Mikhail G. Shapiro

Infrared light excites cells by changing their electrical capacitance.

Optical stimulation has enabled important advances in the study of brain function and other biological processes, and holds promise for medical applications ranging from hearing restoration to cardiac pace making. In particular, pulsed laser stimulation using infrared wavelengths >1.5 μm has therapeutic potential based on its ability to directly stimulate nerves and muscles without any genetic or chemical pre-treatment. However, the mechanism of infrared stimulation has been a mystery, hindering its path to the clinic. Here we show that infrared light excites cells through a novel, highly general electrostatic mechanism. Infrared pulses are absorbed by water, producing a rapid local increase in temperature. This heating reversibly alters the electrical capacitance of the plasma membrane, depolarizing the target cell. This mechanism is fully reversible and requires only the most basic properties of cell membranes. Our findings underscore the generality of pulsed infrared stimulation and its medical potential.

Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamine

The development of molecular probes that allow in vivo imaging of neural signaling processes with high temporal and spatial resolution remains challenging. Here we applied directed evolution techniques to create magnetic resonance imaging (MRI) contrast agents sensitive to the neurotransmitter dopamine. The sensors were derived from the heme domain of the bacterial cytochrome P450-BM3 (BM3h). Ligand binding to a site near BM3hs paramagnetic heme iron led to a drop in MRI signal enhancement and a shift in optical absorbance. Using an absorbance-based screen, we evolved the specificity of BM3h away from its natural ligand and toward dopamine, producing sensors with dissociation constants for dopamine of 3.3–8.9 μM. These molecules were used to image depolarization-triggered neurotransmitter release from PC12 cells and in the brains of live animals. Our results demonstrate the feasibility of molecular-level functional MRI using neural activity–dependent sensors, and our protein engineering approach can be generalized to create probes for other targets.

Genetically encoded reporters for hyperpolarized xenon magnetic resonance imaging

Magnetic resonance imaging (MRI) enables high-resolution non-invasive observation of the anatomy and function of intact organisms. However, previous MRI reporters of key biological processes tied to gene expression have been limited by the inherently low molecular sensitivity of conventional (1)H MRI. This limitation could be overcome through the use of hyperpolarized nuclei, such as in the noble gas xenon, but previous reporters acting on such nuclei have been synthetic. Here, we introduce the first genetically encoded reporters for hyperpolarized (129)Xe MRI. These expressible reporters are based on gas vesicles (GVs), gas-binding protein nanostructures expressed by certain buoyant microorganisms. We show that GVs are capable of chemical exchange saturation transfer interactions with xenon, which enables chemically amplified GV detection at picomolar concentrations (a 100- to 10,000-fold improvement over comparable constructs for (1)H MRI). We demonstrate the use of GVs as heterologously expressed indicators of gene expression and chemically targeted exogenous labels in MRI experiments performed on living cells.

Biogenic Gas Nanostructures as Ultrasonic Molecular Reporters

Ultrasound is among the most widely used non-invasive imaging modalities in biomedicine1, but plays a surprisingly small role in molecular imaging due to a lack of suitable molecular reporters on the nanoscale. Here we introduce a new class of reporters for ultrasound based on genetically encoded gas nanostructures from microorganisms, including bacteria and archaea. Gas vesicles are gas-filled protein-shelled compartments with typical widths of 45–250 nm and lengths of 100–600 nm that exclude water and are permeable to gas2, 3. We show that gas vesicles produce stable ultrasound contrast that is readily detected in vitro and in vivo, that their genetically encoded physical properties enable multiple modes of imaging, and that contrast enhancement through aggregation permits their use as molecular biosensors.

Protein Nanoparticles Engineered to Sense Kinase Activity in MRI

We introduce a family of protein nanoparticles capable of sensing analytes in conjunction with magnetic resonance imaging (MRI). The new sensors are derived from the iron storage protein ferritin (Ft); they are designed and optimized using facile protein engineering methods, and self-assembled in cells harboring specific combinations of DNA coding sequences. As illustration, we show that suitably constructed Ft-based sensors can report activity of the important neural signaling enzyme protein kinase A (PKA). Phosphorylation of the engineered Ft-based nanoparticles by PKA promotes clustering and changes in T(2)-weighted MRI signal.

