Super-resolved image of a transmembrane protein on the surface of a fibroblast: the highest peaks corresponding to 70 detections of these complexes in 50x50nm ² (the image is about 10 microns wide). The technique can achieve 20 frames per second, which allows to reconstruct the trajectories.
Credit: © Gregory. Giannone, Eric. Hosy Florian Levet.
The microscope is the ideal tool for observing the world living on a small scale. What student has not observed the cells of onion skins or chloroplasts between two small pieces of glass? But when we look at smaller objects inside the cell, the diffraction pattern (a) limits the resolution, below 250 nanometers (one quarter of a micron), the objects we wants to observe are only spots because light brushes returned by each point of the object expand and merge.
The detection of single molecules
In recent years, researchers have managed to circumvent this difficulty by a family of new techniques of "super-resolution microscopy" or nanoscopy. These methods are capable of providing images of nanoscale molecular assemblies on the surface of cells, are poised to revolutionize the field of biological cell imaging. What principle? It uses the fact that despite the diffraction, we can precisely locate a point (or molecule) unique, because we know he is in the center of the spot of light that leads us, as soon as we have several points on an object that wants to make a picture, the two tasks overlap and we can not know their exact positions.
The researchers had the idea of using genetic engineering to n 'turn, at a given moment, some random points on the objects they wanted to be imaged on the surface of living cells. A bit like a Christmas garland light bulbs where one after the other and allowing the end to get an idea of the overall shape of the tree, once all the lights were lit.
But these methods have one major drawback: it must be able to turn on and off the molecules one by one, on demand. For this, we use special fluorescent protein introduced into cells by genetic engineering. Two lasers can only turn statistically few molecules at a time. These fluorescent molecules are some points of the image. The operation is repeated for the other points and form, ultimately, the complete picture of molecular assembly to consider. Forced use of specific fluorescent proteins, researchers could not study biological molecules on the surface of cells without genetic modification, nor follow their path for several seconds. Indeed, the fluorescence properties of molecules modified by genetic engineering had mostly well under a second.
Today, the physicists at the Centre de Physique and terrestrial optical molecular biologists and Cell Physiology Laboratory of the synapse (two laboratories CNRS / University of Bordeaux) have developed a new technique to "turn" the molecules: they used the immuno-marking in real time. Living cells are placed in the presence of a fluorescent antibody solution (commercially available, an antibody for each type of object studied), which will bind to them. But instead of putting enough antibodies to bind to the molecules to be studied, it makes a mark dilution under the microscope: the few available antibodies will bind to molecules to study and make them light, then, when they will be extinct , others are already bind to the new antibodies that occur in the sample (the marking is continuous) and will turn to turn.
"Filming" cells with a resolution of 50 nanometers
The technique can save tens of thousands of trajectories of single molecules on a single cell and to study the dynamics at the nanoscale. Researchers have demonstrated its effectiveness on various cell systems (heterologous cells, fibroblasts or neurons in culture) to study different surface molecules (2). The simplicity, adaptability and reliability of this new method can already be considered a large number of studies previously inaccessible by conventional optical microscopy. In particular, it becomes possible to "shoot" the evolution of a single cell, such as a neuron, with a spatial resolution of 50 nanometers for a rate corresponding to the imaging of the video (not 20 frames a second). Bordeaux researchers have already begun the study of the structuring dynamics of neurotransmitter receptors in the synapses of live neurons. There is no doubt that given the large number of teams already interested in this new technique, it should trigger a revolution in the world of biological imaging.
Sources and contacts:
CNRS News Release,
CNRS researchers Laurent Cognet and Daniel Choquet
References: Dynamic super-resolution imaging of endogenous proteins on living cells at ultra-high density, G. Giannone, E. Hosy, F. Levet, A.Constals, K. Schulze, AI Sobolevsky MP Rosconi, E. Gouaux, R.Tampa, D. Choquet and L. Cognet, Biophysical Journal, August 18, 2010.
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