Super-resolved imaging

Summary

In addition to their highly sub-𝜆 dimensions and resonant interaction with light, fluorescent molecules also exhibit stochastic emission properties that are particularly attractive as contrast agents in super-resolved imaging. Indeed, the ability to image and locate each emitter individually means that an image can be reconstructed with a resolution that is limited only by the number of photons emitted by the molecule before it fades.

This super-resolved fluorescence microscopy makes it possible to reach nanometric scales, revolutionizing cellular observation. However, the range of observations is limited in depth and many biases persist, limiting the performance of these microscopes. In collaboration with ISMO and ISCN (CNRS/Université Paris-Saclay) and INP (CNRS/Université Aix Marseille), we have proposed a new approach to obtain the axial position of fluorescent molecules absolutely with nanometric precision [Cabriel et al., Nat. Comm. 2019]. Supercritical emission in the glass slide on which the cells are deposited enables absolute localization, insensitive to axial drift as well as chromaticity issues. This method has made it possible to observe different proteins in the cell cytoskeleton in 3D, the insertion of a fluorescent probe in bacteria, or the position of different proteins in neurons.

We have also introduced a new concept for localizing single fluorescent molecules based on temporally modulated illumination and measurement of the phase of the fluorescence signal induced by this modulation, rather than on localization of the center of the diffraction pattern detected by the camera [Jouchet et al., Nat. Phot. 2021] (figure below). This approach enables a gain in localization accuracy, preserved along the axial direction, providing 3D images with a resolution of 6.8 nm. In addition, this new approach enables the observation of more complex biological samples, such as tumor models or tissues, down to depths of ≈30𝜇𝑚. This imaging method is currently being applied to issues such as oncology to better understand the organization of proteins at the nanometric scale.

Principle of super-resolved imaging with time-modulated illumination (left) and example of an image of mitochondria with sub-30 nm resolution (right).