|Title||High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues.|
|Publication Type||Journal Article|
|Year of Publication||2018|
|Authors||Tsang TKi, Bushong EA, Boassa D, Hu J, Romoli B, Phan S, Dulcis D, Su C-Y, Ellisman MH|
|Date Published||2018 05 11|
|Keywords||Animal Structures, Animals, Brain, Cryopreservation, Drosophila, Imaging, Three-Dimensional, Mice, Microscopy, Electron, Scanning, Sense Organs|
Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native state. However, the applicability of cryofixation is limited by its incompatibility with diaminobenzidine labeling using genetic EM tags and the high-contrast staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation. Here, we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing for quality ultrastructural preservation for diverse EM applications.
|PubMed Central ID||PMC5988420|
|Grant List||R01 DC015519 / DC / NIDCD NIH HHS / United States |
P41 GM103412 / GM / NIGMS NIH HHS / United States
R01GM086197 / GM / NIGMS NIH HHS / United States
R01DC015519 / DC / NIDCD NIH HHS / United States
P41GM103412 / GM / NIGMS NIH HHS / United States
R01 DC016466 / DC / NIDCD NIH HHS / United States
R01 GM086197 / GM / NIGMS NIH HHS / United States
High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues.