Surpassing the physical barriers of the cytoplasmic and nuclear membranes to deliver biomolecules directly into cell nuclei offers opportunities to investigate dynamic processes in living cells. The potential of atomic force microscopy coupled to microfluidics (FluidFM) for volume‐controlled intranuclear delivery is demonstrated, whereby minimally invasive microchanneled probes are remotely driven with high spatiotemporal resolution.
The FluidFM technology uses microchanneled atomic force microscope cantilevers that are fixed to a drilled atomic force microscope cantilevers probeholder. A continuous fluidic circuit is thereby achieved extending from an external liquid reservoir, through the probeholder and the hollow cantilever to the tip aperture. In this way, both overpressure and an underpressure can be applied to the liquid reservoir and hence to the built-in fluidic circuit. We describe in this letter how standard atomic force microscopy in combination with regulated pressure differences inside the microchanneled cantilevers can be used to displace living organisms with micrometric precision in a nondestructive way. The protocol is applicable to both eukaryotic and prokaryotic cells (e.g., mammalian cells, yeasts, and bacteria) in physiological buffer. By means of this procedure, cells can also be transferred from one glass slide to another one or onto an agar medium.
An original method is presented to study single-colloid interaction with a substrate in liquid environment. Colloids, either in solution or adsorbed on a surface, are fixed by suction against the aperture of a microchanneled atomic force microscopy cantilever. Their adhesion to the substrate is measured, followed by their release via a short overpressure surge. Such colloid exchange procedure allows for 1), the quick variation of differently functionalized colloids within the same experiment; 2), the investigation of long-term interactions by leaving the colloids on a surface for a defined time before detaching them; and 3), the inspection of irreversible interactions. After validation of the method by reproducing literature results obtained with traditional colloidal atomic force microscopy, the serial use of colloids with different surface functionalization was shown on a micropatterned surface. Finally, concanavalin A-coated colloids were allowed to adsorb on human embryonic kidney cells and then detached one by one. The adhesion between cells and colloids was up to 60 nN, whereas individual cells adhered with 20 nN to the glass substrate. A cellular elastic modulus of 0.8 kPa was determined using the attached colloid as indenter.
The mechanisms used by viruses to enter and replicate within host cells are subjects of intense investigation. These studies are ultimately aimed at development of new drugs that interfere with these processes. Virus entry and infection are generally monitored by dispensing bulk virus suspensions on layers of cells without accounting for the fate of each virion. Here, we take advantage of the recently developed FluidFM to deposit single vaccinia virions onto individual cells in a controlled manner. While the majority of virions were blocked prior to early gene expression, infection of individual cells increased in a nondeterministic fashion with respect to the number of viruses placed. Microscopic analyses of several stages of the virus lifecycle indicated that this was the result of cooperativity between virions during early stages of infection. These findings highlight the importance of performing controlled virus infection experiments at the single cell level.
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