New Imaging Tools for Cryo-Light Microscopy

How LIGHTNING and TauSense improve targeting for focused-ion-beam (FIB) milling

Projection of a confocal z-stack before and after LIGHTNING applied. Sum159 cells, human breast cancer cells kindly provided by Ievgeniia Zagoriy, Mahamid Group, EMBL Heidelberg, Germany. Blue – Hoechst - indicates nuclei, Green – MitoTracker Green – mitochondria, and red – Bodipy - lipid droplets. Keyvisual-Cancer-cell-under-Cryo_Coral-Cryo_TechNote.jpg

Cryo electron tomography (cryo-ET) is a transmission electron microscopy (TEM) technique dedicated to the imaging of relatively thin volumes (about 200-300 nm thick) using a tilt series. With this technique, the 3D molecular structure of proteins can be revealed.

With the technological advances of the last decade regarding sample preparation using cryo-FIB and cryo-TEM, another milestone was achieved. Hence, the structure of proteins and other biomolecules can now be resolved using cryo-ET in their native environment, inside the cells, with sub-nanometer resolution. For this approach, cells are grown on a TEM grid, vitrified by plunge freezing, and then transferred to a cryo-FIB/SEM to produce a small window (lamella). The lamella can then be imaged in a tilt series with cryo-TEM. The imaged window of the cells must be thin enough to allow the electrons to pass through the sample.

However, to identify target sites in samples, high precision for localization of regions of interest is required. Currently, this is done mainly by cryo-confocal fluorescence microscopy, enabling high precision imaging in 3D. The confocal pinhole eliminates the out-of-focus light, increasing resolution and contrast, particularly in the axial direction.

In the following, it is explained how the LIGHTNING super-resolution technique and TauSense fluorescence lifetime-based tools are used under cryogenic conditions to enable better discrimination and identification of structures of interest with fluorescence microscopy which is performed for subsequent EM steps.

Key Learnings

LIGHTNING super-resolution under cryo conditions

  • How does it work?
  • Deconvolution is well known, but what is special about LIGHTNING?
  • How does LIGHTNING improve the targeting precision with the STELLARIS Cryo confocal microscope?
  • Can I quantify my data after deconvolution with LIGHTNING?

TauSense under cryo conditions

  • What is TauSense?
  • How does TauSense work?
  • TauContrast
  • TauScan
  • TauSeparation
  • TauGating
  • Fully integrated into the Coral Cryo software workflow

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