Detection Techniques

3D High Content Screening

The Opera Phenix system enables for simultaneous image capturing of living cells/organisms in two different fluorescence channels by two separate camera systems. The confocal microscope allows to analyze localization of signals in 3D and provides laser mediated excitation for weak signals. The system set up allows to combine process control via the integrated plate reader with the confocal analysis at the most appropiate time point for detection. 

The system contains a plate handler, cell plate incubator, plate reader with injector (Envision) and the automated microscope. The microscope provides a climate chamber for life cell imaging. Water immersion objectives with up to 63x magnification enhance contrast and intensity of fluorescent labels.

2D High Content Screening

The Cellomics ArrayScan HCS Reader is an automated fluorescence microscopic imaging system for phenotypic screening.
The instrument software provides prebuilt and adjustable image analysis modules (BioApplications) that automatically convert image information into complex sets of numeric data. Up to six different fluorescent cell structures or GFP/YFP fusion proteins can be analyzed. The features reported for each cell include size, shape, amount of fluorescent label, and pattern of fluorescence. Each BioApplication reports cellular data at the individual cell level as well as at the whole well and subpopulation level.

ArrayScan Instruments

Our Unit currently provides three ArrayScan Instruments upgraded for actual standards of provider in 2014 by illumination with diodes, a camera with improved resolution and more robust robotic support to feed plates for analysis. One microscope has been equipped with an Apotome for optical sectioning and provides a live cell chamber for monitoring quantitative kinetic parameters and motility of cells. A brightfield illumination allows to analyse cell motility or morphology without labelling.

High Content Screening assay

High Content Screening assay set up requires essentially to spend sufficient time on proper selection of a cellular reporter system or selection of cellular structures for fluorescent dye mediated decoration. The set up and tuning of parameters for automated object identification (vesicles, filaments or fibers, membranes...) and stable transfer of image data into multi-parameter tables is a very important step in assay set up too. Too strong variations of signals and pixel sizes of structures may prevent usage of automated image analysis and may cause the user to analyze millions of images by eye. Therefore you should contact us already during assay development for support.

Flow Cytrometry (MACSQuant X, 384-well)

The MACSQuant X system provides three lasers for excitation of fluorescent labels from suspension cells. Forward and Sideward-Scatter allow to separate different cell types according to size even for the same surface marker signals. Up to 11 different parameters cam be monitored per probe. The flow-cytometer will be integrated into our FreedomEvo-Workstations for screening of large compound or RNAi libraries.

Label Free Impedance Detection (ACEA)

The xCELLigence system measures impedance-based signals both in label-free cellular analysis and in cell invasion/migration assays.
The Instrument provides real-time cellular analysis for short-term (hours) and long-term (days) assays using label-free, non-invasive impedance measuring. 

Applications include:

  • Compound-mediated and cell-mediated cytotoxicity
  • Cell adhesion and cell spreading
  • Cell proliferation and cell differentiation
  • Receptor-mediated signaling
  • Virus-mediated cytopathogenicity
  • Constant quality control of cells
  • Cell invasion and migration assays

xCELLigence RTCA Instruments monitor cellular responses continuously without exogenous labels by measuring electrical impedance. Microelectrodes integrated in the bottom of specially engineered tissue culture E-Plates measure changes and provide precise quantitative information about the status of cells, including cell number, cell adhesion, cell viability, and cell morphology. Continuous impedance monitoring of cellular response allows researchers to pinpoint important experimental time points for more detailed downstream assays.

Lab Chip 3000 & LabChip® EZ Reader

The off-chip incubation, mobility shift assay uses a microfluidic chip to measure i.e. the conversion of fluorescent phospho-peptide substrate to a dephosphorylated peptide product by phosphatases ore the reverse reaction by kinases. The reaction mixture from a microtiter plate well is introduced through a capillary sipper onto the chip, where the product and substrate are separated by electrophoresis and detected via laser-induced fluorescence. A variety of enzymatic  assays can be performed on a single LabChip 3000 instrument. 

The supported assays for the LabChip 3000 and LabChip® EZ Reader include: 

  • Serine/Threonine Kinases 
  • Tyrosine Kinases 
  • Phosphatases 
  • Proteases 
  • Lipid-modifying Enzymes
  • G-protein coupled receptors (GPCRs)

Surface Plasmon Resonance

The Surface Plasmon Resonance detection system allows automated screening for compounds (even 100 Dalton sized) which bind to proteins immobilized on chips. Different types of chip surfaces allow to couple proteins under mild conditions via His- or GSH- tags, by antibodies or strepravidine.
Specific biological activity of proteins must be controlled by defined binders and non-binders to monitor stability of proteins after repeated regeneration steps for removal of nonspecifically binding compounds.
Kd, plus On and Off rates can be calculated from kinetic data.

Real time kinetic imaging with cells

The FLIPR Tetra system from MD contains in its upper part pipetting modules (384tip head for liquid transfer, washing position for tips plus imaging position and reservoir positions) and at bottom the imaging modules (CCD camera and diode blocks for illumination plus filter wheels). The imaging position can be heated for constant temperature during kinetic measurements.
The system allows to image kinetics in cellular reporter systems in real time and during stimulation by addition of reagents simultanously over a complete plate (hundreds of measurements per minute). The latest system includes luminescence and fluorescence detection with a highly sensitive camera.
Filters (excitation/emission) available:

  • Fluo-4:    470-495 / 515-575
  • Fluo-3:    495-505 / 526-585
  • MemPot: 510-545 / 565-625

Alphascreen

One research focus of Chemical Biology projects at our platform is the implementation of high throughput assays for screening with interacting proteins. For this purpose, we employ the Amplified Luminescence Proximity Homogeneous Assay (ALPHA), a solution based assay, originally developed by Packard Biosciences. As the name implies, ALPHA-screen is a luminescence based proximity assay. One interaction partner is attached to donor beads (Glutathione, Streptavidine), while the other one is coupled to acceptor beads (ProteinA, Ni-chelate), both with a diameter of only about 250 nanometers . A photosensitizer compound is embedded into the donor bead. With this compound, upon illumination with laserlight at a wavelength of 680 nm, ambient oxygen is converted to energy-rich, short-lived singlet oxygen . When no acceptor bead is in proximity, the singlet oxygen decays without producing a signal. If donor and acceptor bead are brought together ( ~ 200 nm) by the biological interaction of the attached biomolecules, the singlet oxygen released by the donor beads initiates a luminescence / fluorescence cascade in the nearby acceptor bead, leading to a highly amplified signal in the 520-620 nm range. The luminescence signal is detected  in a Envision multilabel reader. While the signal for interacting proteins often produces 1.000.000 luminescence units, the background often only shows a few hundred units. Therefore even weak protein-protein interactions can be used for HTS.
False positives acting on the reactive oxygen, the coupling to beads or the chemiluminescence reaction are identified by control peptides or proteins containing fused tags (biotin&His or GST&His).

Fluorescence Readers

Fluorescence polarization, fluorescence intensity readers
The Screening Unit provides 3 Safire2 and one GeniosPro (Tecan AG) plate readers for measurement of fluorescence polarization and intensity beside other read outs like luminescence or absorption. Furthermore the Envision (PerkinElmer) is equipped for luminescence beside its AlphaScreen modul.