The Netherlands Cancer Institute
Plesmanlaan 121 1066 CX Amsterdam
Dr. K. Jalink
0031 (0)20 512 1933
We use advanced microscopy and spectroscopy techniques to study cell signaling events and cytoskeletal dynamics with high spatial and temporal resolution. Our expertise is predominantly in advanced functional imaging and Super Resolution microscopy (STORM/GSDIM). Functional imaging techniques aim to provide information about the function of molecules, rather than just static images of their position within the cell. The most common techniques are Fluorescence Resonance Energy Transfer (FRET), Fluorescence Lifetime Imaging (FLIM), Fluorescence Cross Correlation Spectroscopy (FCCS) and opto-chemistry/opto-genetics. We also develop FRET hard- and software and biosensors for various intracellular messengers, and we develop and apply ‘super-resolution’ light microscopy. These techniques are used in research projects in our group as well as in collaborations within and outside our institute.
High-content screening and functional imaging
Over the last years, we have developed hard- and software to be able to perform microscopy screens at very high resolution: so-called high-content screens. For example, it is possible to screen for proteins involved in shaping the intermediate filament network by knockdown of hundreds of candidate genes, followed by automated analysis of the morphology of the filaments. We also apply functional imaging techniques in medium/high throughput format. Functional imaging approaches such as FRET have traditionally been hard to automate because the FRET signals are often very small and cell-to-cell variability has been significant. This precluded using FRET for screening applications. Two important improvements are now changing that. On the one hand, our longtime experience in constructing FRET sensors has enabled us to devise a generation of sensors that exhibit very robust FRET changes of up to 50%. On the other hand, our developments in wide-field as well as confocal FRET detection have boosted the speed and accuracy with which we can detect FRET changes in single cells (see further). With these changes, we are now able to reliably detect even small changes in cytosolic metabolite concentrations.
Wide-field high-speed FRET detection: single-image Fluorescence Lifetime Imaging Microscopy
FLIM records the fluorescence lifetime of a fluorophore, i.e. the average time that a fluorophore remains in the excited state following excitation. This intrinsically quantitative technique is used to detect the physicochemical properties of the molecular environment of the dye (e.g., pH, ionic strength, radical stress, oxygen levels and more) and to determine FRET efficiency in a robust manner. The conventional FLIM detection approach, Frequency Domain analysis, suffers from drawbacks in that it is slow, requires several (typically 12) individual fluorescent images to be taken in rapid succession, and it tends to produce artifacts when the preparation changes during acquisition (as is the case for living cells).
We published a new paradigm, single-image FLIM (siFLIM; Raspe et al, Nature Methods 2016) that uses a novel camera chip capable of recording two images that are phase-shifted by 180 degrees simultaneously. siFLIM quantitative but up to 30 times faster, it causes less photodamage to the cells and less bleaching to the dye.
Our lab collaborates with industry extensively, aiming to optimize microscopy-related hard and software for the most demanding applications. Exampels include Lambert Instruments (fast FLIM), Leica Mannheim (confocal developments), WillCo Wells (low-drift imaging chambers for STORM/GSDIM super-resolution imaging), OKO labs (microscope incubators) and Leica Wetzlar (Super-Resolution microscopy).
We are associated with the NKI Digital Microscopy Facility who offer access to a wide variety of advanced as well as routine microscopes, including several confocal microscopes, a scanning disk microscope, TIRF setup and wide-field fluorescence microscopes. Our group participates in NL-BioImaging AM (http://www.eurobioimaging.nl). Further information can be found here: http://JalinkLab.nl
- Biomedical & health
- Functional imaging microscopy
- High throughput microscopy
- Computer modelling
- Screen development
- Super resolution Microscopy (STORM/GSDIM)
- Cancer signaling
- Cell adhesion and migration
- GPCR signaling
Expertise and Track Record
The combination of techniques, offering the pipeline of assay development, customizable advanced imaging, routine screening, automated image analysis and advanced visualization is unique. We have a small but very knowledgeable staff that can often accomplish in weeks what would otherwise take many months.
will follow later
We specialize in combining functional microscopy with high-content screening. Our lab has collaborations with industry in developing hard- and software for this purpose.
Horizon 2020 Framework Programme PITN-GA-2014-642687 – BIOGEL
- Leica SP8-SMD, customized for fast FLIM and long-term culture. Hypoxia.
- Home-build frequency domain FLIM setup, 37 degrees, up to 20 FLIM images per second
- Home-build ratio-FRET setup (ultrasensitive)
- Zeiss/PTI ratio-FRET setup
- Leica GSDIM SR setup
- spectrofluorometer, PTI
- spectrometer, Ocean Optics
- plus 10 more instruments at the facility in our institute
We have EuroBioImaging flagship node status since 2014.
Open Science Framework