Ultra-fast cryo-arrest of cells allows observation of molecular organization

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The quality of resolution of a structure or molecule by fluorescence microscopy depends on the amount of light that can be collected from the structure. In fluorescence microscopy, the exposure time can be extended to increase the amount of light detected. However, microscopic structures exhibit random and directed movements. The lengthening of the exposure time thus leads to a blurring of the structures.

Now, researchers have developed technology to stop patterns of molecular activity when observing their dynamics in living cells at any time of interest – within milliseconds – directly on the fluorescence microscope. By doing this, the two fundamental problems of motion blur and photodestruction can be bypassed at the same time.

This work is published in Scientists progress in the article, “Ultrafast cryo-arrest of living cells under a microscope allows multi-scale imaging of out of equilibrium molecular patterns.

Stopping occurs by extremely rapid cooling to temperatures (-196 ° C) where molecular motion is virtually stopped. The arrest must have been very quick for two reasons. First, the microscopic energized patterns that define living cells decay into the dead state if shutdown is too slow. Second, the speed of the arrest had to be faster than the process of ice formation, which would destroy the cells.

Ice formation occurs extremely rapidly in the critical range between 0 ° C and -136 ° C. However, non-intuitively, at very low temperatures (below -136 ° C), ice crystals cannot form because the movement of water molecules is practically stopped. This means that the cooling had to be faster than 100,000 ° C per second.

The researchers took up this technical challenge by developing an ultra-rapid cooling device integrated into a microscope where the cold of liquid nitrogen (-196 ° C) is accelerated under high pressure on a diamond. The same diamond also holds the sample containing the cells on its opposite side. The high pressure bursting in combination with the exceptional thermal conductivity of diamond enabled the high cooling rates needed to shut down cells at -196 ° C in their native configuration. This not only solved the problem of motion blur, but also stopped the photochemical destruction. This opens up the possibility of virtually endless exposure, highlighting molecular patterns that would otherwise be obscured by noise.

Ultrafast cryo-arrest made it possible to use normally destructive high laser powers to analyze native molecular models at resolutions of tens of nanometers that were otherwise invisible. Due to the absence of photodestruction at -196 ° C, the same arrested cells could be observed by different microscopy modalities to measure patterns from the molecular scale to the cellular scale. This new technology has led to the discovery of the nanoscopic co-organization of an oncoprotein and a tumor suppressor protein that protects cells against malignant behavior.

“This is an enabling step for fluorescence microscopy, in particular the combination of super-resolution microscopy and microspectroscopy which allow the mapping of molecular reactions in cells at multiple scales,” said Philippe Bastiaens , PhD, professor and director of
Department of Systemic Cell Biology, Max Plank Institute for Molecular Physiology, Dortmund, Germany. “It will change the way we look at molecular organization and reaction patterns in cells and therefore provide more information about the self-organizing abilities of living matter.”

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