Location: De Sitterzaal/ Oortgebouw
Time: 16:00 hrs
abstract
With correlative microscopy light and electron microscopy methods are combined, allowing a strong synergy between these two kinds of microscopy in the study of cellular processes. Light microscopy (LM)
enables rapid searching for regions of interest in relatively large fields of view with a few 100s of nm resolution exploiting the availability of a wide range of markers. Electron microscopy (EM) exhibits a resolution better than 5 nm over narrow fields of view (several micrometers). Recently, there is a surge of interest in use of these two complementary techniques on one sample. With 3D electron tomography on stained sections of several 100s of nm thick a wealth of information on the 3D cellular architecture of
cells and tissue can be obtained. The staining of membranes and other structures enables the clear imaging of cell morphology and the identification of a great variety cell structures. The fidelity of
interpretation at a molecular resolution is limited because of effects related to chemical fixation, freezesubstitution and/or metal staining during the specimen preparation steps. An alternative specimen preparation approach is to rapidly freeze the samples, This frozen, vitrified samples can be observed with cryo electron microscopy at liquid nitrogen temperatures. The vitrification preserves the molecular structures. In addition, the fluorescence is not quenched, as is the case when metal stains are used
during specimen preparation. Therefore, it would be attractive to apply cryotomography on thin areas of cells, or vitrified sections of cells, to reveal details with a similar resolution as with stained and resinembedded
samples, but without the limitations of stain. Unfortunately, the low contrast and dose sensitivity of vitrified objects poses a practical hurdle to identify areas of interest within the cellular context, thus limiting the range of cell biology applications. To overcome this hurdle, fluorescent
labeling and imaging could be instrumental to identify cellular areas of interest on which cryo electron tomography could be applied. The development of biochemical tools, in combination with dedicated
hardware and software, that will assist researchers to combine different imaging modalities in an effective manner will become an increasingly important issue for correlative microscopy in the coming
few years.An overview will be given of ongoing method developments and applications in the electron microscopy laboratory in our laboratory at the LUMC in Leiden.