Detect puncta panel (3D)

The detect puncta panel includes tools for detecting puncta in your images. Click Puncta in the 3D Environment to enable use of the puncta detection tools.

In multi-color-channel images, you must select a color channel in order to detect puncta. Select the color channel in either the Channel panel in the 3D environment or in the Image Adjustment tool in the 2D window.

Manually detecting individual puncta

Select the channel for the puncta of interest in the Channel panel on the left side of the 3D window. Any puncta detected will be assigned to this color channel.
To model a single punctum, click in the image; the detected punctum is displayed with a color overlay. If Neurolucida 360 software cannot detect a punctum, adjusting the Detection Settings may help.

Detect all puncta automatically by adjusting settings as described below and clicking Detect All.

Puncta-detection panel settings and buttons

Detection Type

Choose either:

All: Use to detect all puncta based on the detection settings you specify.

Proximity: Use to detect puncta based on both the specified detection settings and puncta proximity to other structures. See Proximity Settings.

Detection Settings

The settings in the Detect Puncta panel affect both automatic and manual puncta detection.

Type values into the boxes or use the arrows to incrementally increase/decrease the settings.

Max Detector Diameter: Maximum diameter in microns of potential punctum.
Min Detector Diameter: Minimum diameter in microns of potential punctum. Adjust to ignore objects that are too small to be considered puncta, e.g., background fluorescence/staining.
Measure: Click to measure the size of puncta using the diameter of the circular cursor.
1. Use the mouse scroll wheel + CTRL on the keyboard to adjust the size of the circular cursor to match the maximum or minimum diameter of potential punctum.
2. Click anywhere inside the image to accept the current diameter. The Max/Min Detector Diameter setting will be automatically updated.
Detector Sensitivity: A lower value may result in fewer puncta detected; too high may result in erroneous puncta detection.

Buttons

Preview Seeds: Click to display a preview of puncta modeling using the current puncta-detection settings.
Reset: Resets puncta-detection settings to their default values.

Detection Method

Choose either:

Automatic: Use automatic detection to quickly preview puncta detection so that you can evaluate the effects of modifying puncta-detection settings.
ML-Assisted: Use machine-learning enhanced puncta detection. Once appropriate settings for your images have been established, we recommend using the machine-learning puncta detection method.

Checkboxes

Exclude detection in locations with existing puncta: Check the box to prevent additional puncta being placed in the same location as puncta that were detected in a different channel.
Keep existing puncta: Check the box to detect new puncta without replacing any previously detected puncta in the same channel. Useful if different puncta are modeled well using different detection settings.
Filter image noise: If your image contains nonspecific staining and/or fluorescence, such as shot noise, check the box to minimize its effects on the analysis.

Buttons

Detect All: Automatically detects puncta based on the settings defined in the Detection settings.
Clear Last: Deletes the puncta detected in the previous detection instance.

Options

Center after each manual detection: Check the box to automatically center the window at the last point clicked and avoid the need to pan manually while modeling puncta.

Proximity Settings

If Proximity was selected as the Detection type, you must create one or more Rule Sets to define how you want Neurolucida 360 to look for and detect puncta in your image.

Proximity -based detection is used to detect puncta that are in a certain location relative to, or within a certain distance of other modeled structures, including (trees, spines, varicosities, vessels, somas, and/or puncta in other color channels). Rule Sets can be created to include different structure and location combinations for proximity-based detection. These sets can be saved and reused to provide consistency between different images and experiments. The rule sets can also be saved as part of 3D settings to be used in the batch pipeline for processing entire directories of data.

Proximity Settings:

Rule set: Choose a rule set from the dropdown menu to apply it to an automatic detection.
New: Click to open the Proximity Rules window.
Edit: Choose a rule set from the dropdown menu then click Edit to open the Proximity Rules window. Change the rules as needed in the dropdown menus, then click done. This will overwrite the original rule set.

Proximity Rules window

Use the Proximity Rules window to create or edit rule sets. Enter the name of the rule set, choose options from the Proximity, Trace Object, and Channel dropdown menus, and enter a distance if applicable. Click Add Rule to add another rule to the rule set if desired. Click Done to save the rule set.

Proximity rule set examples

Example: Detect puncta that are either inside somas associated with the red channel, or inside trees associated with the red channel:

Example: Detect puncta that are inside somas associated with the red channel and are also within 1 µm of puncta associated with the green channel:

Example : Detect puncta that are within 2 µm of spines associated with the red channel and are also within 1 µm of puncta associated with the green channel, or puncta that are within 2 µm of spines associated with the red channel and are also within 1 µm of puncta associated with the blue channel:

Choose desired rules from the dropdown menus:

Proximity: Choose location for detections:
- In proximity to: Detect puncta within a user-defined distance of the Trace Object specified below.
- Inside: Detect puncta that are inside the Trace Object specified below. (Distance doesn't apply.)
- Outside: Detect puncta that are outside the Trace Object specified below. (Distance doesn't apply.)
Trace Object: Choose one modeled object type (you can add additional rules to your Rule set to specify proximity to other structures).
Channel: Choose the Trace Object color channel the modeled object should be associated with:
- Any: Includes objects associated with all color channels and objects associated with individual color channels.
- All: Includes objects only associated with all color channels.
- Single: Includes objects only associated with a single color channel. All acquired color channels in the image will be available as a choice in the Channel dropdown menu.
To learn more about associations between traced objects and channels, see Using individual color channels (3D)
Distance (µm): Click in the box under Distance and type in the desired distance in microns. Puncta will only be detected if the distance between the edge of the Trace object and the edge of the puncta fall within the specified distance.

Distance setting is used only when In proximity to was chosen from the Proximity dropdown menu.

Delete rule set: Will delete the rule set selected in the Rule Set dropdown list. You can also click the red X next to a rule to delete it from a rule set.

Add rule: Click to add rules to a rule set. A new row will become visible. Select the new rules from the dropdown menus. In the first column, choose:

And: Requires the puncta to follow both the previous rule and the rule after And.

or
Or: Requires the puncta to follow either the previous rule or the rule just after Or.

Cancel: Changes are discarded and you return to the Detect puncta panel.

Done: Your adjustments are saved and you return to the Detect puncta panel.