Ultrarapid cooling enables the observation of molecular patterns of life


Date:
December 13, 2021
Source:
Max Planck Institute of Molecular Physiology
Summary:
Fluorescence light microscopy has the unique ability to observe
cellular processes over a scale that bridges four orders of
magnitude. Yet, its application to living cells is fundamentally
limited by the very rapid and unceasing movement of molecules that
define its living state. What is more, the interaction of light
with fluorescent probes that enables the observation of molecular
processes causes their very destruction.

Ultrarapid cryo-arrest of cells during live observation on a
microscope now circumvents these fundamental problems. The heart
of the approach is the cooling of living cells with enormous
speeds up to 200,000 DEGC per second to -196 DEGC. This enables an
unprecedented preservation of cellular biomolecules in their natural
arrangement at the moment of arrest. In this low temperature state,
molecular movement and light- induced destruction is stopped,
enabling the observation of molecular patterns of life that are
otherwise invisible.



FULL STORY ========================================================================== Fluorescence light microscopy has the unique ability to observe cellular processes over a scale that bridges four orders of magnitude. Yet,
its application to living cells is fundamentally limited by the very
rapid and unceasing movement of molecules that define its living
state. What is more, the interaction of light with fluorescent probes
that enables the observation of molecular processes causes their very destruction. Ultrarapid cryo-arrest of cells during live observation on
a microscope, as developed in the Department of Systemic Cell Biology
at the Max Planck Institute of Molecular Physiology in Dortmund, now circumvents these fundamental problems. The heart of the approach is the cooling of living cells with enormous speeds up to 200,000 DEGC per second
to -196 DEGC. This enables an unprecedented preservation of cellular biomolecules in their natural arrangement at the moment of arrest. In this
low temperature state, molecular movement and light-induced destruction
is stopped, enabling the observation of molecular patterns of life that
are otherwise invisible.


==========================================================================
The almost 100 trillion cells of our body are alive because they
maintain themselves in a permanently active state by continuous energy consumption. The microscopic patterns that constitute a cell thereby
originate from the ever- dynamic behavior of billions of nanometer-sized biomolecules, like proteins, lipids, nucleic acids and other molecules,
that bustle around in a seemingly unorganized way. To observe how higher
scale organization emerges from this incessant activity, biomolecular
species can be selectively equipped with fluorescent probes. These
fluorescent molecules are photon catalysts: they absorb high energy
photons (e.g. blue light) and subsequently emit lower energy (red-shifted) photons. These photons can be imaged through a microscope to not only
precisely localize the labeled biomolecules, but also report on local
molecular reactions. However, light-induced destruction of the probes
and blurring through the very vital molecular motion are two fundamental problems that hamper observations of how the molecular processes of life generate structure at the cellular scale.

An uncertainty principle to fluorescence microscopy How well a certain structure or molecule is actually resolved by fluorescence microscopy
depends fundamentally on the amount of light that can be collected from
this structure. This is analogous to trying to see the stars in the night skies. Only those stars that are clearly brighter than their surroundings
are visible at first sight. If we photograph the night sky with a long
exposure time, more stars become visible that however become blurred
by the Earth's rotation. Similarly, in fluorescence microscopy exposure
time can be prolonged to increase the amount of detected light. However, microscopic structures never stand still, but exhibit random as well as directed motion. Prolonging the exposure time thereby leads to blurring
of the structures. In this case, however, the movement of small structures
is much faster than the photon catalysis by the fluorophore and therefore
the accuracy cannot be improved by creating better detectors or stronger illumination. Even more, the process of photon catalysis produces toxic radicals, which not only destroy molecular processes and eventually
kill the cells, but also destroy the fluorescent molecule itself. This ultimately limits the amount of light that can be collected from the
probes in the living cells.

The solution is literally very cool Jan Huebinger in the group of Philippe Bastiaens has now developed a technology to arrest molecular activity
patterns during observation of their dynamics in living cells at any
timepoint of interest within milliseconds directly on the fluorescence microscope. By this, both fundamental problems of motional blur and photodestruction can be bypassed at the same time.

The arrest is done by extremely fast cooling to temperatures that
are so cold (-196DEGC), that the molecular movement is virtually
stopped. The arrest had to be very fast for two reasons. First, the
energized microscopic patterns that define living cells disintegrate
into the dead state if the arrest is too slow.

Second, the speed of the arrest had to be faster than the process of ice formation, which would destroy the cells. This can also be observed on a
larger scale, when e.g. tomatoes become very mushy after freezing. Ice formation happens extremely fast in the critical range between 0 DEGC
and -136 DEGC.

However, non-intuitively, at very low temperatures (below -136 DEGC)
ice crystals can actually not form any more, because the motion of
water molecules is also virtually stopped. This means literally, that
cooling had to be faster than 100,000DEGC per second. The researchers
have mastered this technical challenge by developing a ultrarapid
cooling device that is integrated with a microscope where the cold of
liquid nitrogen (-196DEGC) is accelerated under high pressure onto a
diamond. The same diamond also holds the sample containing the cells on
its opposing side. The high pressure burst in combination with exceptional
heat conductance of the diamond allowed to achieve the necessary high
cooling rates to arrest cells at -196DEGC in their native configuration.

This not only solved the problem of motional blur but also stop
photochemical destruction. This opens up the possibility of virtually
infinite exposure, highlighting molecular patterns that are otherwise
obscured in the noise.

Making the invisible visible Ultrarapid cryo-arrest allowed the use of
normally destructive high laser powers to analyze native molecular
patterns at tens-of-nanometer resolutions that were otherwise
invisible. What is more, because of the absence of photodestruction
at -196 DEGC, the same arrested cells could be observed by different
microscopy modalities to measure patterns from the molecular to the
cellular scale. This new technology thereby led to the discovery of
nanoscopic co-organization of an oncoprotein and a tumor suppressor
protein that safeguards cells from exhibiting malignant behavior. "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. 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," says Philippe Bastiaens.

========================================================================== Story Source: Materials provided by Max_Planck_Institute_of_Molecular_Physiology. Note: Content may be edited
for style and length.


========================================================================== Journal Reference:
1. Jan Huebinger, Hernan Grecco, Marti'n E. Masip, Jens Christmann,
Gu"nter
R. Fuhr, Philippe I. H. Bastiaens. Ultrarapid cryo-arrest
of living cells on a microscope enables multiscale imaging of
out-of-equilibrium molecular patterns. Science Advances, 2021; 7
(50) DOI: 10.1126/ sciadv.abk0882 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/12/211213094857.htm

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