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Reference point overview

Is the reference point necessary?

Yes, all data files require a reference point.

The reference point (or point of origin) represents the location on the physical slide or image from which all other points are calculated from.

If the data file has multiple sections represented, this point is (0,0,0) in Cartesian coordinate space for the first section traced in the data file (if there are serial sections).

There is only one reference point per file, even if there are multiple sections and slides.

The reference point:

• Speeds up alignment
• Enables to resume data collection for reproducibility

When do I place my reference point?

Placing the reference point manually is your first step. This step is necessary to enable most commands in Microlucida.

1. Start Microlucida
2. Drive the stage to the desired location (if you're using a microscope)
3. Place the reference point by clicking on the screen
4. The reference point is represented by a small circle

Where do I place my reference point?

Place your reference point on a slide-specific feature or a on a specimen-specific feature. Here are some examples:

Slide-specific: an element of the slide that is easily recognizable

• A "+" sign on a positively charged slide
• The top left corner of the cover glass, etc.

Specimen-specific: anatomical landmark

• The longitudinal fissure on the first section of the first slide
• For NSA slides: the upper left corner of the embedding medium
• For neuron reconstruction (for which there is only one section per data file): a unique point on the section that is visually recognizable at low magnification.
  • Add a fiducial contour easily recognizable at tracing magnification

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