Advanced Electron Microscopy 

The Biochemistry of Mineralized Tissues group is also involved in the Radboudumc Electron Microscopy Center. On this page, you can find more information about the ongoing projects to develop correlative imaging approaches in which fluorescence, electron and Raman microscopy are combined to study biochemical processes in 3D.

Correlative Light and Electron Microscopy

3D Cryo-Correlative Light and Electron Microscopy (cryo-CLEM)

Fluorescence microscopy is a valuable technique to visualize specific structures or components in cell cultures and tissues based on labeling. However, the resolution is limited to several hundreds of nanometers. Therefore, to gain ultrastructural information about regions of interest, fluorescence microscopy can be combined with electron microscopy: correlative light and electron microscopy. We are working on the development of a 3D (cryo)-CLEM workflow in which tissues or cells are fluorescently labeled to visualize markers of interest in live conditions. Next, samples are cryo-preserved or embedded and transferred to 3D (cryo-)Focused Ion Beam SEM (FIB-SEM) or array tomography SEM to analyze the ultrastructure of the identified regions of interest using 3D electron microscopy.

Team members: 
Rona Roverts, Mariska Kea-te Lindert, Ben Joosten

Collaborators:
Zeiss, CryoCapCell 

Correlative Light Microscopy and Liquid-Phase TEM 

Most material-forming processes in biology occur in a liquid environment. To understand these processes, we must observe them in the hydrated state which often remains a challenge. Although cryogenic transmission electron microscopy (cryo-TEM) is commonly used to understand mechanisms of material development in hydrated conditions, it still does not provide dynamic information. Developments in liquid phase electron microscopy (LP-EM) over the last decades have made it possible to combine in-situ observation of material formation with high-resolution chemical information. However, the interaction of electrons with a liquid layer generates a complex sequence of chemical reactions (radiolysis) that influence the reaction. The use of graphene as encapsulating material has become a way to reduce this problem. Finding a robust protocol for the formation of graphene liquid cells (GLCs) still remains a challenge. Our collaboration with VitroTEM, a company that has automated the graphene encapsulating process, aims to make GLC formation more accessible and reliable.

We are developing a novel workflow that combines the advantages of correlative cryogenic light electron microscopy (cryo-CLEM) with LP-EM. This cryo-/LP-CLEM workflow allows for the observation of material formation processes in high resolution by using graphene as window material. It combines the benefits of light and electron microscopy both in vitrified and wet state to be able to image at predetermined time and space.

Team members:
Luco Rutten,  Ben Joosten

Data Analysis of Correlative 3D Imaging

To analyze and visualize the 3D imaging data that are generated by correlative imaging techniques, we use a combination of Matlab scripts and ORS Dragonfly.  An internally developed Matlab-based app is used to process cryo-EM stacks and overlay light and electron microscopy data. Using ORS Dragonly, 3D EM stacks are visualized and machine learning/deep learning models for segmentation are developed. After segmentation, more quantitative analysis can be performed, to further analyze the datasets. 

Team members:
Deniz Daviran, Judith Schaart

Correlative Raman and Electron Microscopy

Correlative Raman and 3D FIB-SEM

Raman microscopy is an optical imaging tool, which provides spectroscopic information on the chemical composition of materials. This method works very similar to fluorescence microscopy, but instead of recording fluorescent labels that are added to the sample, it directly detects the presence of molecules based on their specific interaction with light. In other words, each molecule is its own label! Raman microscopy is able to provide details on the type of proteins, minerals or other components that are present in a tissue, as well as on the 3D structure of the samples. Similar to fluorescence microscopy, the resolution of Raman imaging is limited by the wavelength of light and cannot reveal details of the tissue's ultrastructure.

Ultrastructural details can be provided by high-resolution electron microscopy, where images are generated through the interaction of a material with electrons, to obtain resolutions down to the (sub)nanometer level. We work on correlating Raman and electron microscopy, to superimpose the chemical information provided by Raman microscopy on the 3D structural information from electron microscopy. This combination of imaging techniques will give us new insights into the relation between the composition and structure of (developing) tissues, and lead to better insight into tissue formation, disease progression and eventually lead to better treatment possibilities.

Team members:
Robin van der Meijden, Rona Roverts

Correlative Raman Microscopy and Cryo-TEM 

Cryo-TEM (transmission electron microscopy), assisted by electron diffraction, energy-dispersive X-ray spectroscopy, and electron energy-loss spectroscopy, can determine the evolution of morphology, structure, and chemistry of near-native hydrated samples with subnanometer spatial resolution and second time resolution. Raman microscopy can provide a structural fingerprint to identify organic chemicals and inorganic minerals with spatial resolution (∼1−10 μm). Cryo-Raman can analyze microsized sites in a frozen hydrated sample with a native state and reasonable physiological concentration, unlike the chemical environment change caused by the drying method and high concentrations of chemicals for in-situ liquid samples. Furthermore, cryo-Raman can also identify the organic molecules, which is not easily achieved by cryo-TEM

In this project, we will utilize correlative cryo-TEM-Raman to gather chemical, structure, and morphology of the same regions of samples with the special Finder grid, which can finally enhance our understanding of the interaction between inorganic minerals and organic molecules.

Team members:
Chenglong Li, Robin van der Meijden