Threshold Analysis for Objects

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Overview

The Threshold Analysis for Objects plugin in Microscopy Image Browser (MIB) performs shape and intensity threshold-based analysis of objects in microscopy images, calculating metrics such as size, intensity, or shape.It supports triangulation of object centroids, visualization of results, and export to Excel or MATLAB formats, making it useful for quantitative studies of cellular structures.

GUI Components

Selection of materials and threshold settings

Object of interest use this dropdown to select material of the model for analysis.
Alternatively, it is possible to provide indices of materials using an editbox on the right-hand side of the dropdown.

Color channel to use specify index of the color channel to be used for intensity calculations and thresholding

Threshold policy specify method to threshold the objects:

Absolute provide thresholding value in the Threshold value field. All values that are above this value will be assigned to the Mask layer after press of the Start
Relative allows to choose between Otsu and Median automatic thresholding options. Values above detected threshold will be assigned to the Mask layer. Note! it is possible to modify the threshold value by offsetting it using the Offset value editbox on the right-hand side.

Erode+Dilate additionally applies erosion and dilation operation to minimize thresholding noise and keeping only large thresholded structures.

Application of Erode+Dilate

Object connectivity select connectivity of the object detection, when 4 objects that are touching each other in the diagonal orientation will be split, otherwise (when 8) fused together.

Additional options

Calculate min diameter use this option to measure average thickness of objects. When Highlight as Selection is checked, the point used to measure mean thickness for each object are shown as the Selection layer. Use Shift+C to clear it after evaluation.

Triangulate centroids for multiple objects calculate the triangulated mesh where each edge in this mesh contains distance to the closest other object. This can be useful for analysis of distances between the objects. Remove free boundary facets can be used to remove edges that are connecting objects at the edges of the image producing too large connecting lines.

Remove free boundary facets example

Export results

Make Plot generate a plot with results. It is possible to specify Figure Id to show the plot in a specific MATLAB window and start Autoprint dialog for automatic printing of the figure.

Example of the figure with results

MATLAB Workspace export results to the main MATLAB workspace as ThreshAnalysis and ThreshAnalysisGraph structures.

Fields

ThreshAnalysis has following fields:

    colCh
    materialId
    materialNames
    absoluteThresholdValue
    relativeThresholdMethod
    thresholdOffsetValue
    thresholdPolicy
    ErodeDilate
    pixSize
    datasetName
    modelName
    triangulatePoints
    triangulateRemoveFreeBoundary
    objConnectivity
    objId
    sliceNo
    objMaterialName
    CentroidX
    CentroidY
    FirstAxisLength
    SecondAxisLength
    Eccentricity
    Median
    TotalArea
    SliceName
    ThresholdedArea
    objThresholdValues
    RatioOfAreas
    minDiameter
    minDiameterAverage

graph with properties: ThreshAnalysisGraph is a graph object with the fillowing fields:

    Edges: [39×1 table]
    Nodes: [18×2 table]

Excel save results in Microsoft Excel format .xls

MATLAB format save results in MATLAB format .mat

Triangulation save results of object triangulation in MATLAB format .mat, Amira, or as Excel sheet.

... specify output filename for results.

Regenerate Output Path use this button to regenerate output filename relative to the open dataset. It is useful when processing multiple datasets one after another.

Start calculations

Press the Start button to perform calculations.

Usage

Access the plugin via:
Menu → Plugins → Organelle Analysis → Thres Analysis for Objects.

Follow these steps to analyze objects:

Load Image:

Prepare Model

Create or load a model with segmented objects via Segmentation table -> Load.
Use MIB’s Segmentation tools (e.g., Brush, Threshold, Segment-anything model) to segment objects.

Segmentation model