NMR Hyperpolarization Techniques of Gases

Nuclear spin polarization can be significantly increased through the process of hyperpolarization, leading to an increase in the sensitivity of nuclear magnetic resonance (NMR) experiments by 4-8 orders of magnitude. Hyperpolarized gases, unlike liquids and solids, can often be readily separated and purified from the compounds used to mediate the hyperpolarization processes. These pure hyperpolarized gases enabled many novel MRI applications including the visualization of void spaces, imaging of lung function, and remote detection. Additionally, hyperpolarized gases can be dissolved in liquids and can be used as sensitive molecular probes and reporters. This Minireview covers the fundamentals of the preparation of hyperpolarized gases and focuses on selected applications of interest to biomedicine and materials science.

Tunable thermal bioswitches for in vivo control of microbial therapeutics

Temperature is a unique input signal that could be used by engineered microbial therapeutics to sense and respond to host conditions or spatially targeted external triggers such as focused ultrasound. To enable these possibilities, we present two families of tunable, orthogonal, temperature-dependent transcriptional repressors providing switch-like control of bacterial gene expression at thresholds spanning the biomedically relevant range of 32-46 °C. We integrate these molecular bioswitches into thermal logic circuits and demonstrate their utility in three in vivo microbial therapy scenarios, including spatially precise activation using focused ultrasound, modulation of activity in response to a host fever, and self-destruction after fecal elimination to prevent environmental escape. This technology provides a critical capability for coupling endogenous or applied thermal signals to cellular function in basic research, biomedical and industrial applications.

Non-invasive imaging using reporter genes altering cellular water permeability.

Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging.

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts

The mammalian microbiome has many important roles in health and disease, and genetic engineering is enabling the development of microbial therapeutics and diagnostics. A key determinant of the activity of both natural and engineered microorganisms in vivo is their location within the host organism. However, existing methods for imaging cellular location and function, primarily based on optical reporter genes, have limited deep tissue performance owing to light scattering or require radioactive tracers. Here we introduce acoustic reporter genes, which are genetic constructs that allow bacterial gene expression to be visualized in vivo using ultrasound, a widely available inexpensive technique with deep tissue penetration and high spatial resolution. These constructs are based on gas vesicles, a unique class of gas-filled protein nanostructures that are expressed primarily in water-dwelling photosynthetic organisms as a means to regulate buoyancy. Heterologous expression of engineered gene clusters encoding gas vesicles allows Escherichia coli and Salmonella typhimurium to be imaged noninvasively at volumetric densities below 0.01% with a resolution of less than 100 μm. We demonstrate the imaging of engineered cells in vivo in proof-of-concept models of gastrointestinal and tumour localization, and develop acoustically distinct reporters that enable multiplexed imaging of cellular populations. This technology equips microbial cells with a means to be visualized deep inside mammalian hosts, facilitating the study of the mammalian microbiome and the development of diagnostic and therapeutic cellular agents.

Thermal Mechanisms of Millimeter Wave Stimulation of Excitable Cells

Interactions between millimeter waves (MMWs) and biological systems have received increasing attention due to the growing use of MMW radiation in technologies ranging from experimental medical devices to telecommunications and airport security. Studies have shown that MMW exposure alters cellular function, especially in neurons and muscles. However, the biophysical mechanisms underlying such effects are still poorly understood. Due to the high aqueous absorbance of MMW, thermal mechanisms are likely. However, nonthermal mechanisms based on resonance effects have also been postulated. We studied MMW stimulation in a simplified preparation comprising Xenopus laevis oocytes expressing proteins that underlie membrane excitability. Using electrophysiological recordings simultaneously with 60 GHz stimulation, we observed changes in the kinetics and activity levels of voltage-gated potassium and sodium channels and a sodium-potassium pump that are consistent with a thermal mechanism. Furthermore, we showed that MMW stimulation significantly increased the action potential firing rate in oocytes coexpressing voltage-gated sodium and potassium channels, as predicted by thermal terms in the Hodgkin-Huxley model of neurons. Our results suggest that MMW stimulation produces significant thermally mediated effects on excitable cells via basic thermodynamic mechanisms that must be taken into account in the study and use of MMW radiation in biological systems.