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Service contact (administrators only): "EVO Portal", Perkin Elmer website.
Incubation: Heating and CO2 controller available. No humidity control (use closed samples if necessary).
Objectives: 5x, 20x, 40x (all air).
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Camera: 4.7 Megapixel sCMOS.Incubation: Heating and CO2 controller available. No humidity control (use closed samples if necessary).
Light path/wavelengths:
Excitation windows available:
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Plate loading:
Setup:
ImagingSetting up for imaging:
Evaluate brightness; auto-scale and auto-contrast are always applied.
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info: pick the right focus height for each channel individually.
Do focus finding in other another cell
info: it is possible to disable "use z-stack in test" to trial only single-plane images
info: if you want no stack acquisition upon running the actual experiment, simply select "0 planes"
Overview picture: in the Well Layout "well layout" section, select all interesting wells, ; right click Overview"overview;" preview field: observe the stitched image afterwards.
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Double-check overlap: Pull up the contrast to an extreme value (visually; right-click in the image)
Add Determine whether flat-field correction if will be necessary.
"online-job" = perform image analysis during acquisition
info: Can add layouts (labels) to the output data. This is an additional table.
info: re-use a plate layout (saved, from eg. editor or previous experiment); "Assay" in "define layout" (right-most).
Run experiment
Evaluate that the correct assay is loaded
Name the plate and add keywords
Start!
Select whole plate, right-click, organise images in "realistic" (corresponding to the plate) or "packed" (best for display) layouts
info: The software can do flat-field correction based on acquired images, provided that foreground/background and areas without cells are present. This also works in wells filled with tissue or more-than-confluent cell cultures.
When done: open the lid (settings - Operetta CLS - open) and remove plate/slide holder.
Loose info pieces:
Operetta CLS: z-steps > 100 nm
Fluorescent staining strategy,
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