Mitochondria transplantation between living cells.

Mitochondria and the complex endomembrane system are hallmarks of eukaryotic cells. To date, it has been difficult to manipulate organelle structures within single live cells. We developed a FluidFM-based approach to extract, inject, and transplant organelles from and into living cells with subcellular spatial resolution. The technology combines atomic force microscopy, optical microscopy, and nanofluidics to achieve force and volume control with real-time inspection. We developed dedicated probes that allow minimally invasive entry into cells and optimized fluid flow to extract specific organelles. When extracting single or a defined number of mitochondria, their morphology transforms into a pearls-on-a-string phenotype due to locally applied fluidic forces. We show that the induced transition is calcium independent and results in isolated, intact mitochondria. Upon cell-to-cell transplantation, the transferred mitochondria fuse to the host cells mitochondrial network. Transplantation of healthy and drug-impaired mitochondria into primary keratinocytes allowed monitoring of mitochondrial subpopulation rescue. Fusion with the mitochondrial network of recipient cells occurred 20 minutes after transplantation and continued for over 16 hours. After transfer of mitochondria and cell propagation over generations, donor mitochondrial DNA (mtDNA) was replicated in recipient cells without the need for selection pressure. The approach opens new prospects for the study of organelle physiology and homeostasis, but also for therapy, mechanobiology, and synthetic biology.

AFM cantilever with in situ renewable mercury microelectrode.

We report here first results obtained on a novel, in situ renewable mercury microelectrode integrated into an atomic force microscopy (AFM) cantilever. Our approach is based on a fountain pen probe with appropriate dimensions enabling reversible filling with (nonwetting) mercury under changing the applied pressure at a connected mercury supply in a dedicated experimental setup. The fountain pen probe utilizes a special design with vertical pillars inside the channel to minimize mechanical perturbation. In proof of principle experiments, dropping and hanging mercury drop were observed as a function of the applied pressure at the external mercury supply. Electrical conductivity occurred only through the mercury after filling, and the empty fountain pen probe showed excellent electrical insulation. This was demonstrated by chronoamperometric measurements in the electrolyte and by mechanical and electrical contacting of an ITO substrate with a mercury-filled and empty probe in air. Finally, cyclic voltammetry and square wave voltammetry were done in a static mercury electrode fountain pen configuration, demonstrating the principle usability of the mercury probe for electrochemical studies. Our findings are of fundamental importance as they enable further integration of a renewable mercury electrode probe into an AFM setup, which is the subject of ongoing work.

Wafer-scale fabrication of nanoapertures using corner lithography.

Several submicron probe technologies require the use of apertures to serve as electrical, optical or fluidic probes; for example, writing precisely using an atomic force microscope or near-field sensing of light reflecting from a biological surface. Controlling the size of such apertures below 100 nm is a challenge in fabrication. One way to accomplish this scale is to use high resolution tools such as deep UV or e-beam. However, these tools are wafer-scale and expensive, or only provide series fabrication. For this reason, in this study a versatile method adapted from conventional micromachining is investigated to fabricate protruding apertures on wafer-scale. This approach is called corner lithography and offers control of the size of the aperture with diameter less than 50 nm using a low-budget lithography tool. For example, by tuning the process parameters, an estimated mean size of 44.5 nm and an estimated standard deviation of 2.3 nm are found. The technique is demonstrated--based on a theoretical foundation including a statistical analysis--with the nanofabrication of apertures at the apexes of micromachined pyramids. Besides apertures, the technique enables the construction of wires, slits and dots into versatile three-dimensional structures.

Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes.

Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.