The CLEM Workflow
The further we dive into multimodal imaging, the least it becomes possible to define correlative microscopies.
To cut a long story short, we aim to observe one exact same sample sequentially using various imaging technologies, to ultimately blend them into one single virtual dataset.
To achieve that, the sample must be prepared from the very first step with all the various transformations it will undergo in mind.
We focus our attention on Fluorescence Live Cell Imaging, cryo-immobilization by high pressure freezing, electron microscopy imaging and ultimately, image registration. This is a narrow path, but likely the best to cover the broadest range of multimodal cell and tissue imaging. Want to know more? Let's take an example
Live CLEM-HPF
In this video, we showcase our live CLEM-HPF workflow by capturing a complete experiment, ranging from live cell imaging and cell viability control using a standalone microscope to the integration with our HPM-Live µ for live cell imaging of a sample prior to HPF.
Live Cell Imaging
Life science is about observing changes in a dynamic environment upon stimulation to understand the underlying mechanisms. Live cell imaging is therefore one major technique used to observe these changes as they happen. To make sure we capture the event of interest as we go to the electron microscope (a tool to observe ultrastructure of fixed material), we associate a light microscope to our High Pressure Freezer to achieve a temporal resolution of 1.26second between the last living image and the cryo-immobilization of the sample.
High Pressure Freezing
High Pressure Freezing is a technology that aims to vitrify hydrated specimen up to 200µm in thickness.
To achieve that, we exploit in part the water phase diagram and circumvent the ice transition by placing our object at 2000bar before a rapid cooling.
To read more information on this technology and our solutions, refer to our book chapter recently published here.
We are happy to discuss our technology any time. Contact us!
Freeze Substitution
Once a sample has been vitrified by High Pressure Freezing, it is convenient to warm it back to room temperature for further analysis by electron microscopy.
To prevent damages to the specimen during this warming process, and ensure full use of the material, the sample is dehydrated at low temperatures (-90°C) in presence of solvent, fixators (aldehydes) and contrasting agents (often heavy metal salts). Then the solvent is replaced by a resin that is polymerized while warming the sample to room temperature.
We identified that the existing pool of resins used in volume electron microscopy is not fulfilling the general needs for Volume Correlative Light and Electron Microscopy. This is why we designed a novel resin, starting from a white page, to adress all the challenges associated with volume CLEM. The R221 is our answer to these challenges.
Image registration
Multimodal imaging often requires observing the sample through physically distinct microscopes, or after applying a physical transformation of the sample between two imaging steps.
Image registration is therefore an essential step where accurate superimposition of the various images at distinct magnification will allow targeting the structure of interest and identify precisely the phenotype of interest.
We co-developed eC-CLEM to facilitate the registration of multimodal datasets with high accuracy tracking. Find out more on the dedicated website or the original publication here.