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An Introduction to High-Content Screening Microscopy

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An Introduction to High-Content Screening Microscopy Elena Deligianni Ph.D. Overview of HCS technology What is High Content Screening (HCS)? Automated microscopy to capture images from a multi-well plate or slide and analyse a number of parameters from the image What is High Content Analysis (HCA)? Analysis of the HCS images A powerful image processing software is needed HCA is a multiparametric analysis Compound selection using profiles instead of single read out by calculation of a number of parameters MCF10 cells on laminin Less errors Dr. Christianna Choulaki MCF7 cells on gelatin HCA is a multiparametric analysis High Content Screening Microscopy coupled with High Microplate reader Content Analysis Intensities of DAPI and GFP Parameters for nuclei • Number of nuclei • Intensity of nucleus • Area of nucleus • Roundness of nucleus • Width & length of nucleus • Calculate parameters for single nucleus and mean values per well • Differentiate between nuclei with different morphological characteristics Widefield or Confocal Parameters for gelatin • Intensity Imager • % of degradation Restricted number of Images • Define areas with different % of degradation Advantages of HCSM Operetta Facility is equipped with the HCSM Operetta (Perkin Elmer) • Flexibility C. elegans neuron cells Whole organisms to cells Fixed and live cells Time course experiments Kinetic capabilities Slides and microtitre plates Temperature and CO2 control • Speed Harmony software for Image Analysis Image Segmentation • Detects: Nucleus, Cells, Cytoplasm, MicroNuclei, Object tracking Phenotypic Classification • Intensity, Morphology, Texture, Kinetics, Track Cells Cell Width Cell Length Cell Ratio Width Nucleus Area Nucleus Nucleus Nucleus Length Object No Cell Area [µm²] Cell Roundness [µm] [µm] to Length [µm²] Roundness Width [µm] [µm] 40 342.807 0.417206 8.48611 26.8584 0.315957 149.701 0.997408 10.6974 16.046 Machine learning • Segmentation and classification based on calculated features Open source software used for analysis • BioImageXD, Fiji, EBImage, CellCognition, CellProfiler, Icy Example1: Screening of antimalarial drugs • Malaria is caused by a parasite of the Plasmodium family • Transmitted to people through the bites of infected Anopheles mosquito • Responsible for 500.000 deaths annually while 210 million people are infected (2010-2015 WHO report) • Disease prevails mainly in Africa, South America, India • Progress to control malaria are threatened by 1) insecticide resistance and 2) resistance to artemisinin (the core compound of the best available antimalarial medicines) Example1: Screening of antimalarial drugs Addition of drug in in vitro CTRP-gfp P. cultures berghei strain Zygote Ookinete Calculate Morphology and Intensity Properties Intensity of Cell Intensity of Cell Cell_Length Cell_Ratio Intensity of Cell Object No Cell_Area [µm²] Cell_Roundness Cell_Width [µm] Alexa 488 Alexa 488 [µm] Width to Length Alexa 488 Mean Median Contrast 1 44.6389 0.868422 5.06447 11.16 0.453807 1673.09 1553 0.261203 2 56.4768 0.639592 3.9729 15.5939 0.254772 1557.77 1474 0.213685 Example1: Screening of antimalarial drugs Software is trained to differentiate between zygotes ookinetes & artifacts Drug Mode of Action IC50* Atovaquone Inhibits ATP synthesis 35.6nΜ Lopinavir HIV protease’s inhibitor 30.5μΜ conversion Ritonavir HIV protease’s inhibitor 32.95μΜ Nelfinavir HIV protease’s inhibitor 37.93μΜ Indinavir HIV protease’s inhibitor No effect ookinete Jaspakinolide Increases actin polymerisation No effect of LatrunculinB Inhibits actin polymerisation No effect cytochalasinD Inhibits actin polymerisation 42.42μΜ *triplicate assay Percentage IC50 for Atovaquone. Example2: Evaluating the neuroprotective effect of drugs against oxidative stress in ALS • Amyotrophic lateral sclerosis (ALS) or motor neuron disease (MND) is a disease that causes the death of neurons which control voluntary muscles • The cause is unknown but 10% of cases have a genetic background (SOD1 gene) • Average survival from onset to death is two to four years • No cure for ALS occurs AIM Drug Screening was optimised in 2D cultures in order to be used subsequently to 3D matrix platforms Example2: Evaluating the neuroprotective effect of drugs against oxidative stress in ALS Calcein Hoechst Propidium Iodide Calcein Hoechst Lysotracker Hoechst Selection of fluorescent markers Image segmentation Nuclei segmentation Cytoplasm segmentation and quantification of morphological characteristics Image analysis and statistical evaluation Drug1 Drug2 credits to Dr. Dimitris Tzeranis Example3: Proliferation of nuclei in embryonic mouse brain sutures (credits to Ms. Aggeliki Vogiatzi) TOPRO stained nuclei in brain embryonic sutures BRDU stained nuclei Segmentation of nuclei Segmentation of BrdU nuclei High Content Thank you Screening Facility http://www.imbb.forth.gr/en/facilities- en/imaging/item/3295-high-content-screening- microscopy-unit Thank you! Summary of biological applications with HCSM 1. Changes in cll shape 2. Neurite outgrowth 3. Characterisation of Cell structure cellular organelles 1 2 3 (here peroxisomes) 1. Migration Cell behaviour 2. Colony formation 1 3. Cell tracking/speed 4. Apoptosis 5. Cell cycle characterisation 2 3 4 1. Protein translocation 2. Internalisation 3. RNAi/Drug Drug Effect Screening 1 2 3 4. Cytotoxicity 5. Chemotaxis Images from Perkin Elmer.
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