Time-based single molecule nanolocalization for live cell imaging

Introduction

Despite the tremendous advances in single molecule localization microscopy mainly obtained on fixed cells, a major step is highly needed to transform it into a genuine live-cell nanoscopy. This transformation is essential to understand biological processes in their full multi-scale nature, from the individual molecule, to the cell, to the multicellular tissues.

Current Limitations

Currently, there is no solution to image with an acquisition speed compatible with the dynamics of living cells (>kHz), over large volumes (>10000 μm³), with significant in-depth imaging capability (>10 µm) without compromising the resolution (<10 nm), especially in the axial dimension.

Proposed Solution

I will demonstrate that these challenges can be met without trade-offs by proposing a disruptive approach that requires the entire illumination and detection strategies to be considered anew.

Optical Implementation

Building on my achievements in super-resolution microscopy, I propose a disruptive optical implementation where a full-time coding approach allows for the retrieval of spatial information. This not only meets the needed acquisition rate but also brings the richness of functional information (spectral, lifetime).

Conceptual Breakthrough

In a first conceptual breakthrough, a new sweeping modulated illumination is introduced. Here, each point along the direction of the modulation can be retrieved through a unique frequency, without any ambiguity. By applying this frequency tagging in all dimensions, every single molecule event is described by a unique set of three frequencies.

Paradigm Shift

This uniqueness triggers a second change of paradigm, as emitter positions within a large field of view can now be retrieved in a camera-free setting. Fast acquisition can be performed by a single monodetector, but the power of this temporal approach will be decoupled by the ultimate technical advances of new neuromorphic detections.

Conclusion

This unique in-depth information at the nanoscale will allow us to finally decipher the structural and functional interplay in key biological mechanisms in living cells, yielding unprecedented benefits.