In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells.
Confocal laser scanning microscopy and focused ion beam scanning electron microscopy are two established techniques to obtain three‐dimensional microscopic data of specimen. Since they are based on different interaction mechanism of photons or electrons, they provide complementary information on function and morphology of a sample. Their combination is desirable but difficult since in general sample preparation protocols are different or even exclude each other. We present a workflow for suspension cells, which includes a sample preparation to comply with both imaging methods. Furthermore, a microstructured reference coordinate system is imprinted at the specimen's surface, which is visible for both instruments and serves as a common reference. The specimen is mounted and oriented on a three‐dimensional sample holder to conveniently transfer the sample between both instruments. confocal laser scanning microscopy is used to identify and image target regions, which are revisited and imaged with the electron microscope afterwards. We successfully employed the workflow for virus compartments, formed in HIV‐pulsed cells. The preservation of virus fluorescence during preparation allowed to identify compartments with confocal laser scanning microscopy and to obtain target coordinates with micrometre precision. This allowed accurate guidance of the focused ion beam scanning electron microscopy to resolve ultrastructural details of the compartment.
Keywords: Confocal laser scanning microscopy; correlative light and electron microscopy; fluorescence preservation of embedded samples; focused ion beam; targeted FIB.
© 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.