Functional Steps in the AutoMorphoTrack Workflow

AutoMorphoTrack Package Structure

Representative Outputs Generated by AutoMorphoTrack

Automated detection, lysosomal counting, and mitochondrial morphology classification.

(A) Composite multichannel fluorescence image showing mitochondria and lysosomes, followed by independent detection maps for mitochondria. Scale bar = 10 µm. (B) Each lysosome is labeled and counted per frame, with a corresponding temporal plot illustrating vesicle abundance across the recording. (C) Mitochondrial morphology is classified as elongated or punctate based on area and eccentricity thresholds, shown as a labeled frame (frame 0) and quantitative summary chart. Together, these panels depict the initial detection and morphological analysis workflow implemented in AutoMorphoTrack.

Quantitative shape profiling of organelles.

(A) Continuous shape descriptors extracted for all detected organelles are summarized as parameter distributions showing variability in area, circularity, solidity, eccentricity, and aspect ratio within a single neuron. (B) Comparative violin plots illustrate differences in these shape metrics between mitochondria and lysosomes, providing a continuous morphological quantification beyond categorical classification.

Organelle tracking and motility characterization.

(A) Centroid-based trajectory reconstructions show cumulative lysosomal and mitochondrial movements and a composite overlay of both populations. (B) Quantitative motility metrics are summarized as velocity and displacement distributions. (C) A scatter plot of individual trajectories, together capturing the dynamic range of organelle transport within a neuron.

Mitochondria–lysosome colocalization within single neurons.

(A) Representative overlay highlighting regions of spatial overlap between mitochondria and lysosomes. (B) Quantitative summary plot showing Manders M1/M2 and Pearson r coefficients across time. These analyses quantify the frequency and degree of inter-organelle contact within the cell.

Integrated correlation of morphology, motility, and colocalization metrics.

Comprehensive correlation matrix displaying pairwise Pearson relationships among morphological descriptors, motility parameters, and colocalization indices derived from AutoMorphoTrack outputs. The matrix reveals coordinated trends linking structural and dynamic features within a single neuron.

Lysosome and mitochondrial comparisons between neurons.

(A) Comparative plots showing differences in lysosomal abundance per frame and (B) mitochondrial morphology classification between two neurons. Variations highlight cell-specific differences in vesicle turnover and mitochondrial network organization.

Comparative shape metrics across neurons.

Violin plots comparing (A) circularity, (B) aspect ratio, (C) solidity, (D) eccentricity, and (E) area between Neuron 1 and Neuron 2. Distinct parameter distributions reveal structural heterogeneity and variability in organelle geometry across cells.

Comparative motility parameters between neurons.

Distributions of total displacement (A) and mean velocity (B) for mitochondria and lysosomes from two neurons. Differences demonstrate cell-specific organelle transport dynamics and variability in overall motility behavior.

Comparative colocalization metrics between neurons.

Violin plots showing differences in Manders M1 (A), Manders M2 (B), Pearson r (C), and percent overlap (D) between Neuron 1 and Neuron 2. Divergent distributions indicate variability in mitochondria–lysosome coupling efficiency across cells.

Differential correlation matrix between neurons.

Subtraction map of correlation matrices from Neuron 1 and Neuron 2, highlighting parameters whose inter-relationships differ most strongly between cells. The heatmap summarizes shifts in coordination among morphology, motility, and interaction features, revealing neuron-specific patterns of subcellular organization.

AutoMorphoTrack outputs of other analyzed image stacks.

Functional Steps in the AutoMorphoTrack workflow were run on additional image stacks to verify the validity and applicability of the package. (A) Shows channel isolation, thresholding, and organelle segmentation. (B) Organelle segmentation enables the quantification of lysosomes across multiple frames. (C) The segmented mitochondrial channel is used to quantify mitochondrial morphology (Elongated vs Punctate). (D and E) Analysis of organelle morphology and structural profiling across different measures. (F and G). The trajectory of organelles and the cumulative path taken is quantified and visualized, along with displacement and velocity. (H) The colocalization of mitochondria and lysosomes is visualized and quantified across various approaches. (I) A comparative analysis of the image stack is conducted across all the measures discussed earlier.