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Multi-mode confocal laser scanning microscope


The multi-mode confocal laser scanning microscope (MM-CLSM) is a Leica SP5X-SMD high-end fluorescence microscope. It combines confocal imaging with single molecule detection (SMD) and advanced (time resolved) fluorescence spectroscopy.

The following information can be obtained using this MM-CLSM.

(Co)-localisation: Specific fluorescent probes (chemical dyes or fluorescent proteins technology) allow temporal and spatial colocalisation of (bio)molecules of interest. A range of fluorescent probes can be combined for simultaneous multicolor, multidimensional image acquisition. Specific applications like time-laps imaging or fluorescence recovery after photobleaching (FRAP) can be performed.

3D reconstruction: Optical sectioning (nm resolution) can be achieved using the piezo stage of the confocal microscope, allowing for 3D-image reconstruction of (bio)molecules, organelles and tissues. 3D-image reconstruction can also be performed using simultaneous multi-color image acquisition.

Dynamics using fluorescence fluctuation spectroscopy (FFS): The MM-CLSM has a 'Single Molecule Detection' module that allows for FFS. With FFS, quantification of biomolecules either in vitro or in vivo is possible. Furthermore, in dilute solutions, the diffusion coefficient of a biomolecule can be determined. The interaction between differently labeled molecules can be detected by applying fluorescence crosscorrelation spectroscopy (FCCS).

Fluorescence lifetime imaging microscopy (FLIM): The main feature of the MM-CLSM is the FLIM option. FLIM is a unique and robust technique for the detection of protein interactions in living cells. The FLIM set-up allows for simultaneously monitoring multiple fluorescence lifetimes.

FFS-FLIM: The combination of FFS and fluorescence lifetime acquisition can be used for the detection of interacting molecules at single molecule level. This technique is mostly applicable for in vitro studies.

Technical Details

This MM-CLSM has a unique configuration including:

  • Variable excitation with a supercontinuum tunable white light laser (WLL) source.
  • WLL confocal imaging using up to eight excitation wavelengths simultaneously.
  • WLL tunable pulsed excitation with two wavelengths simultaneously for FLIM applications.
  • Confocal microscope using hybrid detector technology allows for complete, filter free, spectral freedom in imaging.
  • Confocal microscope and time resolved SMD is controlled by the confocal software platform.
  • Automatic 3D-FLIM acquisition using a third parameter like wavelength, time or volume.
  • FLIM acquisition having time resolution (TTS) of 100 ps or better and short detector dead time in combination with a high dynamic range.

Next to the supercontinuum laser, this microscope is also equipped with “standard” argon laser (458, 477, 488, 514 nm) and a pulsed diode laser (440 nm). The microscope operates extreme flexible by the incorporation of an AOBS, AOTF and a 5 channel filter free, spectral HyD detectors.


The MM-CLSM can be used in different research projects varying from signal transduction pathways in planta to protein folding aspects or detection of fast dynamics in photosynthesis. Furthermore, specific applications such as protein interactions (in vitro or in vivo), signal transduction, food processing, single molecule biophysical studies and colloid chemistry are possible.

Complementary Techniques


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Agrotechnology and Food Sciences



Last edited by Oscar de Vos on 2016-05-25