Seitenhistorie

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Tipp
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 planned-to-be-availableavailable (1-10 %). No humidity control: Use closed samples in excessive imaging buffer, if necessary.

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Light/optics: 4x LED light source (can activate 1 source simultaneously), 4x 5x emission filter (UV / green /yellow / red / far-red), Spinning disc optics. 1x top-down-LED (740 nm, for morphology, using transparent-top samples).

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430-500 nm (blue)
500-550 nm (green)
515-580 nm (yellow)
570-650 nm (red)
665-760 nm (far-red)

Tipp

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).

Tipp

Skirt height
Difference between plate-edge height, and imaging surface height
Important value to enter into machine: 0-1.5 mm range allowed.
Can measure the height of unknown plates using the Operetta with the 20x objective.

Edge effects
Prefer not to use the outer rows and outer columns of any given plate.
This because of "edge effects" (thermal, evaporation, different near the plate edge).
Fill those wells with only buffer, for example, and do not use for imaging.

If the edge wells are needed for samples too, make sure to fill the inter-well space, which reduces edge effects in case of 96-well plates (see https://resources.perkinelmer.com/lab-solutions/resources/docs/tch_perform_successful_long_term_live_cell_imaging_in_high-content_analysis_system_013900_01.pdf).

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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Plate layout (right-most). Select wells to measure, click "SELECT". Select FOV, number, position.
Select well-to-test.
Clean plate-bottom with wipe (KimTech wipe or similar) and 70% Ethanol before use.
Load plate with or without lid (place within protrusions).
or: Load slides in holder.
or: Load slide holder into Operetta.

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Tipp

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

Tipp

Rule of thumb for estimated dataset size
The Operetta CLS is only displaying the total size of a dataset if not enough disk space is available. If you want to know the estimated dataset size before you start an experiment, use the following rule of thumb:

Number of wells x fields x channels x z-planes x timepoints x 8 MB = approximate dataset size in MB

Tipp
Flat-field correction 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 eject the lid sample (settings - Operetta CLS - openeject) and remove plate/slide holder. Afterwards "eject" once more to close the system again.

Exporting data

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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