Academic Embedding and Complemetary Expertise

LCAM-FNWI is embedded within the section of Molecular Cytology and Swammerdam Institute for Life Sciences. In this way cell biology (cell culturing, ML-1 & ML-2 labs), molecular biology (cloning, transfection) and image processing (Huijgens, Metamorph, Image J, Matlab, Mathematica etc) are all standard and available for LCAM users. Because the Molecular Cytology group works on optimization of GFP variants and fluorescent sensors, there is extensive spectroscopic and probe expertise on site as well.

LCAM-AMC is embedded in two research groups of the Department of Cell Biology and Histology: Protein Degradation and Aggregation (Eric A.Reits) and DNA Double-strand Break Repair Mechanisms (Przemek Krawczyk and Jan Stap). Acquisition of new equipment and developments are preferably research driven and motivated by research questions that are formulated by these groups. The LCAM also profits from expertise in these groups.

LCAM-NKI consists of the NKI Digital Microscopy Facility, the NKI Robotics and Screening Facility and the Advanced Imaging and Biophysics group (Jalink lab). Embedded in the NKI research institute with cell culturing (ML-1, ML-2 and lentivirus-labs), and full GMO-mouse facility. Large (>25 FTE) local community involved in data analysis and image analysis. In-house construct sequencing facility, full-genome
sequencing facility, peptide synthesis facility, protein production facility, instrument-makers workshop, chemical compound libraries and chemical synthesis group (Dr Ovaa). The NKI also houses the group of Prof. van Herk, one of the world leaders in clinical image analysis and instrumentation.

Speciality of LCAM
Scientists visit LCAM because of its large experience and critical mass on advanced functional live cell microscopy (FRET, FLIM, FCS, FRAP) and its full integration in molecular cell biology & signal transduction.
The uniqueness is that LCAM covers all advanced modes of detection, probe and (FRET) biosensor development, signaling applications, spectroscopy background, hardware adaptations and image processing that is required for these forms of advanced microscopy. A number of techniques were also introduced worldwide by LCAM: Controlled Light Exposure Microscopy (CLEM), Gradual Acceptor Bleaching FRET, TIRF-FRET-FLIM, spinning disk frequency domain FLIM, quadruple color in vivo FCCS, triple lifetime unmixing of spectral identical molecules, algorithms for e.g. confocal FRET (sensitized emission), extremely sensitive instrumentation to detect FRET, and several of the most widely used FRET sensors.
Other highlights are our expertise for Long Time Live Cell Imaging (+120 hr); Correlative Light and Electron Microscopy; UV-A and X-ray Irradiation of Cells; FRAP; Image Analysis and Deconvolution; Cell Sorting. We further mention: Development of super fluorescent proteins (Goedhart/Gadella), FRET sensor technology (K. Jalink, drs J. Klarenbeek, Goedhart & Gadella): we made the brightest fluorescent protein on the planet, our PIP2 sensor was the first FRET sensor for lipids, our recent Epac-based cAMP sensors are the best available by far; Super resolution labeling innovations (dr K. Jalink, prof E. Manders, 2
aio’s); Development of live cell PALM (Crosby, Gadella, Hoebe, Jalink, Manders), Development of live cell fluctuation microscopy (dr Hink). Development of new FRET-FLIM technology (Gadella/Jalink); Controlled light exposure microscopy (E. Manders in collaboration with R.A. Hoebe (AMC), Development of hard- and software for high-content and high-throughput screening microscopy (K. Jalink, dr B. vd Broek, Drs K. Kedziora, 4 programmers); Unified live-cell system for high-throughput, local induction of DNA damage by multiple types of radiations and imaging of ensuing cellular responses
(by J.A. Aten, P. Krawczyk, J. Stap, R.A. Hoebe, C.H. van Oven, J. Verhoeven). Our confocal FRET approach is state of the art and implemented in Leica’s software suite.

User Community
All groups within SILS at the UvA, AMC and NKI use our facilities, and several researchers from outside our institute visit us for experiments. The microscopes can be booked via the internet. The external usage of the microscopes amounts to > 30,000 h/year (> 50%). The expertise covered ranges from mammalian cell biology, yeast genetics and cell biology, zebra fish development work, plant physiology, plant pathology, mouse cancer models (mice) to crystallography and enzymology. Clinical and preclinical groups are also served.

Visiting scientists ~ 50/year, several from abroad. Mostly using FLIM-FRET, Controlled Light Exposure Microscopy, Advanced FCCS, or FRET experiments. All facilities are open access. After a basic introduction course, frequent users are allowed to operate advanced microscope unsupervised. For highly advanced instrumentation technical support staff is available stand-by. All instrumentation is available without costs. Heavy-duty users occasionally share maintenance costs. After an intake meeting new users get advice about experimental set-up, cell culture, etc, starting a pilot experiment, technical, instrument related assistance, help with image restoration and reconstruction, support for analysis, and instructions for laser- and bio-safety.





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