Cooperative Vaccinia Infection Demonstrated at the Single-Cell Level Using FluidFM

Abstract

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.

Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

“…To explore and establish FluidFM for single-bacterium isolation, different parameters were tested and optimized to ensure efficient isolation of a wide range of randomly chosen bacteria from environmental samples. Cantilevers with an aperture diameter of 8 m were used; however, instead of cantilevers with a default channel height of 1 m, as used in previous studies (36,37,51), cantilevers with a channel height of only 0.5 m were used, because they ensured that the targeted bacteria remained confined at the aperture, where they could be easily spotted, and were not sucked into the channel of the probe. Plasma cleaning and coating the cantilever with PLL-g-PEG reduced the chance that a bacterium would irreversibly bind to the cantilever (38).…”

G gene-deficient single-round rabies viruses for neuronal circuit analysis

“…These techniques include using atomic force microscopy in combination with nanofluidics (Stiefel et al, 2012) and using push-pull microfluidic virus delivery (Nguyen et al, 2012). For in vivo applications, single cell two-photon guided electroporation or patch clamp mediated transfection (Kitamura et al, 2008) provide methods to unequivocally map connections of a single neuron (Marshel et al, 2010).…”

Methods for protein delivery into cells: from current approaches to future perspectives

“…Using a pressure controller to regulate delivery, quantum dots and fluorescent dyes can be delivered into cells with minimum disruption to the cytoskeleton and cell morphology [24,25]. An alternative solution is based on the FluidFM platform, which is an adaptation of atomic force microscopy (AFM) in which the injection probe is a hollow AFM cantilever that can be manufactured with a pore diameter ranging from 10 nm to 10 mm [26,38,39]. Upon penetration of the plasma membrane, injection is pressure controlled and has been used to deliver DNA plasmids, fluorescent dyes and vaccinia virus [26,38,39].…”