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).
Light/optics: 4x LED light source (can activate only 1 source at the same time), 4x emission filter (UV / green / red / far-red), Spinning disc optics. 1x top-down-LED (740 nm, for morphology, using transparent-top samples).
Camera: 4.7 Megapixel sCMOS.
Light path/wavelengths:
Excitation windows available:
Detection windows available:
eg. for fluorophores with an excitation peak at 510, use the closest available light source: 475 nm
Plates:
Usage
Around the sample area:
Plate loading:
Setting up for imaging:
Evaluate brightness; auto-scale and auto-contrast are always applied.
No range-indicator (over/under exposure) is available, but a histogram for each channel is displayed
Scaling: Normal mode: Black-to-Colour. Enhanced mode: Black-to-Colour-to-White.
info: right-click on the sample image, "show intensity". Ctrl + Click to add ROIs.
Find focus height: Z-stack!
Start at negative values. Go up to positive. See in "TEST IMAGES" (right of screen)
info: pick the right focus height for each channel individually.
Do focus finding in 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" section, select all interesting wells; right click "overview;" preview field: observe the stitched image afterwards.
The overview picture is automatically downscaled to 200 MB in size; full-resolution stitching is not supported.
Double-check overlap: Pull up the contrast to an extreme value (visually; right-click in the image)
Determine whether flat-field correction 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,
To facilitate auto-detection in subsequent image analysis steps:
a) stain cells with target marker
b) stain cytosol separately (eg. CellMask Blue)
c) stain nuclei separately
Computer
DO NOT SWITCH OFF!
The PC is running a live image data and image analysis server.