Seitenhistorie

Overview:

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Machine start

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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).

Usage

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Machine start

• Depress button (green) on the left side of the machine
• Wait a few minutes until the software displays "device is ready".
• The machine is now ready to use

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 an internal "penta-pattern" of dots, used for high-accuracy positioning of the 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.

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• 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 with or without lid (place within protrusions).
• or: Load slides in holder.
• or: Load slide holder into Operetta.

Setting up for imaging:

• Set up the desired channels, select a well, and take a snapshot. Different z-heights may need to be trialled.
• 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.
• Histogram: if not visible, can be enabled using the arrow under "Channels" to the right of the image.
• 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! "test" is used here, and only applies to the well that was highlighted by clicking, on the right.
• 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.

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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

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Image processing and analysis:

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Loose info pieces:

Z-steps always > 100 nm

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