A method for quantification of vesicular compartments within cells using 3D reconstructed confocal z-stacks: Comparison of ImageJ and Imaris to count early endosomes within basal forebrain cholinergic neurons

Background: Phenotypic changes in vesicular compartments are an early pathological hallmark of many peripheral and central diseases. For example, accurate assessment of early endosome pathology is crucial to the study of Down syndrome (DS) and Alzheimer's disease (AD), as well as other neurological disorders with endosomal-lysosomal pathology. New method: We describe a method for quantification of immunolabeled early endosomes within transmitteridentified basal forebrain cholinergic neurons (BFCNs) using 3-dimensional (3D) reconstructed confocal zstacks employing Imaris software. Results: Quantification of 3D reconstructed z-stacks was performed using two different image analysis programs: ImageJ and Imaris. We found ImageJ consistently overcounted the number of early endosomes present within individual BFCNs. Difficulty separating densely packed early endosomes within defined BFCNs was observed in ImageJ compared to Imaris. Comparison with existing methods: Previous methods quantifying endosomal-lysosomal pathology relied on confocal microscopy images taken in a single plane of focus. Since early endosomes are distributed throughout the soma and neuronal processes of BFCNs, critical insight into the abnormal early endosome phenotype may be lost as a result of analyzing only a single image of the perikaryon. Rather than relying on a representative sampling, this protocol enables precise, direct quantification of all immunolabeled vesicles within a defined cell of interest. Conclusions: Imaris is an ideal program for accurately counting punctate vesicles in the context of dual label confocal microscopy. Superior image resolution and detailed algorithms offered by Imaris make precise and rigorous quantification of individual early endosomes dispersed throughout a BFCN in 3D space readily achievable.

Automated summary

This study presents a method for quantifying immunolabeled early endosomes within basal forebrain cholinergic neurons using 3D reconstructed confocal z-stacks with Imaris software. Comparison of two image analysis programs, ImageJ and Imaris, revealed that Imaris provides superior accuracy and resolution in quantifying early endosomes. The method enables precise and direct quantification of all immunolabeled vesicles within a defined cell of interest.