Ultra-High Resolution Three Dimensional Imaging Using 4pi-Smsn Throughout Whole Cells

Major advances in biology have been tightly linked with innovations in microscopy. A major hurdle over the last ∼100 years is the limited resolution of light microscopy. The advent of single molecule switching nanoscopy (SMSN, also known as PALM/STORM/FPALM) has overcome this fundamental limit by improving the resolution of fluorescence microscopy (250-700 nm) by a factor of ten. This method routinely achieves 20-40 nm lateral resolution and 50-80 nm resolution in the axial direction. While the inferior axial resolution largely restricts the biological discoveries to two-dimensional observations, interferometric SMSN (iPALM or 4Pi-SMSN) achieves unprecedented 10 to 20 nm axial resolution by coherently combining single-molecule emissions in a two opposing-objective setup. It allowed, for the first time, the molecular anatomy of focal adhesions to be mapped with nanometer precision. However, the physical principle of 4Pi geometry limits this approach to isolated structures in thin samples because the single molecule interference pattern repeats every 250 nm in depth. While most of biological processes happen deep in the cellular volume, driving ultra-high resolution imaging deeper into the cell will lead to a new wave of biological discoveries.Here, we present whole-cell 4Pi-SMSN resulting from the confluence of multiple innovations. Our system, for the first time, allowed super-resolution imaging of a ∼10 µm thick sample using 4Pi geometry achieving 10-15 nm resolution throughout the depth. This resolution is 20-50 times higher than conventional microscopy with imaging depth improved by 10-40 fold from the state of art technology of interferometric SMSN. It enables ultra-high resolution three-dimensional imaging for vast majority of the subcellular structures. We demonstrate applications in a range of delicate cellular structures including: bacteriophages, ER, mitochondria, nuclear pore complexes, primary cilia, Golgi complex-associated COPI vesicles, and synaptonemal complexes in whole mouse spermatocytes.