Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures.

ABSTRACT

3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2-50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).