Other Facility Equipment
At the Cell Imaging Facility
The Facility is equipped with various up-to-date computer workstations for image processing. These include several high end workstations with powerful graphics and large amount of RAM and hard drive space dedicated for image processing and analysis. Here is a list of software tools available for the Facility users:
- from Media Cybergenetics
- from SVI
- SoftWoRx from API
3-D image processing and analysis software
- from Bitplane
- from PerkinElmer
2-D image processing and analysis and image acquisition software
- from Molecular devices
- Adobe Photoshop
- Zeiss Axiovision
Some of the computers are equipped with Zeiss LSM software (ZEN) for offline processing of confocal/FCS data.
Micro-injection is one of the techniques to introduce minute amount of substance into live cell/tissue. The Facility is equipped with an Eppondorff micro-injection system mounted on a Zeiss Axiovert 100M fluorescent microscope. It is suitable to inject small amount of substance (e.g., antibody, dye, drug, etc.) into adherent cultured cells. The system is also equipped with a cooled CCD camera (Sensicam) and Metamorph for fluorescence imaging.
Fluorescence is a cyclical process where lifetime of the fluorophore is not only the property of molecule itself but also a function of its environment. In situation such as Fluorescence Resonance Energy Transferring (FRET), the lifetime of donor molecule will be shortened. Therefore, FLIM can be used as a way to measure molecular interactions. Unlike conventional FRET analysis, FLIM measurement is not subjected to fluorescence photobleaching or concentration variations which are difficult to control in live cells.
The FLIM module is an attachment to the Zeiss NLO 510 system which uses time-correlated single photon counting technique to construct the decay curves of fluorophore in every pixel of the image. The system is equipped with a Hamamatsu RS-39 Multi-channel plate detector, a filter wheel and a SPC730 photon-counting board from for photon counting. The system uses a photodiode to obtain synchronization information from the laser pulses to construct the fluorescence decay curve.
Applications
The main application for the setup in biology is for FRET analysis where donor life time can be measured both in presence and absence of acceptor molecules. Then the FRET transfer efficiency can be calculated according to the following formula: -Et=1-tD,A/tD
Where tD,A is the life time of donor molecule in presence of acceptor and tD is the life time of the donor molecules in absence of acceptor.
The system is integrated part of multi-photon microscope. There is no special need for specimen preparation other than that the specimen must be suitable for confocal observation. As all FRET analysis, all control samples are needed (e.g. donor alone, acceptor alone, donor acceptor combined and specimen without any staining for auto-fluorescence).
The system does not have an overly user friendly interface, therefore it is only available by assisted use. Please contact staff for more details.
FCS is a spectroscopic technique for the study of molecular interactions in solution. FCS monitors the random motion of fluorescently labelled molecules inside a defined volume element irradiated by a focused laser beam. These fluctuations provide information on the rate of diffusion (diffusion time) of a particle and this, in turn, is directly dependent on the particle's mass. As a consequence, any increase in the mass of biomolecules, e.g., as a result of an interaction with a second molecule, is readily detected as an increase in the particle's diffusion time.
Applications
Because FCS measurement is made through diffraction limited volume, FCS can be used to study molecule-molecule interactions in living cell. The system can be used to study:
- Receptor-ligand interactions
- Transcription factor-DNA interactions
- Lipid-protein interactions, etc.
The FCS system at the Facility is an integrated part of the Multi-photon and the LSM710/Zeiss LSM NLO systems with all the laser lines for common fluorophores and 2 detectors which enable cross correlation analysis.
