Correlative microscopy

Abstract

In recent years correlative microscopy, combining the power and advantages of different imaging system, e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all the possible combinations of techniques, light and electron microscopy, have made an especially big step forward and are being implemented in more and more research labs. Electron microscopy profits from the high spatial resolution, the direct recognition of the cellular ultrastructure and identification of the organelles. It, however, has two severe limitations: the restricted field of view and the fact that no live imaging can be done. On the other hand light microscopy has the advantage of live imaging, following a fluorescently tagged molecule in real time and at lower magnifications the large field of view facilitates the identification and location of sparse individual cells in a large context, e.g., tissue. The combination of these two imaging techniques appears to be a valuable approach to dissect biological events at a submicrometer level. Light microscopy can be used to follow a labelled protein of interest, or a visible organelle such as mitochondria, in time, then the sample is fixed and the exactly same region is investigated by electron microscopy. The time resolution is dependent on the speed of penetration and fixation when chemical fixatives are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either transfections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence labels in cells on a large section to reduce searching time in the electron microscope. Multiple fluorescence labelling of a series of sections can be correlated with the ultrastructure of the individual sections to get 3D information of the distribution of the marked proteins: array tomography. More and more efforts are put in either converting a fluorescence label into an electron dense product or preserving the fluorescence throughout preparation for the electron microscopy. Here, we will review successful protocols and where possible try to extract common features to better understand the importance of the individual steps in the preparation. Further the new instruments and software, intended to ease correlative light and electron microscopy, are discussed. Last but not least we will detail the approach we have chosen for correlative microscopy.

Influenza A virus ribonucleoproteins modulate host recycling by competing with Rab11 effectors

“…It is widely recognized that the replacement of water with resin, during sample processing for electron microscopy, induces cell deformations. Therefore, a perfect superimposition of confocal and electron microscopy images is simply not attainable (Loussert Fonta and Humbel, 2015). Furthermore, because our protocol required epon resin, inclusion of fiduciary landmarks to help alignment was not feasible.…”

Multi-color electron microscopy by element-guided identification of cells, organelles and molecules

“…Fluorescence microscopy aids to identify biomolecules2345, but lacks structural context. Correlated light microscopy and EM (CLEM)67 allows fluorescence-guided analysis of EM data, but fluorescence retention during EM sample preparation and overlay of images differing order-of-magnitude in resolution may be technically challenging67. In search for a broadly implementable technique to define molecules, organelles and cells at high resolution within mammalian tissue, we decided to implement element-guided identification using energy dispersive X-ray analysis (EDX).…”

Transfer of Anti-Rotavirus Antibodies during Pregnancy and in Milk Following Maternal Vaccination with a Herpes Simplex Virus Type-1 Amplicon Vector

“…It has been reported that RV can become systemic leaving the intestinal route and emerging into the blood [24,25]. As the strain EDIM cannot be propagated in vitro [26], the viral load in the sera of pups was analyzed using a RV specific quantitative real-time polymerase chain reaction (RT-qPCR).…”