Time-based single molecule nanolocalization for live cell imaging
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 allowing to understand biological processe...
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 allowing to understand biological processes in their full multi-scale nature from the individual molecule, to the cell, to the multicellular tissues. Currently, there is no solution to image with an acquisition speed compatible with the dynamics of living cells (>kHz), over a large volumes (>10000 μm3), with a significant in-depth imaging capability (>10 µm) without compromising the resolution (<10 nm) especially in the axial dimension. I will demonstrate that these challenges can be met without trade-off by proposing a disruptive approach which requires the entire illumination and detection strategies to be considered anew.
Building on my achievements in super-resolution microscopy, I propose a disruptive optical implementation where a full-time coding approach allows to retrieve the space information, not only meeting the needed acquisition rate, but also bringing the richness of functional information (spectral, lifetime).
In a first conceptual breakthrough, a new sweeping modulated illumination is introduced, where 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 3 frequencies.
This unicity triggers a second change of paradigm, as emitters positions within a large field of view can now be retrieved in a camera-free setting. Fast acquisition can be performed by single monodetector, but the power of this temporal approach will be decupled by the ultimate technical advances of new neuromorphic detections.
This unique in-depth information at the nanoscale will allow to finally decipher the structural and functional interplay in key biological mechanisms in living cells with unprecedented benefits.ver más
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