MotiQ: an open-source toolbox to quantify the cell motility and morphology of microglia

Abstract

MotiQ is an open-source software for automated quantification of microglial motility and morphology. MotiQ can be applied to in vivo, ex vivo, and in vitro data from confocal, epifluorescence, and two-photon microscopy. MotiQ is not limited to microglia—it can also be applied to other cell types.

Integrating human iPSC‐derived macrophage progenitors into retinal organoids to generate a mature retinal microglial niche

“…We next assayed the changes in MPC morphology over the duration of RO co‐culture by utilizing the MotiQ plugins Cropper, Thresholder, and 2D Analyzer available via ImageJ (Hansen et al, 2022). At 6 weeks PC, IBA1 and CD45 double‐positive MPCs have a higher ramification index, more branch points, and more junctions than at 2 weeks PC, validating that MPCs develop a mature ramified morphology (Figure 3c,d; Supp Figure 2).…”

Novel image analysis tool for rapid screening of cell morphology in preclinical animal models of disease

“…We acknowledge ours is not the first automated tool for assessing morphometric parameters of microglia [ 31 , 32 , 34 , 36 , 38 ]; however, we have expanded on other open-source methods by increasing the throughput of single-cell analysis (e.g., <30 min to identify and quantify ∼500 cells vs. hours to days for existing methods [ 35 , 38 ]) introducing the capability for both fluorescent and chromogenic detection methods, as well as incorporating additional biomarkers of activation. For example, our automated algorithm can provide information about phagocytic activity when multiple fluorophores are used by examining the intensity of the biomarker CD68 in addition to IBA-1 within each cell.…”

“…Existing methods for evaluating complex cell morphologies, like those of microglia, have often relied on manual scoring, which results in subjective and inherently biased data [ 29 , 30 ]. However, other groups have recognized the need for unbiased approaches and as such, have developed standardized methods, such as quantifying 2D (surface area) & 3D (volumetric) characteristics [ [31] , [32] , [33] ], measuring total length and branching complexity of microglial processes [ 34 , 35 ], or clustering microglia into distinct phenotypic subtypes based on expression of various biomarkers [ 36 , 37 ]. The major limitations with these approaches, however, is that some are only partially automated and rely on manual cell selection and/or tracing [ 29 , 30 ], they require high magnification & high-resolution images, which results in low-to medium-throughput analyses [ 32 , 34 , 36 ], and most studies report either fluorescent [ 35 , 36 ] or chromogen [ 38 ] detection methods, but no published method demonstrates the versatility of their tool to analyze morphologies labeled using both detection methods.…”

The GPR84 molecule is a mediator of a subpopulation of retinal microglia that promote TNF/IL‐1α expression via the rho‐ROCK pathway after optic nerve injury

“…Microglial images of the flat‐mounted retinas of Iba1‐EGFP mice were captured with fluorescence microscopy (BZ‐X810) through z stacks. Morphological parameters were assessed with MotiQ, software that is used for automated quantification of microglial motility and morphology (Hansen et al, 2022).…”

“…T A B L E 3 Antibody information for immunohistochemistry. Morphological parameters were assessed with MotiQ, software that is used for automated quantification of microglial motility and morphology (Hansen et al, 2022).…”