ExpertiseIn-house light microscopy expertise and applications. Applications not mentioned might be still possible. Please contact us for more information.
Expertise
In-house light microscopy expertise and applications. Applications not mentioned might be still possible. Please contact us for more information.
Reservation of the instruments occurs through CFMS. Manuals, SOPs and background information can be accessed on the sharepoint site after an access request.
Phase contrast (contrast enhancement method for transmitted light microscopy)
Dark Field
Epi-fluorescence microscopy
Wide field fluorescence microscopy
Confocal fluorescence microscopy
Confocal reflection microscopy
A variety of samples can be used such as fixed or living cells and tissues
Live cell imaging
Several of the microscopes are equipped with a sample heater and CO2 incubator for maintaining optimal conditions for living cells. The spinning disk microscopy or confocal imaging with hybrid detectors allow for gentle illumination schemes. The STED nanoscope can use adaptive illumination schemes to minimize light exposure.
Automated microscopy and kinetic analysis
Multiple images are acquired (and combined) to generate large datasets such as a large field of view or a kinetic timetrace. Imaging coordinates, settings and analysis can be predefined for automation. This can be used to generate images of e.g. a large area, to acquire data of multi-wellplates or tho follow kinetics of the sample.
Co-localization studies
To investigate the presence of two or more molecules in the same area, a co-localization study can be performed. Here each compound is labelled with a different fluorophore and a multicolour image is recorded. Each channel represents one compound. By overlaying the different channels, the co-localization of the compounds within an area limited by the diffraction limit is investigated and can be quantified statistically.
Spectral imaging and unmixing
In spectral imaging, the emission light is split in its components generating a set of images each covering a spectral band. By assembling the intensities of the set of images, a spectral image is reconstructed and for each pixel in the image a spectral signature is known. By unmixing the signals based on reference spectrum, overlapping emission spectra can be separated and the contribution of each spectrum in each pixels is calculated. This technique can be used to amongst others separate fluorescent signals from probes with strongly overlapping emission spectra or strong background signals such as auto-fluorescence.
Deeper light penetration for imaging thick(er) samples:Two-photon microscopy
Two-photon microscopy is an alternative to confocal microscopy for optical sectioning and limiting out-of-focus light. In multi-photon microscopy, molecules are excited with light of a long wavelength (half the energy needed for one photon excitation) and need to absorb multiple photons simultaneously. These events can only occur at high excitation intensities which can be reached in the focal volume of a pulsed laser, thereby eliminating out-of-focus excitation and fluorescence. Two photon microscopy is advantageous for optical sectioning in thick samples.
Fluorescence lifetime imaging microscopy (FLIM)
In fluorescence lifetime imaging microscopy (FLIM) the fluorescence lifetime is used instead of the spectral property of fluorescence. Fluorescence lifetime is a measure of how long a fluorescent molecule or fluorophore remains in its excited state before returning to the ground state by emitting a fluorescence photon. The decay of a fluorophore does not always occurs at exact the same time after excitation but a distribution of decay times is observed resulting in a characteristic time constant. This decay is dependent on the fluorophore and is highly sensitivity to the molecular environment and changes in molecular conformation. Flim can therefore be used as an alternative to separate fluorophores, to study cellular metabolism using autofluorescent molecular imaging, to monitor microenvironmental parameters (such as temperature, viscosity, pH, and ion concentration), using FLIM-based sensors and to study protein–protein interactions using Förster resonance energy transfer (FRET) sensors.
A recent review for more information can be found here.
FRET is a photophysical process where energy from one fluorophore (donor) is transferred to a second fluorophore (acceptor). The efficiency of this process dependents among others on the distance between the two fluorophores. Therefore by determination of the FRET efficiency, information about the (relative) distance between the two fluorophores can be obtain. Based on this principle, many fluorescence indicators are developed to record cellular processes.
Calcium imaging
Calcium signals, the (local) change in calcium concentration, plays an important role in many biological processes and accurate timing and location are crucial. By the use of fluorescence microscopy and a calcium indicator, changes in calcium concentrations can be recorded. To do this, an optimal choice of the wide range of indicators as well as fast and sensitive fluorescence imaging methods are crucial.
Diffusion studies
Fluorescence correlation spectroscopy: FCS
In this technique, fluorescent fluctuations caused by diffusion of fluorescent particles through the focal volume are recorded. Information such as diffusion coefficients, concentrations, co-diffusion can be determined by analysis of these fluctuations. In-house analysis software was developed to analyse encapsulation efficiency which can be applied in different biological fluids.
