Fluorescence imaging of biomolecules within living cells can suffer from low resolution due to the molecules’ constant, rapid movement, resulting in motion blur that worsens with longer exposure times. Additionally, the intense light used to excite fluorescent molecules can kill cells, destroy the molecules of interest and lead to photobleaching, making observations of intricate biomolecular processes even more difficult. Researchers at the Max Planck Institute of Molecular Physiology have proposed a solution that addresses both of these common problems through the rapid cooling of living cells in a manner that ceases motion, prevents photobleaching and preserves biomolecules in their native configuration during reactions.
The method, known as ultrarapid cryo-arrest, involves cooling living cells to -196°C at speeds of up to 200,000°C per second, using a high pressure burst of liquid nitrogen on the opposite side of a diamond heat exchanger. The rapid cooling prevents the formation of ice crystals, eliminates motion blur and prevents photobleaching, allowing for both longer exposure times and more intense light to be used when imaging the molecular processes suspended in time. The result is much clearer, brighter images than what can be achieved at room temperature.
The researchers used this method to observe the spatial patterns of an oncoprotein and related tumor suppressor protein at nanoscale resolution. By comparing cryo-arrested cells that were exposed or not exposed to epidermal growth factor, the team discovered a previously unknown co-organization pattern between the oncoprotein and tumor suppressor. Additionally, the cryo-arrested cells could be observed using different microscopy modalities to observe the same process at different scales and put the molecular patterns into a larger context. This research was published in Science Advances.
“This is an enabling step for fluorescence microscopy, especially the combination of super-resolution microscopy and microspectroscopy that allow the mapping of molecular reactions in cells at multiple scales,” said study co-author Philippe Bastiaens. “It will change the way we observe molecular organization and reaction patterns in cells and therefore provide more insight in the self-organizing capabilities of living matter.”
Photo: Fluorescence microscopy of an oncoprotein and corresponding tumor-suppressor in a living cell before cryo-arrest (left) and super-resolution image obtained under cryo-arrest (right). Credit: Max Planck Institute of Molecular Physiology