STW Microscopy valley

microscopy valley projects

1    Super Resolution Optical Microscopy integrated in a TEM

2    Live‐cell imaging with integrated in‐situ scanning electron and 3D confocal microscopy

3    3-D Integrated CLEM by CLSM-FIB-SEM

4    Integrated Raman-FIB-SEM

5    Cryo-electron tomography with cryo-optical super resolution: New tools to see

      nanomachines in cells

6    Novel genetic biosensors for CLEM

1    Super Resolution Optical Microscopy integrated in a TEM

In this project we will develop and apply methods and techniques to combine high resolution resolution light microscopy with transmission electron microscopy (TEM).  Three groups will work closely together focusing on different aspects. The Koster team will focus on the development of super-resolution correlative microscopy approaches in general, and their implementation and optimization for integrated light and transmission electron microscopy in particular. For this part of the project 1 PhD position and 1 post-doc position (1 year) is available. The Gerritsen team  will focus on the technical realization and characterization of a concise super-resolution optical system that will be built in a TEM and that is based on a the commercial iCorr module by FEI company. For this part of the project 1 PhD position is available. The Klumperman team will focus on the development of optimal specimen preparation procedures for integrated light and electron microscopy for the Koster and Gerritsen teams. For this position, 1 technical assistant (2 years) position is available.

Project leader    Prof.dr.ir. A.J. Koster, Leiden University Medical Center, Molecular Cell Biology
                          a.j.koster@lumc.nl,  https://electronmicroscopy.lumc.nl

Collaborators    Prof.dr. Hans C. Gerritsen, Physics Department, Utrecht University
                         Prof.dr. Judith Klumperman, Cell Biology Dept. Utrecht Medical Center

Positions           2x PhD student, 1x Postdoc, 1x Technician

2    Live‐cell imaging with integrated in‐situ scanning electron

      and 3D confocal microscopy

In this project we will integrate 3D confocal fluorescence microscopy in a scanning electron microscope. The confocal microscope allows for imaging dynamic behavior in live cells contained in a small microfluidic sample holder with electron and light transparent windows. The scanning electron microscope can then be used at any designated moment following live-cell observations.

Project leader     Dr. Ir. J P. Hoogenboom, Delft University, Department of Imaging Science and Technology
                            j.p.hoogenboom@tudelft.nl, http://www.tnw.tudelft.nl/en/

Positions            1x PhD student, 1x Technician

3    3-D Integrated CLEM by CLSM-FIB-SEM

This project is focused on the development of 3-D correlative microscopy using a dual beam system. The commercial dual beam is capable of producing 3-D high resolution images by repetitively removing thin layers by ion-beam milling and recording SEM images (slice and view). Here, a confocal fluorescence microscope will be integrated in the dual beam system to rapidly locate regions of interest in 3-D. This will significantly speedup the 3-D correlative imaging workflow.

Project leader     Prof.dr. Hans C. Gerritsen, Utrecht University, Physics Department
                           H.C.Gerritsen@uu.nl,  UtrechtBiophysics.nl

Collaborators      Prof.dr. Judith Klumperman, Cell Biology Dept. Utrecht Medical Center
                           Dr. W.H. Müller, Biology Dept, Science Faculty, Utrecht University

Positions             2x PhD student, 1x Postdoc, 1x Technician

4    Integrated Raman-FIB-SEM

Scanning electron microscopy (SEM) and focussed ion beam (FIB) enable visualization and material processing in repeatable cycles of operation. Inclusion of a Raman micro-spectrometer in a FIB-SEM system enables detailed optical spectroscopic material analysis at selected moments during material processing. The integration of a compact and modular Raman system with a FIB-SEM from the company FEI in the MESA+ institute at the University of Twente will be prepared. Raman imaging in FIB-SEM will be developed. An aim will be to improve spatial resolution to decrease the resolution gap between FIB-SEM and optical microscopy. This new analytical platform will explore diverse applications in material - and biological sciences. Applications may range from carbon materials and catalysis to plasmonic materials and may also include work on biological cells in interaction with materials. The analysis of multimodal labels, i.e. labels suitable for both optical and electronic tracking, will be of immediate general interest for biological applications. The work will be partially performed in collaboration with program partners. HybriScan Technologies b.v., a Dutch company in integrated SEM & Raman spectroscopy, is supporting this project.

Project leader     Dr. C. Otto, University of Twente, Applied Physics, Medical Cell Biophysics
                            c.otto@utwente.nl, http://www.utwente.nl/tnw/mcbp/

Collaboratror      Prof. dr. L.W.M.M. Terstappen

Positions            1x PhD student, 1x Postdoc, 1x Technician

5    Cryo-electron tomography with cryo-optical super resolution: New tools to see

      nanomachines in cells

We aim to develop correlative microscopy techniques based on cryo-super resolution light and cryo-electron tomography microscopy for visualizing nanomachines in thin cryo-samples. A cryo super-resolution light microscopy will be setup to image vitrified 200nm thick cells. Photo-activatable fluorescent proteins will be employed to image with super resolution a variety of cellular structures (<50 nm lateral and <100 nm axial optical resolution). Here, the FEI CorrSight and the ‘MAPS’ software will be used to navigate between cryo-LM and cryo EM images.

In addition the recently acquired Krios Titan cryo-TEM at the NeCEN (www.necen.nl) will be used for correlative imaging and tomography of subcellular sites.  Novel image processing software will be employed for improved detection and analysis of ‘tagged’ nanomachines in the crowded cellular environment of immune cells and bacteria. Both biochemical and genetic approaches will be combined with high-resolution cryo LM and cryo-ET to image the 3D structure of a new type VII secretion system that we are investigating (van der Wel, Cell 2007; Sani, PLoS Pathog 2010; Abdallah, J Immunol. 2011; Houben, Cell Microbiol. 2012) .

Project leader    Prof.dr. P.J. Peters, Netherlands Cancer Institute
                          P.Peters@NKI.nl, www.nki.nl/research/peters,  www.necen.nl,  www.kavli.tudelft.nl

Collaborator       Dr. K. Jalink (NKI‐AVL), Netherlands Cancer Institute

Position              1x PhD student

6    Novel genetic biosensors for CLEM

We combine electron microscopy, including nanotomy, with the fluorescent toolbox [PDF] and develop and implement probes for
correlative microscopy in clinical diagnosis and cell biology. In microscopy valley, emphasis will be an further development and application of probes,  both genetically-encoded as well as affinity-based, for use in systems developed in the other Microscopy Valley projects, as well as generic use.  Application will be in diverse biomedical projects, ranging from fundamental cell biology to research in Type 1 Diabetes.

Project leader     Dr. B.N.G. Giepmans, University Medical Center Groningen, Dept. of Cell Biology
                           b.n.g.giepmans@umcg.nl, www.cellbiology.nl

Collaborator       Dr. Ir. J P. Hoogenboom, Delft University, Department of Imaging Science and Technology

Positions             1x PhD student, 1x Technician