A regular pattern with quantum dots (red) printed on an elastic silicon substrate (grey). Cells (green) can move the substrate. Microscopy observation of the deformation takes place from below. (Visualisations: Bergert et al. Nature Communications 2016, edited)

Until now, the fluorescent reference points could be only randomly embedded in the substrate material in TFM applications. The researchers under Poulikakos have now succeeded to orderly arrange these points in a regular grid pattern on a silicon substrate for the first time. To accomplish this, they used Nanodrip, a 3D nano-printing technology developed a few years ago in the laboratory of ETH professor Poulikakos.

The regular and clearly defined arrangement of the orientation points offers advantages. “We no longer have to remove cells and compare before and after images. Instead, we can determine the forces using a single microscopy image,” says Aldo Ferrari, a senior assistant in Poulikakos’ group. This allows scientists to observe cells over a longer period of time and, among other applications, to repeatedly measure the cell’s forces at various times.

Cooperation between of several research groups

The technical development was possible thanks to the close cooperation of many ETH researchers: Scientists from the laboratory of ETH professor Edoardo Mazza determined the physical properties of the silicone substrate and developed numerical models which allowed them to calculate the forces that corresponded to each deformation of the surface. ETH professor Olga Sorkine-Hornung and Daniele Panozzo, a professor at the New York University, contributed to the computer-assisted calculations of the effective displacement of the points in the microscopy images.

Moreover, the scientists used blue, green and red luminescent quantum dots as fluorescent dyes obtained from the group under ETH professor and quantum dot expert David Norris. Quantum dots are nanostructures made of semiconductor material with a customised geometry.

More accurate and in 3D

The new technique has further advantages: It is more accurate than previous methods, and it is possible for the first time to combine traction force microscopy (and therefore cellular force measurements) with immunohistochemistry, a common cellular biological technique that uses fluorescent antibodies to make specific cell components visible. “In a single microscopy image, we can simultaneously display both the presence of a particular protein and the forces acting, allowing us to identify interrelationships,” says Ferrrari. “This enables new types of experiments in cell biology.”

Finally, with this development it is also possible to determine cell-generated forces in three rather than two dimensions. “We use confocal microscopy, which allows us to record multiple images of the silicon substrate and the cell, layer by layer, and assemble them into a 3D image with the assistance of a computer,” says Ferrari.

Application in cancer research

“The new system is easy to use and ready for implementation,” says Poulikakos. The software developed for it is open source, and the ETH researchers have made it available to colleagues free of charge. Interested scientists must, however, be able to use nanoprinting technology in the laboratory in order to produce the quantum dot silicon substrate.

The new system can be used in cell biology and biomedical research to study the movements of cells or to measure interactions between cells and implants. Poulikakos’ group, for instance, is collaborating with researchers from Politecnico di Milano to explore how the activities of individual genes, the mobility of cells of a particular type of carcinoma and the thereby acting forces are interrelated.