Next Generation Correlative Cryo-Light and Electron Microscopy
- Abstract number
- 341
- Presentation Form
- Submitted Talk
- Corresponding Email
- [email protected]
- Session
- Stream 6 (Frontiers): Correlative Imaging of Organelle Organization and Architecture
- Authors
- Dr Chris Parmenter (1)
- Affiliations
-
1. University of Nottingham
- Keywords
Cryogenic, SEM, FIB, CLEM, cryo-light, Correlative
- Abstract text
Summary
Building on the fundamental principle of correlative light and electron microscopy (CLEM) we present hardware solutions to enable correlative cryo-microscopy. This emerging field is fraught with barriers, as the hardware platforms and sample handling requirements of light and electron microscopy techniques are commonly not compatible. We present our attempts to overcome some of these challenges.
Introduction
Correlative light and electron microscopy (CLEM) is becoming a well-established approach to enhance understanding of cells and tissues by combining traditional information from light microscopy such as fluorescence localized to cellular components (nucleus, membranes, proteins or cell scaffold) with electron microscopy data/images that tend to have higher magnification. Much work has been done to establish the application of these techniques to investigate the same area i.e. correlation. Cryogenic electron microscopy and cryogenic light microscopy have been shown to offer the study of samples under near to native state conditions. By combining these concepts of CLEM and cryo-light/EM it is possible to get the best understanding of a sample. Handling samples under cryogenic conditions has limitations including that samples easily contaminate (frost) and can be damaged through handling.
Methods/Materials
Cryo-TEM grids were frozen using a Gatan CP3 plunge freezer (Gatan, USA). Light microscopy was performed on an upright Nikon widefield fluorescence microscope (E400, Nikon) fitted with an Aurox laser-free confocal system (Clarity, Aurox). To enable cryo-light microscopy the standard sample stage was replaced with a Linkam CMS 196 cryo-stage (Linkam). Electron microscopy was performed on an FEI Quanta 3D SEM (FEI, USA) with a Quorum 3010 cryo-system (Quorum, Loughton, UK).
Results and Discussion
Cells were plunge frozen and transferred to a grid storage box. This box was moved to the CMS 196 and the TEM grid loaded into the cryo-bridge. This allowed cryo-light microscopy to be performed, including both overview images (x4 objective) and high-mag images of individual cells (x100 objective). To relocate the cells to the cryo-SEM stage, it was necessary to transfer the sample back to the cryo-TEM box and move the box to the cryo-SEM loading stage. The TEM grid can then be removed from the box and mounted to the cryo-SEM shuttle, a total of 6 steps. Once secure, it is moved to the cryo-stage of the SEM. At every point, there is a chance of damaging the sample, and it is impossible to maintain sample orientation. To overcome this, we developed a sample holder that can be used in both the CMS196 and also easily relocate to the cryo-shuttle. This requires only 4 steps and maintains the sample geometry. This small but important step forward lays the groundwork for 3D cryo-volume imaging and cryo-lift-out using FIB-SEM at cryo-conditions and correlative imaging between platforms.
Conclusion
Correlative cryo-CLEM has huge potential for biology and beyond. There are a number of challenges that have to be overcome around sample handling, particularly maintaining sample orientation and limiting sample contamination. We have demonstrated a practical solution to overcome these challenges.