Fluorescence Microscopy

Fluorescence microscopy allows to image living cells with high spatial and temporal resolution. The core facility is equipped with a confocal and a widefield microscope. The LSM880 Zeiss confocal microscope, besides imaging of optical sections (z stacks) and time lapse measurements (up to several days), allows to acquire spectrally resolved images. The Nikon Ti-E widefield microscope, equipped with a highly sensitive EMCCD camera, is used for automatic multi-position imaging on multi-well plates. Cage incubators are available on both instruments allowing image acquisition at physiologically relevant temperatures.

The microscopes can be used, together with experienced staff, also for measuring protein-protein interactions and protein diffusion with advanced imaging approaches such as Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS). Our instruments are optimized to measure FRET with several approaches: acceptor photobleaching FRET (also in automatic way in multi-well plates), ratiometric FRET (both at the single cell and population level) and fluorescence lifetime imaging FRET.

Several groups in the institute are equipped with both basic and advanced microscopes. The list and the contact persons are available under the section “Microscopes in research groups”.

Equipment

Nikon Ti-E inverted widefield fluorescence microscope
Xcite exacte ultrastable fluorescence light source. 515 nm, 532 nm, and 594 nm laser lines (dedicated to acceptor photobleachhing FRET).
Perfect Focus System. NIS elements and Nikon Jobs software for automatic image acquisition and analysis. Andor iXON897 EMCCD camera.
Incubator (OkoLab)

Zeiss LSM880 Laser Scanning Confocal Microscope
Laser lines (CW): 440nm, 457 nm, 488 nm, 514 nm, 543 nm, 594 nm, 633 nm. Laser lines (pulsed): 440 nm, 488-640 nm (tunable laser).
Spectral detector: 32 channels GaAsP PMTs and 2 MA-PMTs.
Definite Focus. Fluorescence lifetime imaging microscopy module (ISS).
Incubator (PeCon).

 

Applications

  • Confocal imaging
  • Automatic imaging in multi-well plates
  • Spectral Imaging
  • Acceptor photobleaching FRET
  • Ratiometric FRET
  • Fluorescence lifetime imaging microscopy
  • Fluorescence correlation and cross-correlation spectroscopy for measurement of protein diffusion and protein-protein interactions
  • Photon Counting Histogram
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