionally, there are significantly more data points for analysis with PeakForce QNM than with force volume. Together this allows for a much lower error standard error of the mean (assuming the variations in the measurement are statistically independent, SEM=σ/√n) and more accurate results. For an average image capture time of 5 minutes PeakForce QNM can capture nine 256x256-pixel images per sample type, providing 589,824 force curves, each analyzed to obtain a modulus data point. In contrast, force volume can only obtain 32x32-pixel images in the same amount of time, resulting in 9,216 data points.23 Even if the standard deviations of the two samples were the same, the PeakForce QNM case would have a standard error of the mean of about eight times lower than that for force volume.
  • For this type of measurement, PeakForce QNM is much more relevant than force volume in terms of resolution, quality, and amount of delivered information, and thus offers great perspectives in cell mechanics investigation.
    The mechanical properties of biological samples often affect their structure and functional activity and are, hence, very important to biologists. Force volume has been accepted since the mid-1990s as a powerful tool for measuring and mapping the mechanical properties of biological samples. Force volume is optimized for mapping with low ramp rates (~0.5–10Hz) and relatively low resolution. PeakForce QNM improves upon force volume in terms of resolution and speed (with ramp rates ~250Hz–2KHz), making it more practical to collect and analyze much more data for better detail and statistics. Together, force volume and PeakForce QNM provide new opportunities for comparisons of material response at over about four orders of magnitude of ramp rate in air or liquid environments. In addition, the new features of NanoScope and NanoScope Analysis offer the user a maximum of ease-of-use and flexibility to collect, process, and analyze the thousands of force curves in a typical force volume or PeakForce QNM map.
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    By Bede Pittenger, Andrea Slade, Alexandre Berquand (Bruker Nano Surfaces); Pascale Milani, Arezki Boudaoud, and Olivier Hamant (Ecole Normale Superieure de Lyon, France); Special thanks to Manfred Radmacher (University of Bremen, Germany)

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