Note: The Exocytosis Detection + Vesicle Tracking recipe has been merged into the new Exocytosis Detection recipe in Aivia 7. For information about the new Exocytosis Detection recipe, please go here.


Recipe Description


The Exocytosis Event Detection recipes in Aivia detects exocytotic events in total internal reflection fluorescent (TIRF) microscopy images. There are two (2) versions of the recipes: one for event detection only; the other for detecting events and tracking vesicles associated with exocytosis. The recipe 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

Parameters and their descriptions are summarized in the table below.

Preset Group
Parameter Name
Min Value
Max Value
Description
Vesicle Detection
Vesicle Threshold
(with Vesicle Tracking only)
0
65,535
Adjusts the detection sensitivity for vesicle detection; a lower value will detect more vesicles
Vesicle Detection
Min Vesicle Size
(with Vesicle Tracking only)
0
65,535
Specifies the minimum size of a detected object to be included in the analysis results based on the area (in pixels) of the detected objects
Event
Detection
Event Intensity Change (counts)
0
65,535
Adjusts the detection sensitivity for event detection based on the amount of intensity change (in number of gray levels) at the event from baseline; a lower value will detect more events
Event Detection
Transience
0
100
Specifies the range of duration for the events by computing a normalized ratio for the difference between maximum and average intensities to the difference between the average and minimum intensities; a lower value will enable detection of events with longer durations
Event Detection
Bright Detection Frame (Enable Bright Detection only)
0
65,535
Specifies the maximum number of frames for an event with extended intensity increase in its surrounding regions may last; this parameter is used only when Enable Bright Detection is selected
Event Detection
Enable \ Disable Duplicate Events Removal
Toggles the removal of detected events that occurs in a given region in successive time frames
Event Filter
Event Size Filter
0
5,000
Specifies the minimum size of a detected event to be included in the analysis results based on the area of the detected event
Event Filter
Event Mean Intensity Filter
0
65,535
Specifies the minimum threshold for mean intensity of the detected event region to be included in the analysis results
Event Filter
Event Total Intensity Filter (Enable Event Total Intensity Filter only)
0
1 x 1018
Specifies the minimum threshold for the total intensity of the detected event region to be included in the analysis results

Presets

There are two preset groups in the Exocytosis Event Detection-only recipe: Event Detection and Event Filter. The Exocytosis Event Detection with Vesicle Tracking has one additional preset group: Vesicle Detection. Each group has three pre-configured parameter grouping to help you get started on the analysis. The default preset values are as follows:

Vesicle Detection (Vesicle Tracking only)

Parameter Name
Dim
Medium
Bright
Vesicle Threshold
15
25
40
Min Vesicle Size
4
4
4

Event Detection

Parameter Name
Gross
Short
Long
Event Intensity Change (counts)
2,000
2,000
2,000
Transience
0 - 20
0 - 20
0 -40
Bright Detection Frame
0
2
5

Event Filter

Parameter Name
Sml-Bright
Med-Med
Large-Dim
Event Size Filter
3
15
50
Event Mean Intensity Filter
5,000
3,000
1,000
Event Total Intensity Filter
15,000
45,000
50,000

Measurements


The Evocytosis Event Detection recipe generates intensity measurements for each detected event and, in the with Vesicle Tracking recipe option, for each detected particle plus track measurements for the particles. You can add additional measurements to the analysis results by using the Measurement Tool in Aivia. The measurements generated by the recipe are as follows.

Measurement
Events
Vesicle Tracks
Mean Intensity
x
x
Total Intensity
x
x
Area

x
Total Time

x
First Frame

x
Last Frame

x
X

x
Y

x
Velocity Magnitude (instantaneous)

x
Additional events measurements

  • Total Events Detected
  • First Event Frame
  • Has Relation (boolean)
  • Events Detected on Track (boolean)
  • Event on Frame (instaneous, boolean)
  • Total Events in Image (Summary)


Tutorial


Before beginning the tutorial, please download the Exocytosis Event Detection Demo Image (ExocytosisDemo.zip). For information on how to select presets or modify parameter values, please refer to the tutorial on how to use the Recipe Console. You can download the image directly here.
  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 Exo Event Detection + Vesicle Tracking recipe
  3. Select the following preset for each preset group; if you are using the Exo Event Detection recipe, ignore the Vesicle Detection preset settings:
    • 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 in tact
    • Event Intensity Change (counts): 1,000
  6. Click the From beginning button or press the F4 key on your keyboard to begin applying the recipe to the image

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

Results

Exocytosis Event Detection (blue) + Vesicle Tracking (green) results

Image Credits

Takashi Tsuboi, University of Tokyo