New microscopy techniques for 3D super resolution imaging – TRIDIMIC

Our understanding of living matter requires measurement tools having an increasing accuracy and, at the same time, sufficient speed, robustness and reliability so that a large number of conditions can be screened in a reasonable time. This notion of screening is essential to finding new drugs and also for academic researchers who can then base its investigation strategy on systematic approaches therefore allowing out of the box discoveries to be made. Over the past 2 decades, light microscopy has undergone a revolution, initiated by the appearance of cameras that can measure cell components with a good dynamic range. The microscope has then become a tool for measuring intracellular concentrations. In recent years a second stage has been reached with the detection of single molecules in the cell, allowing their counting and localization in 2 dimensions with a precision of a few tens of nanometers.

In this project, Imagine Optic, the worldwide leader in adaptive optics (AO), and two academic teams from the Physics and Biology departments at Ecole Normale Superieure (Paris) have joined forces to develop a new imaging technique for the 3D localization of single particles in fixed or living cells, with an isotropic resolution of a few nanometers. This first proof of concept will provide the foundation for the industrialization and commercialization of a system.

Over the past 5 years, several techniques to gain a factor of 10 in the lateral resolution on a living biological specimen have been developed. However, the axial resolution offered by these different techniques is generally an order of magnitude below the lateral resolution. AO allows for the corrections of the residual aberrations of the optical system of the microscope and those generated by the sample itself. In addition, it permits a precise reshaping of the Point Spread Function of a microscope (PSF) with a well-defined asymmetry along the optical axis, and thus allows a very precise axial position determination.

We therefore propose to implement an AO system on a standard single molecule imaging microscope (we will develop an independent module that is inserted between the microscope and camera). We also will develop software tools for correcting optical aberrations, the PSF reshaping and image processing required for 3D localization.

We will validate our developments on three biological systems that are representative of a variety of configurations in which the super-resolution 3D imaging of single molecules is applicable. First, we will measure the membrane trafficking of neurotransmitter receptors in 3 dimensions. Next, we will use our technology to map the nuclear organization of RNA polymerase II. Finally, we will develop a large-scale imaging approach to count c-Fos mRNA molecules in a population of cells so as to understand how the normal regulation of this RNA is involved in cancer development.

Our knowledge of the function (or dysfunction) of a cell is more and more often related to direct observation of morphology and molecular distribution.Thus, from a societal perspective, nanoscale 3D imaging represents a new and powerful tool toward understanding the biological mechanisms behind diseases and thus ultimately leading to the development of new drugs. Whether in basic or applied research, our new 3D imaging technique will potentially be useful for biomedical diagnostics and industrial activity in pharmacology, cosmetics and biotechnology