Fluorescence Recovery after Photobleaching: FRAP
Fluorescent molecules are photobleached in a region of interest (ROI). Due to diffusion of fluorescent particles into this ROI and diffusion of bleached particles out of the ROI, fluorescence in the ROI recovers over time. By analysis of the fluorescence recovery curves, the macroscopic diffusion can be explored. Diffusion coefficients can be determined making use of in-house analysis software.
Single particle tracking: SPT
By detection and localization of particles in consecutive frames, trajectories can be reconstructed describing the movement of single particles. Measurements can be both performed on our spinning disk microscope and nanosight. By analysing the trajectories, motional behaviour can be analysed. This information can be used for e.g. nanoparticle characterisation (diffusion coefficients, size distributions and concentrations) employing Nanoparticle Tracking Analysis (NTA) (NanoSight) or in-house analysis software .
Laser capture microdissection
Laser capture microdissection (LCM), also called microdissection, laser microdissection, or laser-assisted microdissection, is a method for isolating specific cells of interest from microscopic regions of tissue/cells/organisms (dissection on a microscopic scale with the help of a laser). LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. In LCM, the sample is first visualised by light microscopy to find a region of interest. This region of interest is than cut out of the sample using a powerful UV laser beam. Thereafter the dissected region is collected in a non-contact way to prevent any contamination by catapulting the sample using a UVa laser pulse for about 1 nanosecond minimizing heat transfer and ensuring gentle isolation. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling.
Advantages of laser capture microdissection
Obtaining pure cell populations without the risk of contamination
Genome, transcriptome and proteome analysis after isolation possible
Can be performed on different sample types such as snap frozen or formalin fixed paraffin-embedded tissue sections, cytospins, cell smears, chromosome preparations and live cells.
Can be combined with fluorescence staining to visualize particular cells or cell organelles of interest
Micromanipulation
The size of objects observed under the microscope are often in the range of several micrometers. Their small size limits fine manipulations of these objects. A micromanipulator allows manipulation with a precision on the sub micrometer scale, making it possible to perform among others microinjection and patch clamp experiments.
Microinjection
To deliver foreign material such as fluorescent dyes, proteins or DNA to a cell, microinjection can be used. Therefore a very sharp glass pipette is inserted into the cell and the materials are injected directly into the cell. The whole process is guided by observation under the microscope.
Combined patch clamp and epi-fluorescence imaging for single channel studies
Patch clamp is a method used in electrophysiology to record currents of a small set or a single ion channel. In the laboratory of prof. Luc Leybaert a microscopy setup is developed for recording epi-fluorescence signals of a single cell while performing a patch clamp experiment on a single channel. For more information we recommend to contact the staff.
Confocal imaging with simultaneous photo stimulation
Light irradiation can be applied to a region of interest while imaging a wider field of view. This can be used for confined stimulation of the sample with light as for example with applications using photoactivatable probes or uncaging dyes.
UV activation of caged compounds
Cages compounds are used when molecules such as second messengers, need to be released in a specific location, time and amplitude. A molecule is kept biologically inert by modification with a photo-removable protecting group. Irradiation of the sample with an uncaging beam results in the release of the trapped molecule, permitting targeted perturbation of a biological process. More information can be found in the following articles:
Decrock E., De Bock M., Wang N., Bol M., Gadicherla A.K., Leybaert L. (2015), Flash photolysis of caged IP3 to trigger intercellular Ca2+ waves. Cold Spring Harb Protoc, 3:289-92. doi: 10.1101/pdb.prot076570.
Leybaert L., Sanderson MJ. (2001), Intercellular calcium signaling and flash photolysis of caged compounds. A sensitive method to evaluate gap junctional coupling. Methods Mol Biol. 154:407-30.
Image analysis
The microscopy centre provides guidance for basic image analysis via FIJI. FIJI is open source free software for scientific image analysis which bundles plugins for various applications in the life sciences.
For projects requiring novel approaches in image processing, collaboration with IPI is possible. IPI, the Image Processing and Interpretation Research Group , performs state of the art research in the field of digital image and video processing for a wide range of applications such as:
Applications not mentioned in this list might be still possible on the instruments or one of our affiliated partners might be able to help you. Please contact us for more information.
