Aneesh Tazhe Veetil

We thank all the authors, collaborators, and friends, particularly @Swathi for her efforts in building this probe from scratch. We thank @krishnanyamuna.bsky.social for the inputs, @tifrhyderabad.bsky.social, @DAE India and @India Alliance for the funding and kind support.

Microglial labeling using MITIGATE has worked in our hands in cultured microglia, larval zebrafish and mice. MITIGATE will be useful for visualizing microglial dynamics within primary human tissues and in human cerebral organoids without eliciting an immune response.

We extended the MITIGATE technology for biotinylation of surface proteins on microglia using the micro-mapping technology pioneered by the McMillan group and Jim Wells's lab.

Further, we have used the MITIGATE platform for tracking single microglia and their interaction with neurons in co-culture using photoactivatable fluorophores tethered to the microglial surface.

We have deployed MITIGATE to label microglia in larval zebrafish brains and studied microglia (green)-pathogen (red) interaction in wild-type fish.

MITIGATE technology is spectrally tunable to any color, thus making it suitable for deep-tissue imaging. It offers excellent specificity to microglia (red); astrocytes (green) and neurons were not labeled by MITIGATE)

Here is a first look at microglia (red) from a mouse brain using MITIGATE.

We named this new microglial imaging technology MITIGATE (Microglial Imaging Through Intrinsic GPCR-Assisted Tethering of Exogenous molecules).

The solution: We have developed a chemical tool to visualize and manipulate microglia by covalently targeting fluorophores to the most abundant surface marker of microglia, the P2RY12 receptor, which is a GPCR.

Also, genetic technologies show leaky expression in other cell types in the brain (e.g., neurons and astrocytes). Non-specificity is a big issue.

The research problem we addressed in this study—live visualization of microglia in wild-type organisms using genetic technologies (e.g., GFP) is challenging because they become activated upon viral transduction of fluorescent marker proteins (e.g., GFP).

GPCR-targeted imaging and manipulation of homeostatic microglia in living systems Microglia, the resident innate immune cells of the brain, are known to perform key roles such as synaptic pruning, apoptotic debris removal, and pathogen defense in the central nervous system. Microglial mutations are directly linked to many neurodevelopmental (e.g., schizophrenia) and neurodegenerative (e.g., Alzheimer's disease) disorders, indicating the diagnostic and therapeutic potential of microglia for treating these conditions. Currently, we lack robust molecular tools to specifically image and manipulate microglia in vivo, which presents a major hurdle in our understanding of the brain-wide functions of these cells during the early onset of brain diseases. Here, we describe a molecular technology for imaging and manipulation of homeostatic microglia in live organisms (e.g., in zebrafish and mice) by covalently targeting the purinergic receptor, P2RY12. Using this technology, we imaged microglia-pathogen interactions in the larval zebrafish brain and revealed various morphological states of microglia in the adult mouse brain. We further expanded the microglia labelling approach to single-microglia tracking and microglial surfaceome mapping using photoactivatable fluorophores and photoproximity labelling, respectively. We anticipate the use of this universal tool for studying microglial biology across species to reveal the dynamics and polarization of resting microglia into a reactive state found in many neurodegenerative diseases. ### Competing Interest Statement The authors have declared no competing interest.

Today, we report the first independent study from our lab describing a new technology for live imaging and manipulation of homeostatic microglia (brain-resident macrophages) in the brain.
www.biorxiv.org/content/10.1...