High-throughput microscopy is becoming an important and widely-used research tool to screen libraries of small molecules and conventional drugs for their efficacy in reverting pathological phenotypes at a cell-based level.

High Content Image Analysis (HCA) allows the rapid assessment of key cellular pathways by analyzing large number of cells in a quantitative manner for their response to exogenous and intracellular stimuli. Large panels of compounds/siRNAs can be studied in a single HCS assay and multiple cellular parameters can be analyzed simultaneously.

Among the parameters that can be measured are neurite outgrowth, cell shape and proliferation, transport efficiency, the phosphorylation state of given markers, cytoskeletal defects, stages of the cell cycle, nuclear morphology, apoptosis and ploidy, mitotic spindle structures, motility etc.

Projects

Several groups within and outside the CMNS are using the HTS/HCA Facility to set-up their favorite cell-based assay for screening siRNA or drug libraries, as well as for in-depth High Content Image analysis (HCA). At the moment the facility is hosting high content screening and image analysis for projects financed by AIRC, Telethon and by the EU-Cilia.

Information, instrument availability and support

Contact the Head of the facility Dr. Antonella Ragnini-Wilson.
We will happy to help you to devise and establish your favorite cell-based assay using our Facility.

 

HTS/HCA Facility microscope set ups

The Wide-Field HTS/HCA Facility provides the ScanR life Science image station (Olympus) for high throughput screening approaches and an in-built Authomated Microscopy station Nikon for automated time lapse acquisition on multiwell plates and image analysis. All microscopes can be loaded with various well plate formats and can analyse fluorescent or chromatically-stained (BrightField) samples.

 

ScanR life Science image station (Olympus):

  • Inverted microscope: Olympus IX81 with motorised Z axis
  • Infrared laser autofocus (Hardware and software controlled
  • Illumination: MT20E lamp for multifluorescence imaging
  • Objectives: SuperApocromatic 20x, 40x and 60x numerical aperture 0.75 and O.90 respectively for short distance work with plastic and glass bottom multiwell plates (0.15-02 mm), DIC and Nomarski configuration
  • Sensor: Digital camera CCD  QE  µm 1344 x 1024 pixel, binning, FireWireTM (IEEE-1394) interface and trigger
  • Exitation/Emission Filters:  High velocity filter wheel turret equipped with DAPI/FITC/TRIC, GFP, CFP, RFP, YFP filter cube. Additional Exitation/Emission Filter combinations can be added on request. There is also the possibility to examine chromatically stained specimens(Brightfield) as well as to use differential interference contrast (DIC)
  • Probe handling: Multiwell plates, objects slides, adapters for various plate formats
  • Robotic plate loading: Hamilton Microlab SWAP
  • Software:  ScanR data acquisition; ScanR Analysis Software: two licenses located in separate computer workstations allowing analysis to be performed in a remote place while acquisition is running

 

Wide Field Authomated microscopy station for time lapse microscopy:

  • Inverted microscope: NIKON ECLIPSE TE2000 inverted microscope.
  • Illumination: XENON lamp for multifluorescence imaging.
  • Sensor: CCD CoolSNAP ES Monochrome (Photometrics) QE  µm 1344 x 1024 pixel, binning, FireWireTM (IEEE-1394) interface and trigger.
  • Stage: motorised microscope stage System ProScan™ (PRIOR-Scientific)X,Y,Z,  
  • Microscope Motorization control:  MetaMorph™ System (MolecularDevice)
  • Objectives: PlanApo 100x/1.44, 60x/1.4 oil and Nikon 10X/O.25, PlanFluor 20X/045, PanFluor 60X/0.85
  • Exitation/Emission Filters High speed filter wheels equipped with Curv-o-matic (OMEGA Optical) filters for DAPI, YFP, CFP, GFP, RFP authomated acquisition. Manual Filter wheel equipped with Blue excitation B-2A, Green Exitation G-2A, Red excitation Cy5 HYQ filter block
  • DIC and Nomarski configuration 100x, 60x A.N 1.4 oil CFI planapo NIKON.
  • Additional Software: META Serie 7.0 for image processing, analysis implemented with multidimensional imaging, Co-localization and Overlay Fluor option; AQI-X AutoDeblur Gold CWF with Autovisualise alignment plus option(MediaCybernwetics) for 2D and 3D reconstruction, deconvolution and image processing is available in separate computer workstations
  • Data Archive system:  Local hard disc storage drive (80GB); intranet storage 250GB and NAS 2.25 TeraByte

 

Additional equipment HTS sample handling

  • Multiwell plate and liquid handling: FREEDOM EVO 150 (TECAN) equipped with a 96-tip multichannel (MCA) arm, 2 independent pipetting (LiHa) arms, 96 pin tool fixture and Hydroflex™ multiwell plate washing station.