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Fluorescence microscopes have the unique ability to observe cellular processes on scales spanning four orders of magnitude. However, its application in living cells is fundamentally limited by the very rapid and constant movement of the molecules that define its survival state. More importantly, the interaction of light and fluorescent probes can observe molecular processes, leading to their destruction. The Department of Systems Cell Biology at the Max Planck Institute for Molecular Physiology in Dortmund performs ultra-fast cryo-capture of cells during real-time observation under the microscope, which can now circumvent these basic problems. The core of this method is to cool living cells to -196 °C at a rate of up to 200,000 °C per second. This allows cell biomolecules to be preserved in an unprecedented way in their natural arrangement when captured. The Max Planck Institute wrote in a press release that in this low temperature state, molecular motion and damage caused by light cease, so that molecular patterns of life can be observed, otherwise these patterns would be invisible.

The nearly 100 trillion cells in our body are alive because they keep themselves permanently active through continuous energy consumption. Therefore, the microscopic patterns that make up cells are derived from the constant dynamic behavior of billions of nanometer-scale biomolecules (such as proteins, lipids, nucleic acids, and other molecules) that run around in seemingly disorganized ways.

In order to observe how this endless activity produces larger-scale tissues, biomolecular species can be selectively equipped with fluorescent probes. These fluorescent molecules are photonic catalysts: they absorb high-energy photons (such as blue light) and then emit low-energy (red-shifted) photons. These photons can be imaged through a microscope, which not only allows precise positioning of labeled biomolecules, but also reports local molecular reactions. However, the destruction of light-induced probes and the blurring caused by very important molecular motions are two fundamental problems that hinder the observation of how the molecular processes of life produce structures on the cellular scale.  

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The extent to which a fluorescence microscope actually resolves a structure or molecule fundamentally depends on the amount of light that can be collected from that structure. This is similar to trying to see the stars in the night sky. Only those stars that are significantly brighter than the surrounding environment are visible at first glance. If we shoot the night sky with a long exposure time, we will see more stars, but they will become blurred due to the rotation of the earth.

Similarly, under a fluorescence microscope, the exposure time can be extended to increase the amount of light detected. However, the microstructure never stands still, but exhibits random and directional movement. Extend the exposure time and cause the structure to be blurred. However, in this case, the movement of the small structure is much faster than the photon catalysis of the fluorophore, so the accuracy cannot be improved by creating a better detector or stronger illumination. What's more, the photon catalysis process produces toxic free radicals, which not only destroy the molecular process, and ultimately kill the cell, but also destroy the fluorescent molecule itself. This ultimately limits the amount of light that can be collected from the probes in living cells.

Jan Huebinger of the Philippe Bastiaens group has now developed a technique that can capture molecular activity patterns while observing the dynamics of living cells at any time of interest in a few milliseconds directly on a fluorescence microscope. In this way, the basic problems of motion blur and light destruction can be bypassed at the same time.

By cooling extremely quickly to such a cold temperature (-196°C), the molecular motion is practically stopped, thus achieving stagnation. The arrest must be very fast for two reasons. First, if the arrest is too slow, the vibrant microscopic patterns that define living cells will be broken down into dead states.

Second, the speed of stagnation must be faster than the process of freezing, which destroys the cells. This can also be observed on a larger scale, for example tomatoes become very mushy after freezing. In the critical range between 0 °C and -136 °C, ice formation occurs very quickly. However, non-intuitively, at very low temperatures (below -136 °C), ice crystals can actually no longer form because the movement of water molecules has almost stopped. This means that, literally, the cooling rate must be faster than 100,000°C per second.

The researchers overcome this technical challenge by developing an ultra-fast cooling device integrated with the microscope to cool liquid nitrogen (-196°C) onto the diamond under high pressure. The same diamond also fixes a sample containing cells on its opposite side. The combination of high pressure burst and diamond's excellent thermal conductivity allows the high cooling rate necessary to fix the cells in the original configuration at -196°C. This not only solves the problem of motion blur, but also prevents photochemical damage. This opens up almost unlimited exposure possibilities, highlighting molecular patterns that are masked in noise.

Ultra-fast cryo-capture allows the use of high laser powers that are often destructive to analyze natural molecular patterns with a resolution of tens of nanometers that would otherwise be invisible. More importantly, since there is no light damage at -196 °C, the same stagnant cells can be observed through different microscope methods to measure patterns from molecular to cellular scale.

Therefore, this new technology has led to the discovery of a nano-scale synergistic organization of oncoproteins and tumor suppressor proteins that can protect cells from exhibiting malignant behavior. "This is an advantageous step for fluorescence microscopy, especially the combination of super-resolution microscopy and microscopic spectroscopy, which can map molecular reactions in cells on multiple scales. It will change the way we observe molecular organization and reaction patterns in cells. Way, so that we have a deeper understanding of the self-organizing ability of living matter," said Philippe Bastiaens.

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