Glia Cell Morphology Analysis Using the Fiji GliaMorph Toolkit

Abstract

Glial cells are the support cells of the nervous system. Glial cells typically have elaborate morphologies that facilitate close contacts with neighboring neurons, synapses, and the vasculature. In the retina, Müller glia (MG) are the principal glial cell type that supports neuronal function by providing a myriad of supportive functions via intricate cell morphologies and precise contacts. Thus, complex glial morphology is critical for glial function, but remains challenging to resolve at a sub‐cellular level or reproducibly quantify in complex tissues. To address this issue, we developed GliaMorph as a Fiji‐based macro toolkit that allows 3D glial cell morphology analysis in the developing and mature retina. As GliaMorph is implemented in a modular fashion, here we present guides to (a) setup of GliaMorph, (b) data understanding in 3D, including z‐axis intensity decay and signal‐to‐noise ratio, (c) pre‐processing data to enhance image quality, (d) performing and examining image segmentation, and (e) 3D quantification of MG features, including apicobasal texture analysis. To allow easier application, GliaMorph tools are supported with graphical user interfaces where appropriate, and example data are publicly available to facilitate adoption. Further, GliaMorph can be modified to meet users’ morphological analysis needs for other glial or neuronal shapes. Finally, this article provides users with an in‐depth understanding of data requirements and the workflow of GliaMorph. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Download and installation of GliaMorph components including example data Basic Protocol 2: Understanding data properties and quality 3D—essential for subsequent analysis and capturing data property issues early Basic Protocol 3: Pre‐processing AiryScan microscopy data for analysis Alternate Protocol: Pre‐processing confocal microscopy data for analysis Basic Protocol 4: Segmentation of glial cells Basic Protocol 5: 3D quantification of glial cell morphology

GliaMorph: a modular image analysis toolkit to quantify Müller glial cell morphology

“…Implementing codes as macros with graphical user interfaces allows direct use and alteration of code, even by users without any coding experience. This is supported by the online availability of code ( ), example data (Zenodo: ), YouTube tutorials ( ), as well as a step-by-step user guide Kugler et al (2023) .…”

“…Manual measurements of retina size and MG cell bodies were performed using a line ROI (for details, see workflow document in Kugler et al, 2023 ). Manual measurements of intensity distributions were performed using a line ROI and plotting intensity values from maximum intensity projections (MIPs).…”

Bone-derived PDGF-BB enhances hippocampal non-specific transcytosis through microglia-endothelial crosstalk in HFD-induced metabolic syndrome

“…Microglial morphology analysis was performed as previously described [ 28 ]. Briefly, morphological analyses of microglia were performed using ImageJ software (National Institutes of Health, USA).…”