Focus Stacking (Combination of Focal Planes)

Overview

Depending on the preparation, it is often not possible to bring all parts of a specimen into focus simultaneously.

To address this, multiple images are captured at different focal depths and combined into a single image using a technique commonly known as focus stacking (or focus merging).

This produces a final image in which structures across multiple focal planes are simultaneously in focus.

When This Method is Used

Focus stacking is used selectively, specifically where a single focal plane does not adequately represent the structure:

• The specimen has significant depth or uneven surface structure
• Important features lie on different focal planes

Unless otherwise stated, plates on this site are derived from single focal plane observations. The plate description will state when focus stacking has been used to create the image

Method

Image acquisition

• The specimen is observed under transmitted light
• A series of images is captured while adjusting the fine focus through the specimen
• Each image represents a different focal depth
• Care is taken to:
  • Maintain consistent illumination
  • Avoid movement of the specimen
  • Capture sufficient overlap between focal planes

Image processing (Affinity Photo 2)

Focus stacking is performed using Affinity Photo 2:

• Open “File → New Focus Merge”
• In the “New Focus Merge” dialog
  • Click Add and select the image stack
  • Click OK to run the merge
  • The software automatically:
    • Aligns the images
    • Combines the sharpest regions from each frame

Post-processing

• The merged image is reviewed, with minor adjustments being applied in the Develop Persona, such as:
  • White balance
  • Exposure
  • Contrast

All adjustments are applied conservatively, with the aim of:

• Preserving observed structure
• Avoiding introduction of artefacts
• Maintaining a faithful representation of the specimen

Notes on interpretation

Focus-stacked images should be interpreted as a visual synthesis of multiple focal observations, rather than a literal single-plane view. They are intended to improve clarity and completeness, not to alter or enhance structural features, and are only used only where necessary.