Operetta CLS

Summary:

Service contact (administrators only): "EVO Portal", Perkin Elmer website.

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.

Incubation: Heating and CO₂ controller available. No humidity control (use closed samples if necessary).

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
"skirt 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.
When correctly entered, objectives will never touch the plate bottom (if still; plate will be pushed upwards).
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).
Edge effects: Fill those wells with only buffer, for example.

Around the sample area:

If the table is not powered, eject/re-insert he plate holder (software). Do not manually move table.
Plate/sample holder is mounted on steel tracks: DO NOT CLEAN/TOUCH (if dirty, call service)
Plate/sample holder contains a "penta-pattern" of dots, used internally for high-accuracy positioning of sample.
Red dot on objectives needs to match the red dot on the microscope. Objectives can be manually lifted out of the system.
With open lid, the machine counts as a class 3B laser. Close the door firmly.
Emergency release hatch button (lid open) is on the lower right of the machine.

Plate loading:

Start Harmony software

Log-in with user account: personal account, with personal settings

User accounts can only delete own data. Administrators can delete all.

info: The software is process-oriented; (re)configuration and data generation are alternating.

Setup:

Choose plate. Nomenclature = number of wells, manufacturer, type/name of plate

Light path/wavelengths:

Excitation windows available:

365 nm (340-380 nm)
475 nm (460-490 nm)
550 nm (535-565 nm)
630 nm (615-645 nm)

Detection windows available:

430-520 nm (blue)
500-550 nm (green)
570-650 nm (red)
665-760 nm (far-red)

eg. for fluorophores with an excitation peak at 510, use the closest available light source: 475 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.