Software Webinar

Whole C. elegans worms were fixed by HPF and embryonic cells in metaphase were imaged in situ by cryo-fluorescence microscopy. The screened worms were then heavy-metal stained by freeze substitution, resin-embedded and sectioned so that the same volume could be located and imaged at high resolution, with high contrast, by the Crossbeam. Using this workflow, the targeted metaphase could be successfully reconstructed. Additionally, this approach allowed serendipitous discoveries: an adjacent intriguing punctate fluorescence signal was able to be correlated to a putative autophagosome.

Thus, cryo-fluorescence microscopy of high-pressure frozen thick samples can be used to trap and image transient cellular structures in a near-to-native state; appropriate processing and subsequent correlative volume EM imaging then allows the reconstruction of these targeted architectures at high resolution and in 3D.

_{Courtesy of Kedar Narayan, National Cancer Institute / NIH and Frederick National Laboratory for Cancer Research, USA}

Once cellular structures such as spindle pole bodies are identified in the LSM system, the superior imaging quality of ZEISS Crossbeam allows targeting and analyzing the ultrastructure using cryogenic volume imaging. Even with low acceleration voltages, Crossbeam enables high-contrast imaging of non-stained, vitrified samples while protecting the sample from damage. High-resolution images acquired with the LSM and high-contrast images from the Crossbeam facilitate a precise image overlay. Once the region of interest is relocated in the Crossbeam using ZEN Connect, 3D datasets of the identified cells were acquired. Two spindle pole bodies were targeted within the correlative volume. The orientation of individual microtubules becomes clearly visible in the high-contrast images according to the cutting direction of the FIB. Further cell compartments could be identified in the 3D volume.