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The Valrose Biology Institute is now equipped with an optical microscope with molecular resolution

Physicists, mathematicians and biologists joined forces to develop the prototype of a photonic microscope. The instrument took the “Multi-Angle-TIRF” technique and stretched it beyond its technical limits. The microscope can be used to observe and measure processes at an axial resolution of about 30 nm and is compatible with live samples, which opens up a whole range of possibilities for cell culture imaging.


Publication : 15/03/2019
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Optical microscopes are essential tools in biology for observing samples and understanding the phenomena that occur in them. But even though instruments were designed with increasingly higher resolutions, they were reaching the theoretical limit (the "diffraction limit" established by Ernst Abbe in 1873).

Starting in the 80s, different "super-resolution" techniques were developed (StED, PALM/StORM or SIM) to exceed (or rather “circumvent”) this diffraction limit. Over the years, these different techniques kept on improving and now present the enormous advantage of offering very high performances in both lateral resolution (up to 20 nm for a 2D image) and axial resolution (up to 100 nm for a 3D image). Unfortunately, when using these techniques, enormous quantities of energy are projected on the sample and it must also be stained with very specific fluorescent markers, making it difficult to be used with live samples.
For some applications, it is more crucial to be able to improve the axial resolution, and simultaneously examine several molecules using standard fluorescent molecules or directly fluorescent proteins. 

A precision technique based on one of the light's properties 

The MA-TIRF (multiple-angle total internal reflection fluorescence) method is particularly suited in this context. Initiated in the 90s, this technique uses one of the properties of light, the "evanescent wave". When a sample is illuminated from a very oblique light source, the evanescent wave only penetrates a few hundred nm (this is the standard TIRF principle). By playing on the angle of illumination, penetration can be made to vary. And by precisely combining several angles of illumination, additional information can be obtained. But this requires a very specific reconstruction algorithm. 

The prototype that was developed has a resolution 15 times higher than that of an optical microscope. Biologists, physicists and mathematicians collaborated to develop the microscope prototype and the associated reconstruction algorithm that makes it possible to obtain a 3D view of cells with an axial resolution of 30 nm. The microscope is also compatible with the use of three simultaneous markers and the observation of live samples, opening up new perspectives in very high-resolution imaging. 

This microscope is sufficiently precise to rebuild samples with a thickness of up to almost 500 nm and measure an extremely slight difference between 2 equivalent markers on the same cell. Finally, in addition to offering better axial resolution, its algorithm also improves lateral resolution by integrating a deconvolution process. 

Researchers used this prototype to gain a wider understanding of how cells grow and interact with their direct environment (the "extracellular matrix"). By studying integrins (anchor molecules that bind the cell to its matrix) and associated molecules, they were able to demonstrate the instrument's performances by confirming the orientation of this anchor molecule and measuring its length (approximately 70 nm). The device's high resolution also made it possible to dissociate the proportion of integrins truly bound to the cellular membrane from those present in the vesicles inside the cell and to observe the thickening of the matrix. These results pave the way for very high-resolution studies of a large number of biological processes, especially in the vicinity of the cell membrane. 

 Schema Schaub

Figure a. Standard TIRF image, where the actin (in magenta), the extracellular part (in red) cannot be dissociated from the intracellular part (in green) of the integrin 51. Figure b. The same sample reconstructed in 3D by MA- TIRF where the extracellular marking (red) can clearly be observed under the intracellular marking (green). Figure c. Measurement of the distance between the extracellular and intracellular parts gives ~ 80 nm.
© Sebastien Schaub