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Microfluidic cryofixation for correlative microscopy

Significance Statement

Recent years have seen enormous advances in cellular imaging by fluorescence, electron, and X-ray microscopy. Correlative microscopy aims to combine two or more of these techniques to better understand the complex links between structure and function within cells. While imaging of the same specimen by complementary microscopy techniques has already provided many new insights into cellular processes, not all imaging technologies can 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. Fixation is a prerequisite for EM, as samples must be cut into thin sections in order to be penetrated by the electron beam. Traditional chemical fixation is known to introduce artifacts at the nanometer scale and provides low temporal resolution. In contrast, cryo-fixation is widely regarded as the gold standard in stabilizing biological samples for ultramicroscopy. However, the two standard cryofixation methods, high-pressure freezing and plunge freezing, cannot take place during live imaging, involve critical transfer steps, and often result in a loss of spatial registration and temporal synchrony. Vital to cryo-preservation of cell structure with molecular detail is vitrification, i.e. the freezing of water in a glass-like, non-crystalline state. At atmospheric pressure and low cryoprotectant concentration, the high cooling rates required for vitrification can only be attained with picoliter-sized samples. In this work we prove the viability of a microfluidic concept that can bridge the gap between live cell imaging and cryofixation for a wide class of applications in cell biology and other fields. The new technique relies on microfluidics for the cryofixation of cells directly in the light microscope (no transfer step required) and with millisecond time resolution. Ice crystallization is suppressed by ultra-rapid cooling of a suspended, electrically heated microchannel that equilibrates quickly with a liquid nitrogen chilled support when heating is interrupted. Additionally, our microfluidic system allows the circulation of media and rapid exchange of samples, making it ideal for a wide range of cell culture systems and some small model organisms. Of particular interest would be the study of cellular processes with millisecond dynamics, and the capture of transient cellular events immediately after stimulation by light, electricity, or drugs. Microfluidic cryo-fixation will enable precise temporal correlation of live imaging and cell stimulation with post-fixation ultrastructural studies including optical nanoscopy, electron microscopy, or X-ray tomography.

Microfluidic cryofixation for correlative microscopy. Global Medical Discovery











Mejia YX1, Feindt H2, Zhang D1, Steltenkamp S2, Burg TP1. Lab Chip. 2014 Sep 7;14(17):3281-4.

1. Max Planck Institute for Biophysical Chemistry, 37077 Goettingen, Germany. [email protected]

2. Micro Systems Technology (MST), Center of Advanced European Studies and Research (caesar), 53175 Bonn, Germany.


Cryofixation yields outstanding ultrastructural preservation of cells for electron microscopy, but current methods disrupt live cell imaging. Here we demonstrate a microfluidic approach that enables cryofixation to be performed directly in the light microscope with millisecond time resolution and at atmospheric pressure. This will provide a link between imaging/stimulation of live cells and post-fixation optical, electron, or X-ray microscopy.

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