Correlating cryo-super resolution radial fluctuations and dual-axis cryo-scanning transmission electron tomography to bridge the light-electron resolution gap.

[Display omitted] • Applying SRRF nanoscopy under cryo condition. • Cryo-scanning transmission Electron tomography (CSTET) allows imaging of whole organelles and their interaction in 850 nm areas of intact and near-native cells. • Combining dual-axis CSTET with deconvolution results in visually isotropic resolution also in the axial direction. Visualization of organelles and their interactions with other features in the native cell remains a challenge in modern biology. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access 3D volumes on the scale of 1 micron with a resolution of nanometers, making it ideal for this task. Here we introduce two relevant advances: (a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and (b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy. Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to-isotropic resolution in the reconstruction without averaging. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell. [ABSTRACT FROM AUTHOR]