![]() When the screen involves less than a few dozen plates or the user wants to obtain a qualitative estimation of the imaging quality, the screener can select these plates for review using the Plate Viewer tool ( Fig. Cellprofiler analyst machine learning tools download#We have provided an example QC pipeline as supplementary material, for use with a set of HCS images available for download at. In either case, the QC pipeline with the selected metrics can be saved for reuse in a later experiment. However, for the large number of images typically generated by an HCS experiment, measurements are best deposited into a database using the Export to Database module. For a small number of images, the measurements extracted by the QC pipeline may be viewed in Excel or a similar spreadsheet program. As described above, we recommend PLLS to detect blur and PM to detect saturation artifacts. The Measure Image Quality module includes the PLLS, focus score, and PM the Measure Texture module measures the image correlation at multiple spatial scales and the Measure Image Intensity module evaluates the mean, median, standard deviation, and minimum and maximum image intensities. We implemented the metrics described above in CellProfiler, allowing analysis modules to be assembled into an automated pipeline. Even though focusing was performed automatically, two plates contained extensive focus artifacts and were discarded from the screen for these plates, each image was manually annotated as in- or out-of-focus. For additional validation, we used images from a large phenotypic screen obtained with the protocol above, along with the addition of a MitoTracker (Invitrogen, Carlsbad, CA) mitochondrial stain and the acquisition of four sites per well. The automated microscope was then programmed to collect a z-stack of 32 images ( z = 0 at the optimal focal plane, 16 images above the focal plane, 16 below) with 2 µm between slices. For each site, the optimal focus was found using laser autofocusing on the Hoechst channel. ![]() The 384-well plates containing U2OS cells stained with Hoechst 33342 and Alexa Fluor 594 phalloidin markers were imaged with an exposure of ms for Hoechst and phalloidin, respectively, at 20× magnification and 2× binning. ![]() HCS microscopy images were acquired using an ImageXpress Micro automated cellular imaging system (Molecular Devices, Sunnyvale, CA). ![]()
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