Introduction
This guide of the Center for Microscopy and Image Analysis explains how to count nuclei in HCS images acquired with the HCS - MD ImageXpress Confocal HT.ai and export the results.
NOTE 1: It is made for single-timepoint, multi-site, and multi-well acquisition for one channel (DAPI). Please adapt it to your own needs if you have different acquisition settings.
Note 2: Think beforehand how to design your plate. The data will be sorted by well A1-A12, B1-B12 etc.
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Start the "MetaXpress" software either on Special VM B or on the dedicated Special MetaXpress VM.
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Login with the same name and password as on the machine.
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Go to "Screening" and select "Review Plate Data [DB]".
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In the "Review Plate Data" window, click "Select Plate" to choose your plate to be analyzed.
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Your plates are first ordered according to the acquisition date. Double-click to open the folder.
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The different plates acquired on that day will appear below.
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"Select" will open the respective plate data.
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The plate format shows up. "-" indicates that these wells were imaged.
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If you have run an analysis on this plate before, you can hide the values (Low Pressure) by unclicking "Show values" in the "Display tab".
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For a montage, mark the corresponding wells, define the sites per well and select a channel for display.
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For a high-resolution image of one site in a well, right-click on the corresponding well.
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In this example, a previous analysis has been performed on the plate.
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Select your channel to be displayed and analyzed.
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In the "Run Analysis" tab, under "Analysis" select "<Count Nuclei>".
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If you already have saved settings, you may proceed to the next step.
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To create your own settings, click on "Edit List" and "New Settings" with your specification.
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Click on "Configure Settings" to adjust the segmentation parameters.
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Choose your channel for the segmentation: "DAPI".
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Choose how to display the results (we recommend "New").
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In the drawing toolbar, choose "Line" to measure the width of a few nuclei by clicking on one end of the cell and by placing the mouse over the diameter and getting the distance.
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Measure a few cells and use these values for the "Approximate min./max. width" for segmentation.
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Hover over regions of cells and background and check the intensity in order to choose the segmentation parameter "Intensity above local background".
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Click "Test Run" to test the input settings.
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When the analysis is done for your test image, an overlay of the segmentation will be shown in red together with your grayscale image. Additionally, a color-coded segmentation image and cell-by-cell results will be shown.
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Show/hide the overlay to judge the quality of the results.
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If necessary, go back and adjust the input parameters to improve the segmentation.
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If you are happy with the result, click "Save Settings".
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In order to check a different image of the same plate, go to the "Review Plate Data" tab and right-click on a different well. Do a "Test Run" again and judge the result.
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You can now run the segmentation algorithm either onthe whole plate or on on a selection.
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If you want to run on a selection, select the corresponding wells by right-click (will appear green) and choose the sites per well.
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In the "Display tab", choose for "Image Overlay": "Show cell Segmentation".
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Use this button to show/hide the segmentation and judge the quality of the algorithm.
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Go to the "Measurement tab" and choose the "Analysis" (if multiple were run on the same plate).
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Choose the type of "Analysis" to be displayed.
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Click "Show Heat Map" to display your results as a heat map.
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For data export, go to "Screening" and select "Plate Data Utilities [DB]".
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Select "Export Measurements".
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Select "Image Measurements" and click "OK".
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The measurements are first sorted according to analysis date for the plate (Measurement Set) and then for the plate (Name [Plate Info]).
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Either double-click on the measurement or use the arrow to make it appear in the "Query" on the right.
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Click "Next".
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Click "Finish".
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In "Rows" select "Well Name" with the Type "Image Measurement" by double-clicking so it appears on the right.
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In "Columns" double-click on "Total Nuclei" to get the number of nuclei per image.
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Click "OK".
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Select your export folder.
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Check "Export all measurements to one file" and define a File Name.
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Select "Tab Delimited (Data only)".
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Click "OK".
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You will get a notification that the export was successful.
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In the "Data" tab, click "From Text/CSV".
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Open the ".txt"-file generated in Step 9.
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Check whether the format is correctly recognized and click "Load".
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A new tab will appear with your well information and the number of detected nuclei.
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To start referencing the data, go back to the "Sheet1", click on cell "A2" and type "=".
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Go to the data sheet (test2), and click on the first cell (A2) with a well name, with the reference appearing in the formula bar.
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Press Enter to make the reference.
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Repeat the same procedure for the counted nuclei by typing "=" in cell "B2" and referencing it to "C2" (Total Nuclei (CountNuclei)!) in data sheet.
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Mark Cells "A2&B2" and extend the referencing by grabbing and dragging the lower right corner of the marked cells down to the maximum number of sites measured.
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Specify the number of sites per well and number of wells.
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By grabbing the lower right corner of the two marked cells H2 and J2, drag to extend to your maximum well number.
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Type in the number of wells to be averaged, the corresponding mean and standard deviation will appear and the histogram will fill itself.
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Here you can adjust your x-axis label.
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