Aivia Software

Exocytosis Detection

Owned by Hoyin Lai
Last updated: Jan 28, 2023 by Aiden Maloney-Bertelli (Unlicensed)Version comment
3 min read

The Exocytosis Detection recipe in Aivia detects exocytotic events in total internal reflection fluorescent (TIRF) microscopy images.

This recipe provides an option for tracking vesicles associated with exocytosis and is tunable for the detection of small, point exocytosis events (e.g., kiss-and-run) as well as large, diffuse events (e.g., full fusion).

Parameters and Presets

Parameters

Recipe parameters for Exocytosis Detection and their descriptions are summarized in the table below.

Preset Group

Parameter Name

Min Value

Max Value

Description

Preset Group

Parameter Name

Min Value

Max Value

Description

Vesicle Detection

Vesicle Threshold

(Vesicle Tracking only)

1

255

Adjusts the sensitivity for vesicle detection; a lower value will detect more vesicles

Min Vesicle Size

(Vesicle Tracking only)

0

100 px2 or µm2

Specifies the minimum size of a detected object to be included in the analysis results based on the areas of the detected objects

Event Detection

 

Event Intensity Change

0

255 (8-bit)

65,535 (16-bit)

Adjusts the sensitivity for event detection based on the size of the intensity change (in number of gray levels) at the event from baseline; a lower value will detect more events

Transience

0

100

Specifies the range of duration for the events by computing a normalized ratio for the difference between the maximum and average intensities to the difference between the average and minimum intensities; a lower value will enable detection of events with longer durations

Bright Detection Frame

(Enable Bright Detection only)

0

255 (8-bit)

65,535 (16-bit)

Specifies the maximum number of frames that an event with extended intensity increase in its surrounding regions may last; this parameter is used only when Enable Bright Detection is selected

Duplicate Events Removal

Not applicable

Toggles the removal of detected events that occur in a given region in successive time frames

Event Filter

Event Size Filter

0

1,000 px2 or µm2

Specifies the minimum size of a detected event to be included in the analysis results based on the area of the detected event

Event Mean Intensity Filter

0

255 (8-bit)

65,535 (16-bit)

Specifies the minimum threshold for mean intensity of a detected event region to be included in the analysis results

Event Total Intensity Filter

(Enable Event Total Intensity Filter only)

0

1 x 1018

Specifies the minimum threshold for the total intensity of a detected event region to be included in the analysis results

 

Presets

There are three preset groups in the recipe: Vesicle Detection, Event Detection and Event Filter. Each group has three pre-configured parameter groupings to help you get started on the analysis. The default preset values are as follows:

 

Vesicle Detection

Parameter Name

Dim

Medium

Bright

Vesicle Threshold

15

25

40

Min Vesicle Size

0.05 px2 or µm2

0.05 px2 or µm2

0.05 px2 or µm2



 

Event Detection

Parameter Name

Gross

Short

Long

Event Intensity Change

8 (8-bit)
1,966 (16-bit)

8 (8-bit)
1,966 (16-bit)

8 (8-bit)
1,966 (16-bit)

Transience

0 - 40

0 - 40

0 - 20

Bright Detection Frame

0

2

5



 

Event Filter

Parameter Name

Sm-Bright

Medium

Large-Dim

Event Size Filter

0.05 px2 or µm2

0.15 px2 or µm2

0.5 px2 or µm2

Event Mean Intensity Filter

19 (8-bit)
4,981 (16-bit)

12 (8-bit)
3,002 (16-bit)

5 (8-bit)
1,311 (16-bit)

Event Total Intensity Filter

15,000

45,000

50,000



 

Tutorial

Before beginning the tutorial, please download the Exocytosis Detection Demo image. For information on how to select presets or modify parameter values, please refer to the tutorial on how to use the Recipe Console.

  1. Unzip the demo file and load the demo image, “ExocytosisDemo.tif,” into Aivia.

  2. In the Recipe Console, click on the Recipe selection dropdown menu and select the Exocytosis Detection recipe.

  3. Select the following presets for each preset group:

    • Vesicle Detection: Medium

    • Event Detection: Gross

    • Event Filter: Large-Dim

  4. Click on the caret to the left of the Event Detection preset group to show the preset parameters.

  5. Modify the parameter value as follows, leaving the other parameters intact:

    • Event Intensity Change (counts): 1,000

  6. Click the Start button or press the F4 key on your keyboard to begin applying the recipe to the image.

The detected particle tracks and outlines for the events will be overlaid on the image.

 

Results

 

Exocytosis Detection tutorial results

 

Measurements

The Exocytosis Detection recipe generates intensity and position measurements for each detected event and, if Vesicle Tracking is enabled, for each detected particle in addition to morphology and advanced count measurements for the particles. You can add additional measurements to the analysis results with the Measurement Tool in Aivia and view measurement definitions on the Measurement Definitions page. The measurements generated by the recipe are given in the table below.

Object Set

Morphology

Intensity

Position

Advanced

Object Set

Morphology

Intensity

Position

Advanced

Events

None

  • Pixel-based Mean Intensity

  • Pixel-based Total Intensity

  • Centroid X

  • Centroid Y

None

Vesicle Tracks

  • Area

  • Pixel-based Mean Intensity

  • Pixel-based Total Intensity

  • Total Time

  • First Frame

  • Last Frame

  • Velocity Magnitude

  • Event on Frame

  • Total Events

 

Image Credits

Takashi Tsuboi, University of Tokyo, Tokyo, Japan