Operetta CLS

Overview:

Incubation: Heating available. CO₂ controller planned-to-be-available. No humidity control: Use closed samples in excessive imaging buffer, 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:

• 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)

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).
• 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.

Usage

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.

• 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.

• 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.
• 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)
• Pick the right focus height for each channel individually.
• Next, to be certain, do focus finding in another cell. Possible plate-tilting may be revealed, and needs inspection.

• 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
• You can add layouts (labels) to the output data. This is an additional table.
• Re-use a plate layout (saved, from eg. editor or previous experiment); "Assay" in "define layout" (right-most).

Run the 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

• When done: open the lid (settings - Operetta CLS - open) and remove plate/slide holder.

Image processing and analysis:

Loose info pieces:

Z-steps always > 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 attached to the microscope:

DO NOT SWITCH OFF!

The PC is running a live image data and image analysis server.