Equipment

Fluorescence is a powerful analytical tool that combines the magnifying properties of light microscopy with the visualisation of fluorescence to monitor live real-time protein interactions, at steady state or after cellular ligand challenge.

Laser confocal microscopes have different imaging modes (e.g. conventional confocal fluorescent intensity imaging vs fluorescence life time imaging) and can provide quantitative data to support your fluorescence research hypothesis.

Its important that you look at the specification of the microscope instrument you want to work with to ensure that you can optimally excite and image your fluorescent dye labels before commencing your fluorescence research work.

Example specifications of the Zeiss 880 confocal microscope based at the Cellular Analysis Facility and Leica SP8 confocal instrument situated in the Bower building are listed below.

 

Zeiss 880 LSCM

The system is equipped with a high-resolution galvo scanner and 3 channel QUASOR detector unit which is wavelenghth tuneable for custom spectral collection of emission light. The QUASOR consists of two side spectral green and red PMTs and a central GaAsP detector. A transmitted light PMT is available for bright field imaging. Images can be recorded up to a maximum 16 bit depth dynamic range. The red sensitive side PMT and GaAsP detector can also record fluorescence as a read out of photons which is more advantageous than analog integration read out because much higher signal-to noise-ratios can be obtained as a no secondary analog circuitry exists in the detector readout path. This system does not have an Airy Scan II 32 channel spatial detector fitted so has no super-resolution capability.

To use this instrument with training email the Cellular Analysis Facility: mvlssrf@glasgow.ac.uk / Anthony Dornan.  

If you need to perform confocal imaging with a super-resolution detector or other super-resolution modes (Single Molecule Localisation Microscopy-STORM/PALM, 3D Lattice SIM²) contact the Cellular Analysis Facility by email: mvlssrf@glasgow.ac.uk / Anthony Dornan.

 

Leica TCS SP8 LSCM

The confocal has two tuneable spectral Hybrid detectors and a spectral tuneable PMT detector. The HyDs can be operated in photon counting or conventional analog mode to record images up to 16 bit depth format. A dedicated transmitted light PMT is also available for acquiring images in bright field format. Pulsed lasers (440 & 470 nm, pulse frequency range 0.3125 to 40 MHz) can be used to perform fluorescence life time imaging including FRET measurement. This system does not have any confocal super-resolution capability.

To enquire about the use of this instrument with training to perform confocal or fluorescence life time imaging contact Prof. Michael Blatt by email: 

 

These instruments have supported our novel fluorescence work developed within the School of Molecular Biosciences:

-Spatial Intensity Distribution and Fluorescence Intensity Fluctuation Spectrometry analysis to quantify the monomeric or homo-oligomerisation state of receptor proteins tagged with mEGFP at their  C terminus.

-Intensity based Fluorescence Resonance Energy Transfer (FRET) imaging.

-Fluorescence Lifetime FRET imaging.

-Fluo-8 calcium imaging.

-FRAP measurement to quantify the diffusion rate and mobile state of fluorescently tagged proteins.