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).
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:
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
Choose objective. Start at eg. 20x / 0.4 NA
Choose operational mode (opt-mode). Confocal = spinning disc mode, with pinholes, 5% transmission of light, both ways. Epi = non-confocal, 100% transmission of light, both ways.
Binning: (makes image size <4.7 Megapixel) → signal x4, noise x2, resolution significantly reduced.
Live preview: eg. Show current image during stack acquisition. off = evaluate manually after setting up acquisition.
Click "new" → new experiment.
Set up individual channels for your fluorophores.
info: Arrowheads under menus always reveal more detailed settings.
Plate layout (right-most). Select wells to measure, click "SELECT". Select FOV, number, position.
Select well-to-test.
Clean plate-bottom with KimTech wipe and 70% Ethanol before use.
Load plate (place within protrusions).
or: Load slides in holder.
or: Load slide holder into Operetta.
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 other 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)
Add flat-field correction if necessary
Loose info pieces:
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.