Improved-Throughput Traction Microscopy Based on Fluorescence Micropattern for Manual Microscopy

<div><p>Traction force microscopy (TFM) is a quantitative technique for measuring cellular traction force, which is important in understanding cellular mechanotransduction processes. Traditional TFM has a significant limitation in that it has a low measurement throughput, commonly one per TFM dish, due to a lack of cell position information. To obtain enough cellular traction force data, an onerous workload is required including numerous TFM dish preparations and heavy cell-seeding activities, creating further difficulty in achieving identical experimental conditions among batches. In this paper, we present an improved-throughput TFM method using the well-developed microcontact printing technique and chemical modifications of linking microbeads to the gel surface to address these limitations. Chemically linking the microbeads to the gel surface has no significant influence on cell proliferation, morphology, cytoskeleton, and adhesion. Multiple pairs of force loaded and null force fluorescence images can be easily acquired by means of manual microscope with the aid of a fluorescence micropattern made by microcontact printing. Furthermore, keeping the micropattern separate from cells by using gels effectively eliminates the potential negative effect of the micropattern on the cells. This novel design greatly improves the analysis throughput of traditional TFM from one to at least twenty cells per petri dish without losing unique advantages, including a high spatial resolution of traction measurements. This newly developed method will boost the investigation of cell-matrix mechanical interactions.</p></div

Schematic diagram showing the fabrication of the improved-throughput TFM device.

<p><b>A)</b> The procedure of microcontact printing on the coverslip and the structure of the new designed device. <b>B)</b> Fluorescence image of the micropattern on the cover slip before seeding cells. <b>C)</b> The micropattern observed through the culture medium after seeding cells. <b>D)</b> The microbeads on the surface of the PAA gels observed by the fluorescence microscope. <b>E)</b> The image of microbeads inside gels.</p

Experimental validation of beads on surface as an indicator of substrate deformation.

<p><b>A)</b> Phase-contrast image of HeLa cell on the PAA gels (beads were both mixed inside and linked on surface of the gels, as illustrated in the inset). <b>B)</b> Scatter plot of RMSD computed for 22 cells utilizing fluorescence images of the beads on surface (vertical axis) and beads inside (horizontal axis). <b>C)</b> Fluorescence image of beads on surface. <b>D)</b> Fluorescence image of beads inside gels. <b>E)</b> Displacement field was calculated using the fluorescence image of beads on surface before and after cell removal by NaOH solution. <b>F)</b> Displacement field was calculated using the fluorescence image of beads inside before and after cell removal by NaOH solution. The solid white line stood for the cell outline in the both <b>E)</b> and <b>F)</b>.</p

Immunostaining of cells on substrate of different topography.

<p><b>A)</b> Representative immunofluorescence confocal microscopic images of the F-actin (red) of cells on substrate with beads inside. <b>B)</b> Representative immunofluorescence confocal microscopic images of the F-actin (red) of cells on substrate with beads on surface. <b>C)</b> Statistical quantification of the mean fluorescence intensity of actin within the HeLa cells on substrate with different positioned beads (n = 28 for beads inside, n = 34 for beads on surface). <b>D)</b> Representative immunofluorescence confocal microscopic images of the vinculin (green) with beads inside. <b>E)</b> Representative immunofluorescence confocal microscopic images of the vinculin (green) with beads on surface. <b>F)</b> Comparison of total vinculin area on PAA gels with beads inside and beads on the gel surface (n = 22 for the former, n = 20 for the latter). Bars represent mean ± standard deviation. Two-tailed t-test was performed for statistical analysis in both <b>C)</b> and <b>F)</b>.</p

Eliminating the stage shift in the improved-throughput TFM.

<p><b>A)</b> Apparent stage shift in the original displacement field. <b>B)</b> The actual displacements caused by HeLa cells after correction by the image processing algorithm.</p

Multiple pairs of NF and FL fluorescence images.

<p><b>A)</b> Many FL images had been captured before cell detachment while the NF image of only the last cell was captured after detaching all cells, as other cells could not be found in the traditional TFM. <b>B)</b> Utilizing coordinate system, multiple pairs of NF and FL images were captured in sequence by going back to the original position in the improved-throughput TFM. The circle stands for the PAA substrate. The small rectangle in the circle represents the view field using the 40× objective.</p

The results of improved-throughput measurements.

<p>Each panel was composed of a colorimetric bar, traction force field and phase-contrast image. The recovered traction fields of <b>A</b>, <b>B</b>, <b>C</b>, <b>D</b>, <b>E</b>, <b>F</b>, <b>G</b> and <b>H</b> were only a part of the total traction force fields in one petri dish.</p

Results of MTT assay on substrate with beads inside and on surface.

<p>The optical density at wavelength of 570 nm of each sample at 24 h, 48 h, and 72 h was detected. Bars represent mean ± standard deviation. Two-tailed t-test was performed for statistical comparisons (n = 3, *represents <i>p</i>>>0.05).</p