An Operetta CLS (PerkinElmer) high-content imaging device is available at ZIK plasmatis. These types of devices are fully housed microscopes without oculars being primarily used in the pharmaceutical industry for screening campaigns of large compound libraries. Owing to its high speed motorized table (2-parametric 384-well plate measurement in <8min) and the flexible optics (8 excitation LEDs, 8 emission channels), such technology is increasingly used in research laboratories. Primarily cells in multiwell plates (6-well to 1536-well), 3D tumor spheroids and organoid cultures in 96-well round bottom plates, and tissue sections on microscopy slides (up to 4 simultaneously) are analyzed in our lab. Laser-based autofocus provides the necessary z-precision across all measurements per plate or slide. Digital phase contrast (DPC) allows the determination of a cell’s cytosol without the use of fluorescent labels. Air (1.25x, 5x, 20x, 40x) and water immersion objectives (20x, 40x) are available, with the latter providing automatic water replenishment for long (time) series measurements. Because of the climate chamber (37°C, 5% CO2), time series experiments can easily be performed over the course of days. Fluorescence images can be acquired either in epifluorescence mode or with a spinning disc in true-point confocal mode. The 16-bit sCMOS camera with 4.7 megapixels has a resolution of 50nm. The system is FRET-ready. Additionally, its PreScan-ReScan capability allows automatic re-imaging of cells of interest, for example: in lysosome-labeled cells, find the 10% brightest cells when taking a picture at 5x and re-image only these 10% at 20x for detailed analysis. Example 2: Find DAPI-positive regions (tissue) on microscopy slides at 1.25x and re-image only tissue at 5x.
Across all projects, about 1 million images (2-3 terabytes) are recorded each year at ZIK plasmatis. The IT infrastructure necessary for handling and storing this amount of data is available. Algorithm-based image analysis is performed exclusively quantitatively, without observer bias in Harmony software (PerkinElmer). Manual creation of regions of interests (ROI) is not possible. Once an algorithm-based analysis strategy has been set up, it is applied to all images of an experiment - as in FACS analysis of several samples - and image information is transformed into numbers. Again similar to FACS, parameters of subpopulations can be analyzed: are mitochondria in cells of the G2 but not G1 cell cycle phase tending to be more peri-nuclear or rather close to the plasma membrane? Previously tackled questions of the group ZIK plasmatis "plasma-redox-effects" or in cooperation with our cooperation partners include, for instance the quantitative detection of the following parameters: cell size, nuclear size, apoptosis and terminal cell death, mitochondrial superoxide production and membrane potential, length-to-width ratio, number of tumor cell extrusions <2μm, real-time phagocytosis kinetics of fluorescently-labeled Staphylococcus aureus, average tumor cell-immune cell proximity including clustering, average migration rate of tumor cells and immune cells considering cell division rates, cell death in three-dimensional Candida albicans biofilms, quantification of dendrites, verification of single cells per well after single cell sorting by FACS, micronucleus detection, quantification of neutrophil extracellular traps (NETs) with a pre-defined intensity-to-size ratio (exclusion of dead, non-NETing neutrophils), degradation of antigen in professional antigen-presenting cells, number of tumor-resident macrophages in whole tissue sections, time-resolved analysis of dead cells in tumor spheroids, differential contribution of two differently-labeled cell populations to formation of tumor spheroids, and machine-learning algorithms for the morphological description of two populations from over 300 morphometric parameters. The raw data (images) can be exported to third-party software for analysis; however, it is recommended to analyze the data with the device-specific image acquisition and analysis software Harmony (PerkinElmer). Four Harmony licenses on 30-core computers are available for quantitative image analysis.
Contact: Dr. Sander Bekeschus, sander.bekeschusinp-greifswaldde