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Service contact (administrators only): "EVO Portal", Perkin Elmer website. |
Incubation: Heating is available (37-42 °C; system temperature of about 25 °C without heating activated). CO2 controller available (1-10 %). No humidity control: Use closed samples in excessive imaging buffer, if necessary.
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What about my specific excitation line? For fluorophores with an excitation peak at for example 510, use the closest available light source: 475 nm See https://searchlight.semrock.com/ for excitation/emission spectra of many fluorescent dyes and proteins. When testing a new assay, make sure to include enough controls to verify that no crosstalk appears. |
Plates
- Manufacturer comments on plate usage: https://www.perkinelmer.com/uk/lab-products-and-services/application-support-knowledgebase/microplates/microplates-knowledge-base.html
- Machine takes "all" available cover-slip bottomed plates.
- But, do not use Techno Plastic Products (yellow stripe); which have plate-vents and cannot be supported by the holder.
- may have a glass or plastic "coverslip" bottom
- for good cell-attachment, can coat the bottom with poly-L-lysine
- When correctly entered, objectives will never touch the plate bottom (if still; plate will be pushed upwards).
Usage
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Machine start
- Depress button (green) on the left side of the machine
- Wait a few minutes until the software displays "device is ready".
- The machine is now ready to use
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Z-stacks yes/no? It is possible to disable "use z-stack in test", to test with only single-plane images If you want no stack acquisition upon running the actual experiment, simply select "0 planes" "Selecting" wells and higlighting highlighting wells Clicking on wells higlights highlights them, and points at wells to be used in test images Selecting wells (higlightinghighlighting, then clicking "select") determines which wells to use in the experiment. |
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- Evaluate that the correct assay is loaded.
- Name the plate and add keywords.
- → Start! And observe live-preview images during acquisition (when enabled)
- Select the whole plate, right-click, organise organize images in "realistic" (corresponding to the plate) or "packed" (best for display) layouts
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- In the "settings" menu up top, data management can be used to export data
- Export your data as an "archive" for later re-analysis using Harmony, eg. on the CAi image-analysis PC
- Export your data as "measurement + associated data" to save results as TIFF files, alongside already-performed measurement outcomes, and re-analysis eg. using CellProfiler. Be aware that the exported raw images are not flat-field corrected, so it might be necessary to correct the images before the analysis.
- Connect to the CAi shared network drive to store the data, or bring your own data-carrier (USB).
- After the session, delete your data from the Operetta computer.
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Loose info pieces:
Z-steps always > 100 500 nm
Fluorescent staining strategy:
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