What are Out-of-Focus Objects and what should I do if I see them in my Cytos™ run?

Question

What are Out-of-Focus Objects (OOFOs) and what should I do if I see them in a Cytos run on the AVITI24™ system?

Answer

Out-of-Focus Objects (OOFOs) are optical imaging artifacts that appear as a diffuse haze, bright gradients, or localized bright patches in one or more imaging channels in CytoCanvas™ images from Cytos runs on AVITI24. OOFOs are caused by stray light scattering off particulates or residue along the imaging path outside of the Cytos flow cell. The biological sample and the inside of the Cytos flow cell are not the source of the OOFOs. To avoid OOFOs, carefully inspect and clean flow cells before loading, and clean the nest if you see any debris or residue.

Symptoms

Cytos run images with any of the following imaging artifacts can indicate the presence of OOFOs:

• Diffuse haze or glow across part of the field, with a gradient that brightens toward one edge
• Localized bright spot or patch, fixed across tiles, recurring in the same position run after run
• Localized bright spot or patch that appears in a different position on each run
• Aberrations across the field of view, consistent on the same side of the instrument
• Elevated background across an entire channel, uniform, with no gradient and no fixed bright feature
• Sharp-edged bright objects that move with the stage

Considerations

What are OOFOs?

Figure 1. Example images of Out-of-focus objects (OOFOs). Top row shows OOFOs marked in red circles. Bottom row shows additional unmarked examples of OOFOs.

Out-of-Focus Objects (OOFOs) are optical imaging artifacts that appear as a diffuse haze, bright gradients, or localized bright patches in one or more imaging channels in CytoCanvas images from a Cytos run (Figure 1). OOFOs are not caused by anything in the biological sample and do not occur inside the flow cell.

Sources of OOFOs include contamination on the nest or flow cell exterior. For example, residue, permanent marker marks, or any material or smudges carried over from sample preparation can cause OOFOs. They have been observed in all channels and across Cytos runs with a variety of cell types.

See the Troubleshooting section of the user guide and the knowledge base article on cell culture techniques for optimal Cytos images if you suspect an issue within the flow cell (such as unsuccessful seeding or sample preparation).

Types of OOFOs

OOFOs occur on or around the outside of the flow cell:

• Positionally Stable OOFOs appear in the same position across multiple Cytos runs. This indicates that the source is on something that does not change between runs, such as the nest (especially on surfaces below the flow cell), or the optical path itself.
• Variable OOFOs appear in a different position from one run to the next. This indicates that the source is something that travels with the consumable or with handling, such as dust on the flow cell exterior, alcohol film, fingerprints, or debris picked up between flow cell assembly and loading.

Impact of OOFOs on Data Quality

The imaging systems that generate Cytos images and data are generally not sensitive to the diffuse background caused by OOFOs. While OOFOs raise overall background, they do not create false polonies. The diffuse haze does not mimic cellular morphology and therefore does not confound segmentation. However, if an OOFO is locally severe enough to approach saturation over part of the field of view, the background rises in the affected region. This could depress the detection of low-abundance targets for cells under the severe OOFO.

How to Check for OOFOs in a Cytos Run

Review your Cytos run images. An OOFO rarely covers the whole flow cell, so you can compare the affected wells against unaffected wells as needed.

Additionally, review the QC report generated as part of the run output. The Cell Count metrics indicate the number of cells within a well that are free of imaging artifact regions such as unusual intensity or low counts.

How to Resolve and Prevent OOFOs

Most OOFOs are reduced or resolved by cleaning the flow cell and nest. If OOFOs persist after cleaning, contact your Field Application Scientist or Support.

Clean the Flow Cell

Review the Clean the Assembled Flow Cell section of the Cytos User Guide for detailed instructions. In addition, consider the following tips and recommendations:

• After wiping the assembled flow cell surface with an isopropyl wipe, do not allow flow cell to air dry. Instead, use a dry, lint-free wipe to immediately wipe the flow cell to prevent streaks on the flow cell.
• When using canned air to remove remaining dust, be careful to hold upright and avoid blowing contaminants onto the surface.
• Load the flow cell immediately after cleaning so that it does not accumulate dust.

Clean the Nest

If OOFOs are observed, clean the nest before the next run. Figure 2 shows images of contaminated nest surfaces that require cleaning. The Load the Flow Cell section of the Cytos user guide provides instructions to wipe down the nest before loading the flow cell. Consider the following tips and recommendations:

• Ensure careful handling when interacting with the nest. See the user guide for details.
• Remove hard contaminants from the nest surface before applying any pressure to it or loading a flow cell.
• Clear any loose debris by carefully pushing it off into a wipe or blowing it away with canned air.
• Wipe any sticky debris with an isopropyl wipe, followed by a dry lint-free wipe.

Figure 2. Carefully clean nest surfaces. (A) and (B) show contaminated nest surfaces that are common sources of OOFOs.

• Visually inspect the nest area and the stage below the flow cell for remaining dust, residue, or flecks. Ensure the surface has dried completely before loading a flow cell.