Seitenhistorie

...

• 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.
• 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.

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).
• info: using "Test Images", and marking regions, right-click and "use as background for well" for a visual aid for selecting further regions.

Run experiment

• 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 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

...