Calcium-responsive contrast agents for magnetic resonance imaging (MRI) offer a promising approach for noninvasive brain-wide monitoring of neural activity at any arbitrary depth. Current examples of MRI-based calcium probes involve synthetic molecules and nanoparticles, which cannot be used to examine calcium signaling in a genetically encoded form. Here, we describe a new MRI sensor for calcium, based entirely on a naturally occurring calcium-binding protein known as calprotectin. Calciumbinding causes calprotectin to sequester manganese ions, thereby limiting Mn 2+ enhanced paramagnetic relaxation of nearby water molecules. We demonstrate that this mechanism allows calprotectin to alter T 1 and T 2 based MRI signals in response to biologically relevant calcium concentrations. The resulting response amplitude, i.e., change in relaxation time, is comparable to existing MRI-based calcium sensors as well as other reported protein-based MRI sensors. As a preliminary demonstration of its biological applicability, we used calprotectin to detect calcium in a lysed hippocampal cell preparation as well as in intact Chinese hamster ovary cells treated with a calcium ionophore. Calprotectin thus represents a promising path toward noninvasive imaging of calcium signaling by combining the molecular and cellular specificity of genetically encodable tools with the ability of MRI to image through scattering tissue of any size and depth.

Calcium ions represent one of the key second messengers accompanying neural activity and synaptic signaling. Accordingly, dynamic imaging of calcium fluctuations in living organisms represents a cornerstone technology for discovering neural mechanisms that underlie memory, determine behavior, and modulate emotional states as well as how these mechanisms are perturbed by neurological disease and brain injury. While optical technologies are well established for high resolution imaging of calcium dynamics, physical limits on light penetration hinder their application for whole-brain imaging in intact vertebrates. Unlike optics, magnetic resonance imaging (MRI) enables noninvasive large-scale imaging across vertebrates of all sizes. This has motivated the development of several sensors that leverage innovative physicochemical mechanisms to sensitize MRI contrast to intracellular and extracellular changes in calcium. Here, we review the current state-of-the-art in MRI-based calcium sensors, focusing on fundamental aspects of sensor performance, in vivo applications, and challenges related to sensitivity. We also highlight how innovations at the intersection of reporter gene technology and gene delivery open potential opportunities for mapping calcium activity in genetically targeted cells, complementing the benefits of small molecule probes and nanoparticle sensors.

Imaging transgene expression in live tissues requires reporters that are detectable with deeply penetrant modalities, such as magnetic resonance imaging (MRI). Here, we show that LSAqp1, a water channel engineered from aquaporin-1, can be used to create background-free, drug-gated, and multiplex images of gene expression using MRI. LSAqp1 is a fusion protein composed of aquaporin-1 and a degradation tag that is sensitive to a cell-permeable ligand, which allows for dynamic small molecule modulation of MRI signals. LSAqp1 improves specificity for imaging gene expression by allowing reporter signals to be conditionally activated and distinguished from the tissue background by difference imaging. In addition, by engineering destabilized aquaporin-1 variants with different ligand requirements, it is possible to image distinct cell types simultaneously. Finally, we expressed LSAqp1 in a tumor model and showed successful in vivo imaging of gene expression without background activity. LSAqp1 provides a conceptually unique approach to accurately measure gene expression in living organisms by combining the physics of water diffusion and biotechnology tools to control protein stability.

Calcium-responsive contrast agents for magnetic resonance imaging (MRI) offer an attractive approach to noninvasively image neural activity with wide coverage in deep brain regions. However, current MRI sensors for calcium are based on synthetic architectures fundamentally incompatible with genetic technologies for in vivo delivery and targeting. Here, we present a protein-based MRI sensor for calcium, derived from a calcium-binding protein known as calprotectin. Calcium-binding causes calprotectin to sequester manganese. We demonstrate that this mechanism allows calprotectin to alter T1 and T2 weighted contrast in response to biologically relevant calcium concentrations. Corresponding changes in relaxation times are comparable to synthetic calcium sensors and exceed those of previous protein-based MRI sensors for other neurochemical targets. The biological applicability of calprotectin was established by detecting calcium in lysates prepared from a neuronal cell line. Calprotectin thus represents a promising path towards imaging neural activity by combining the benefits of MRI and protein sensors.