Classify Objects

The Classify Objects command evaluates every object against a set of shape criteria and, optionally, an intersection criterion. Each object either matches (passes every enabled criterion) or does not, and the selected match handling mode decides what happens to the object’s class labels in each case - add a class, strip a class, or reclassify entirely.

This is where raw segmentation output (“every connected group of bright pixels”) turns into biological meaning (“this is a nucleus, this is an EV - that speck isn’t”) - the criteria exist because segmentation almost always over-detects, picking up noise, debris, and imaging artifacts alongside genuine objects. Because match handling is itself configurable, the same command also covers narrowing an already-named population and reclassifying objects based on what they overlap.

Input Selection

Parameter	Description
Input Classes	Restrict evaluation to objects that already carry every one of these classes. Leave empty to evaluate every object regardless of its current class

Objects created by Extract Objects carry their segmentation class as an object class from the start, so a fresh, unfiltered population can be selected here the same way as an already-named class from an earlier Classify Objects step. See Object Classes for how that numbering works and why listing more than one class here narrows the population rather than widening it.

Shape Criteria

An object matches only if it satisfies every criterion below - they combine as a logical AND, not a choice of one.

All shape-based criteria accept the field’s maximum representable value as “no limit” for the upper bound and 0 for “no lower limit” - there is no dedicated “disabled” sentinel, so leaving a bound at its default effectively disables it.

Criterion	Description
Min area / Max area	Object area, in the chosen Size unit
Min circularity / Max circularity	Range 0.0-1.0; 1.0 = perfect circle
Min solidity / Max solidity	Ratio of area to convex hull area (0-1)
Min aspect ratio / Max aspect ratio	Fitted-ellipse major/minor axis ratio
Min eccentricity / Max eccentricity	Elongation from fitted ellipse (0 = circle, 1 = line segment)
Min Feret / Max Feret	Bounding-box diagonal, in the chosen Size unit
Allow edge touching	If disabled, objects that touch the image border fail the match

Size unit

Shape criteria that depend on physical size (area, Feret) use a single Size unit shared by both:

• Pixels (px) - absolute pixel count
• Nanometres (nm) - converted using the pixel size from image metadata

There is no separate intensity filter in Classify Objects - mean/min/max/sum intensity per channel are recorded as metrics on every object, but they aren’t part of the match criteria here.

Intersection Criterion

An optional, additional criterion evaluated alongside the shape criteria above - an object must satisfy this too, not instead.

Criterion	Description
Intersecting With	An object class to test overlap against. When set, the object only matches if it also overlaps at least one object of this class by at least Min intersection area
Min intersection area	Minimum overlap area, in the chosen Size unit. Has no effect while Intersecting With is unset

Unlike the shape criteria, this one is disabled entirely by leaving Intersecting With unset (no sentinel value needed) rather than by relaxing a threshold. It corresponds to the Intersection Count metric - counting how many objects from another class overlap a given object - used here as a pass/fail gate rather than just a recorded value.

Match Handling

Every object is evaluated once against the criteria above to get a single match / no match result. Match Handling then decides what that result does to the object’s classes:

Mode (GUI label)	On match	On non-match
Add class on match	Add Output Tag to the object’s existing classes	No change
Add class on mismatch	No change	Add Output Tag to the object’s existing classes
Remove output class on match	Remove Output Tag from the object	No change
Remove output class on mismatch	No change	Remove Output Tag from the object
Remove objects matching criteria	Clear all classes from the object	No change
Keep objects matching criteria (default)	No change	Clear all classes from the object
Reclassify on match	Clear all classes, then add Output Tag	No change
Reclassify on mismatch	No change	Clear all classes, then add Output Tag

Note

Two further modes - Remove class on match and Remove class on mismatch - are hidden from the command picker but still valid in project files (e.g. hand-edited or migrated from an older version): they strip every class listed in Input Classes (not the output class) from the object, without touching any other class it carries. “Clear all classes” removes an object’s classification entirely - it still exists (its geometry and metrics are computed), but with no class assigned it won’t appear in any class-filtered results, count, or export.

Choosing a mode

• First-time classification (segmentation class → a real named class) and reclassifying by overlap (moving objects into another class based on what they intersect) are both worked through in the examples below.
• Narrowing an already-named population with a stricter filter, without renaming it: leave Output Tag unset and use the default Keep objects matching criteria - objects that fail the new criteria are dropped; objects that pass keep their existing class untouched.
• Tagging without removing anything else: use Add class on match to layer an additional class onto objects that already carry others, e.g. flagging objects that also satisfy a secondary criterion.

Example: Spot detection

Input Classes:   (empty - segmentation class from Extract Objects)
Min area:        3 px²
Min circularity:  0.1
Allow edge touching: true
Match Handling:  Reclassify on match
Output Tag:      ch1@spot

Objects larger than 3 px² with any circularity are reclassified as ch1@spot; everything else keeps only its segmentation class and is dropped from all named-class results.

Example: Reclassify by overlap

Input Classes:      ch1@spot
Intersecting With:   tetraspeck@spot
Min intersection area: 1 px²
Match Handling:      Reclassify on match
Output Tag:          tetraspeck@spot

Any ch1@spot object that overlaps a tetraspeck@spot calibration bead is moved into the tetraspeck@spot class, removing it from the spot count - the pattern used in Spot Count to exclude bead artefacts.