Molecular Cell Biology 4

Microscopic imaging and technology (5.03.04)

Principal investigators

Prof. Dr H.J. Tanke, Prof. Dr A.J. Koster, Dr K. Szuhai, Dr P. Corstjens, Dr J. Vrolijk, Dr R.W. Dirks, Dr C.R. Jost, Dr. Ir. F.G.A. Faas, Dr. M. Barcena

Aim and focus

The aim of this research program is to develop and/or adapt microscopic imaging methods, instrumentation and fluorescent labeling technologies to study the molecular composition of tissues, cells and subcellular compartments. The program involves both light and electron microscopy and supports projects within the realm of both cell and structural biology.

Examples include the use of two-photon or confocal laser scanning microscopy to study molecular dynamics in living cells using FRET/FRAP techniques, time-lapse fluorescence imaging of living cells, two-photon intravital microscopy of small laboratory animals, digital fluorescence workstations to perform 48 colour FISH karyotyping in case of malignancies and pre/postnatal abnormalities. For high-resolution (nm-scale) ultrastructural studies, the core electron microscopic imaging tools include (cryo) transmission electron microscopy (TEM) with energy-filtering, (cryo) scanning electron microscopy (SEM) and electron tomography for the 3D imaging of cellular organelles and macromolecules. Ongoing technical developments aim at improving specimen preparation and labeling techniques and advancing further automation of methodologies. A key focus is correlative light-electron microscopy, which allows the combination of high-resolution imaging by electron microscopy with live-cell dynamic imaging by fluorescence light microscopy. In addition, the group develops new fluorescent reporter molecules (such as luminescent crystals that are used in point-of care-tests using lab-on-a-chip devices), cytochemical staining procedures and dedicated software for cell and structural analysis. Applications of microscopic imaging and technology range from fundamental cell biological studies, imaging the cellular ultrastructure and/or dynamics with electron microscopy or live cell microscopy to diagnostic applications of multi-colour FISH and fluorescence reporter technology in case of oncology, genetics and infectious diseases. 

Position in international context

The group has an international reputation due to its pioneering role in the development of multicolour FISH techniques, fluorescence microscopy and microscopy automation, cryo-electron tomography and correlative microscopy. Consequently, the group has organized many EMBO workshops (on FISH technology), Marie Curie courses (on genomic profiling), EMBO courses (on electron tomography) and was part of the 3D EM European Network of Excellence (LSHG-CT-2004-502828). In addition, the Section electron microscopy participates in the European Euro-Bioimaging initiative aimed organizing open-access microscopy infrastructure on a European level. This section is also associated with Instruct, a European ESFRI program aimed at organizing access to structural biology imaging infrastructure. In March 2012 the electron microscopy section will organize the next Instruct meeting in Leiden.

Content / highlights / achievements

  • Discovery of a new role of the NFATc2 gene in a Ewing sarcoma variant (with Department of Pathology)
  • Development of lab-on-a-chip systems for point of care (POC) diagnosis
  • An intravital multi-photon microscopy system has been made available for investigation of small laboratory animals and is currently used by several LUMC groups.
  • Room-temperature dual-axis tilt electron tomography of cryo-immobilized SARS-virus infected vero cells (with Department of Virology);
  • Construction of a light-microscope system enabling fluorescence imaging of cryofixed biological specimens prior to observation with cryo-electron microscopy (e.g. von Willebrand Factor with the Thrombosis and Hemostasis group).
  • Insight in the structure and replication of coronavirussen (see Knoops et al, and Barcena et al)); (collaboration with Department of Virology)
  • In 2010 a patent on an integrated light-electron microscope of which Bram Koster is one of the inventors was filed for the American market. A prototype of that instrument was installed in 2010 in the section EM.
  • Application of COBRA technology in a study by the Medema group (Utrecht) showing that segregation errors can cause translocations and whole-chromosome instability in tumorigenesis (jointly published in Science 2011 30; 1895-8).

Future themes

Several approaches to combine fluorescence light microscopy with high-resolution cryo-electron tomography are in development, focusing on identifying and imaging molecular assemblies in situ without chemical or gold labeling pretreatments. The group has been involved in the development of a prototype “integrated Light and Electron Microscope-iLEM” platform (to be produced by the company FEI). Last year MCB has acquired a CW-STED microscope to explore the utility of super-resolution light microscopy for the LUMC. 

Cohesion within LUMC

The light and electron microscopy facilities are extensively used for collaborative projects within the LUMC. Important themes of collaborative research are genetics, pathology, oncology, hematology and infectious diseases.