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Microtechnologies for biomolecular complex analysis and cryo-microscopy

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Micro- and nanofluidic technologies provide exciting opportunities to study the structure and function of biological systems at the level of cells, organelles, and molecular machines.

In the first part of this seminar, I will describe the use of micromechanical resonators with embedded fluidic channels of only 10 pL volume for the label-free, mass-based measurement of protein aggregation kinetics. The sensitivity of the method is greatly enhanced by a fluctuation analysis termed mass correlation spectroscopy (MCS). This technique has been used to monitor the formation of insulin amyloids from monomers to mature fibrils. While, during MCS measurements, molecules are dispersed free in solution, embedded channel resonators also can be used in a surface-based mode analogous to the quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). In this mode, the devices are advantageous due to their low sample consumption, wide dynamic range, and reaction limited kinetic measurements. The benefits and limitations of nanomechanical mass measurements and other nanofluidic techniques for the analysis of large biomolecular complexes will be discussed.

In the second part of the talk, I will focus on work of our group towards imaging dynamic cellular events by correlative microscopy using microfluidic cryofixation. Recent years have seen enormous progress in cellular imaging by fluorescence, electron, and X-ray microscopy, but many of these advanced imaging technologies cannot be readily combined. In particular, the correlation between live-cell imaging and electron microscopy (EM) remains challenging due to a lack of adequate fixation technology. Cryofixation is widely regarded as the gold standard in stabilizing biological samples for ultramicroscopy. Unfortunately, all current methods of cryofixation require samples to be removed from the light-microscope before freezing. This transfer often results in a loss of spatial registration and strongly limits temporal resolution. We recently introduced and validated a new concept for the cryofixation of cells directly in the light microscope and with millisecond time resolution. Formation of crystalline ice is suppressed by the high cooling rate (~10^4 K/s), which is enabled by placing the sample in a microfluidic channel embedded inside a thin polymer foil of low thermal mass. We expect that, in the future, this new concept can help to bridge the gap between live-cell imaging and cryofixation for a wide class of applications in cell biology and other fields. In particular, the method should enable precise temporal correlation of live-cell imaging and cell stimulation with post-fixation ultrastructural studies by means of optical nanoscopy, electron microscopy, or X-ray tomography.

This talk is part of the Biophysical Seminars series.

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