The power of microfluidics for dynamic time-lapse experiments: On demand webinar

Increasingly, scientists recognize the need to observe cellular changes over extended periods of time to truly understand biological systems. In this on-demand webinar, Dr. Enrique Rojas, Assistant Professor of Biology at New York University, and Sunil Desai, Research Technology Specialist at MilliporeSigma, discuss microfluidics as a tool for dynamic time-lapse experiments and explain how to explore single-cell biophysics with a simple, robust microfluidics system.

In particular, Rojas describes his application subjecting bacteria to rapid oscillatory osmotic shocks for studying the biomechanical basis of cell growth and division. In gram-negative strains, Rojas demonstrates the outer membrane’s significance for sustaining mechanical perturbations and growing in the presence of antibiotics. Additionally, Rojas discusses the ecology of bacteria-bacteriophage interactions and the genetic basis of cell growth.

Watch this webinar on demand and read on for highlights from the Q&A session at the end of the live webinar.

Q: How are the mechanical forces used by the bacteria for their benefit?

ER: In the case Staphylococcus aureus, one way to think about it is that the turgor pressure is a consequence of being small. A lot of material inside a small volume alters the turgor pressure. This is just a by-product of being small, it is therefore likely that being small is selected for by bacteria. This means they have an enormous force that they can exploit to achieve certain physiological processes. In the case of cell division in Staphylococcus aureus, the bacteria have evolved to use turgor pressure to drive daughter cell separation, which could be viewed as a way to save productions of enzymes. In other words, you don't have to generate as many enzymes to degrade the very thick, gram-positive cell wall as you would have to if you didn't exploit turgor pressure to drive cell division.

A publication (Rojas 2017: Cell systems) shown in one of the slides describes how, in Bacillus subtilis, turgor pressure is used to drive mechanical expansion of the cell wall during cell growth. These are two examples of how turgor pressure is exploited for subcellular physiology.

Q: Can the hypoxic condition be induced/changed acutely, i.e., in minutes?

SD: The hypoxic condition can be induced very rapidly. I'm not sure it can happen on a minute timescale. I know it happens within a few hours. The time it takes would depend on how much media you’re using. We do work with microvolumes, so we do have it optimized in that sense. But I am not sure that we can achieve it on a minute timescale.

ER: It is shorter than ours because we have a small volume for the gas we change. The gas volume under the manifold is just 40cc, 40mm, and then counting all the difference from the gas tank to Onyx system and then Onyx system to the plate is minimal. I would say the gas change may not be able to exchange the gas tank within one minute, but definitely under half an hour, then you should be able to see the gas contents differ.

Q: What possibility is there to perform correlative microscopy? Can the microfluidic carry a fixative at the end of an experiment and then distract through reflected light microscopy from above or by SEM?

ER: I am not familiar with a correlative microscope, but all the inverted microscopes are compatible with our system. We have performed immunocytochemistry on it. And then we use paraformaldehyde (PFA) as our fixation regions and then fixture cells and introduce secondary antibody and perform the staining. The fluoric and microscopes and the confocal microscopes have been proven to work on our platform.

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