Relatively new is the use of AFM in cell biology. We wrote about this recently in a Spotlight that described a novel method to probe the mechanical properties of living and dead bacteria via AFM indentation experimentations ("Dead or alive – nanotechnology technique tells the difference ").
Researchers in Switzerland have now demonstrated novel cell biology applications using hollow force-controlled AFM cantilevers – a new device they have called FluidFM.
"The core of the invention is to have fixed already existing microchanneled cantilevers to an opportunely drilled AFM probeholder" Tomaso Zambelli tells Nanowerk. "In this way, the FluidFM is not restricted to air but can work in liquid environments. Since it combines a nanofluidics circuit, every soluble agent can be added to the solution to be dispensed. Moreover, the force feedback allows to approach very soft objects like cells without damaging them."
As cell biology is moving towards single cell technologies and applications, single cell injection or extraction techniques are in high demand. Apart from this, however, the FluidFM could also be used for nanofabrication applications such as depositing a conductive polymer wire between to microelectrodes, or to etch ultrafine structures out of solid materials using acids as the spray agent. The team has reported their findings in a recent paper in Nano Letters ("FluidFM: Combining Atomic Force Microscopy and Nanofluidics in a Universal Liquid Delivery System for Single Cell Applications and Beyond").
Staining living neuroblastoma cells by gentle contact on the cell membrane. (a) Diagram showing the staining procedure by gentle contact (not to scale). The hollow tip is maintained in contact with the cell membrane thanks to the force feedback (set point of less than 1 nN). The active agents dissolved in the solution of the microchannel spontaneously diffuse across the membrane into the cytoplasm. (b) Superposition of a differential interference contrast image and of the corresponding fluorescent one of a cell after staining with CellTracker green. The microchannelled cantilever filled with CellTracker green is positioned over the cell in the red circle using the optical microscope. The tip is then brought into gentle contact with the cell membrane by the AFM force feedback system and left there for 15 min, before taking the fluorescent and phase contrast image
Zambelli originally realized that the technology of the atomic force microscope that is normally used only to image cells could be transformed into a microinjection system. The result of the development by Zambelli and his colleagues in the Laboratory of Biosensors and Bioelectronics at the Institute of Biomedical Technology at ETH Zurich and in the Swiss Center for Electronics and Microtechnology (CSEM) in Neuchâtel was the "fluid force microscope", currently the smallest automated nanosyringe currently in existence.
"Our FluidFM even operates under water or in other liquids – a precondition for being able to use the instrument to study cells" says Zambelli.
The force detection system of the FluidFM is so sensitive that the interactions between tip and sample can be reduced to the piconewton range, thereby allowing to bring the hollow cantilever into gentle but close contact with cells without puncturing or damaging the cell membrane.
On the other hand, if membrane perforation for intracellular injection is desired, this is simply achieved by selecting a higher force set point taking advantage of the extremely sharp tip (radius of curvature on the order of tens of nanometers).
To enable solutions to be injected into the cell through the needle, scientists at CSEM installed a microchannel in the cantilever. Substances such as medicinal active ingredients, DNA, and RNA can be injected into a cell through the tip. At the same time, samples can also be taken from a cell through the needle for subsequent analysis.
According to Zambelli, while this approach is similar to microinjection using glass pipettes, there are a number of essential differences.
"Microinjection uses optical microscopy to control the position of the glass pipette tip both in the xy plane and in the z direction (via image focusing)" he explains. "As consequence of the limited resolution of optical microscopy, subcellular domains cannot be addressed and tip contact with the cell membrane cannot be discriminated from tip penetration of the membrane. Cells are often lethally damaged and skilled personnel are required for microinjection."
"The limited resolution of this method and the absence of mechanical information contrast strongly with the high resolution imaging and the direct control of applied forces that are possible with AFM. Precise force feedback reduces potential damage to the cell; the cantilever geometry minimizes both the normal contact forces on the cell and the lateral vibrations of the tip that can tear the cell membrane during microinjection; the spatial resolution is determined by the submicrometer aperture so that injection into subcellular domains becomes easily achievable."
Experiments conducted by the Swiss team demonstrate the potential of the FluidFM in the field of single-cell biology through precise stimulation of selected cell domains with whatever soluble agents at a well-defined time.
"We confidently expect that the inclusion of an electrode in the microfluidics circuit will allow a similar approach toward patch-clamping with force controlled gigaseal formation," says Zambelli. "We will also explore other strategies at the single-cell level, such as the controlled perforation of the cell membrane for local extraction of cytoplasm
"Zambelli and his colleagues are convinced that their technology has great commercial potential. Rejecting offers from well-known manufacturers of atomic force microscopes for the sale of the patent for the FluidFM, they have founded Cytosurge LLC, a company dedicated to commercially develop the instrument.
Today, Zambelli's laboratory contains two prototypes of the instrument, which are being tested in collaboration with biologists.
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