Scientific practice

VIPP makes intermediate outputs easier to see; it cannot decide whether those outputs answer a biological question correctly. A defensible workflow combines visible processing with representative data, independent references, and a frozen evaluation procedure.

Minimum analysis lifecycle

flowchart TD
  Q["Define the biological quantity"] --> D["Select representative development data"]
  D --> T["Build and tune while inspecting intermediates"]
  T --> R["Freeze workflow, software, and decision rules"]
  R --> V["Evaluate on held-out references"]
  V --> B["Run the collection"]
  B --> A["Audit outputs and report exclusions"]

Four questions to ask

1. What is the target? A nucleus, punctum, branch, event, overlap, or intensity summary needs an operational definition.
2. What can vary? Include acquisition day, operator, treatment, tissue, signal level, artifact, and biological heterogeneity in representative data.
3. What is independent evidence? Reference annotations, calibrated phantoms, known synthetic truth, or a justified external method should not be the same output used to tune the workflow.
4. What would make the workflow fail? Predefine failure/QC criteria before seeing all experimental results.

Continue by decision

• Choose 2D or 3D before building spatial nodes.
• Tune without fooling yourself before a parameter sweep.
• Validate a workflow before drawing biological conclusions.
• Report a VIPP analysis before sharing or publishing.

Inspection is necessary, not sufficient

A plausible overlay can still be biased, inconsistent, or wrong outside the inspected field. Conversely, a strong aggregate metric can hide systematic boundary errors. Use visual and quantitative evidence together.