Super-Resolution Single Molecule Localization Microscopy with Fluorescence Lifetime Imaging – SiMpLeLIFe

How to image molecules in living cells? How to spy on the details of their interactions, organization and dynamics at the molecular scale, their intricate dance to create and maintain the cellular structure and function?

When we use an optical system, from a phone camera to an advanced research microscopy system, the wavelike nature of light imposes the resolution limit at which we can observe the fine details of an object. In the case of last-generation classical fluorescence microscopes, this limit lays at around 200 nanometers. While this is well below the typical size of a cell, it is still not enough to observe the molecules that form that cell, responsible of the cell survival and fate. It is then impossible to understand how these biomolecules organize in the cell, how they interact with each other, and what is the rhythm of this complex yet defined choreography that dictates the subcellular structure and dynamics.

Fortunately, optical microscopy has been recently revolutionized by the advent of a new set of techniques capable of bypass such a fundamental resolution limit, recognized with the Nobel Prize in Chemistry 2014. In particular, we know now how to separate the light emission of fluorescent molecules in time, making them blink one after the other. Thanks to this trick, we can reconstruct images with a detail in the order of tens of nanometers. With this new approach, called super-resolution microscopy or nanoscopy, we have greatly advanced in our knowledge of the organization and the molecular dynamics inside living cells, and how the influence of one on the other are indivisible.

The goal of this research proposal is to develop a device that will allow us to image single molecules at the same time that we measure the speed at which they emit light. Indeed, this rate of photons emission is influenced by the local environment of the molecule, as well as by the presence of other interacting molecules in a vicinity of less than 10 nanometers. With this type of measurements, we will be able to study not only the organization and dynamics of these biomolecules but also directly visualize their interactions.

We will use this pioneering imaging technique to the study of neuronal plasticity; that is the ability of neurons to enhance or inhibit their communication, mechanism at the basis of processes like learning and memory. A better comprehension of these mechanisms at the molecular level will therefore lead us to a better understanding of our brain, as well as the origin of certain neurodegenerative diseases.