High Content Analysis

Perkin Elmer FLIM-Opera

The Opera is a new generation confocal microplate imager providing solutions for fully automated simultaneous high speed and high resolution screening. The system has environmental control and a fluorescence lifetime imaging (FLIM) capability.

The system can be used to perform selections based on cell morphology, protein-protein interactions, protein-localization etc.  The range of morphological alterations and biochemical processes we will be able to interrogate will be extremely broad due to the open architecture of the instrumentation and the use of novel biophysical approaches to imaging, including fluorescence lifetime imaging.

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High resolution screening

The water-immersion objectives of the Opera allow high resolution high-content screening. This is combined with confocal imaging, where the image degrading out of focus blur is blocked, meaning that the Opera has can achieve spatial resolution sufficient to perform screening of morphological changes in yeast organelles.

Yeast expressing targeted forms of eGFP and mRFP

A. Chameleon Ti:Saffire pulsed IR laser; B: frequency doubling unit; C: fast gated ICCD; D: picosecond delay unit.

FLIM-FRET screening

The FLIM-upgrade to the MBF Opera includes the addition of a pulsed violet-laser (IR-laser plus frequency doubler); fast gated intensified camera and integrated electronics.

Protein-protein interactions can be screened by exploiting fluorescence resonance energy transfer - FRET between donor and acceptor fluorophores conjugated to protein pairs on interest.

FLIM is the best way to quantify FRET and intermolecular FRET between two proteins on interest needs to be correlated with the acceptor concentration (i.e. intensity). The PE FLIM-Opera is the only truly integrated commercial system available that can perform high-resolution confocal steady state imaging as well as concurrent confocal FLIM.

4D screening

The inherent optical sectioning of a spinning disk confocal allows us to acquire fast z-series of the objects to be analysed. The Opera at MBF is upgraded with temperature, CO2 and humidity control in the imaging chamber. This allows acquisition of multi-dimensional times series with high temporal resolution in a single 384 well plate.

Lower temporal resolution with more plates can be achieved with a robotic incubator which is integrated to the Opera with a Twister robot. This allows multiple plates to be removed from the incubator, screened and placed back to continue their growth prior to re-screening.