Biology subjects

Shapiro, M. G.

Publications and source records attributed to Shapiro, M. G..

At least 19 recordsLinked to original sources

bioRxiv2026

Longitudinal imaging of single cells in opaque tissue with cell chirp ultrasound

Small numbers of cells can determine the fate of an entire organism. For example, metastatic cancer cells seed distant organs, therapy-resistant clones repopulate tumors, and T cells infiltrate and expand in tissues with matching antigens. Visualizing the activity of such cells in living organisms requires highly sensitive, specific and repeatable imaging in optically opaque tissues. Here we introduce cell chirp, an ultrasound imaging method for real-time imaging of single cells in living organs inaccessible to light. Cell chirp exploits the harmonic response of genetically encoded gas vesicles to frequency-coded ultrasound to sensitively and specifically visualize single cells in scattering background. We first develop this method in tissue-mimicking phantoms, demonstrating the ability to quantitatively detect single cells as validated by optical cell counts. We then establish the ability of cell chirp to image thousands of genetically labeled cells within a million-cell background tissue, track reporter gene expression over days, and suppress background signals by more than two orders of magnitude. Finally, we use cell chirp to track the fate of antigen-positive or antigen-negative tumor-cell minorities during weeks-long T-cell engager treatments. Cell chirp reveals the dynamics of sparsely distributed, genetically defined cells in opaque tissues.

bioengineering↗

bioRxiv2026

Cytosolic MagLOV Magnetofluorescence in Mammalian Cell Lines and Primary Neurons

Magnetic fields can influence the outcome of photochemical reactions through the radical pair mechanism, but whether this sensitivity extends to standard experimental conditions in mammalian cells has remained unclear. Here, we quantify the cytosolic magnetofluorescence of MagLOV, a genetically encoded, flavin-binding fluorescent protein, in mammalian cells, validated against extensive artifact controls. Excitation intensity and magnetic field strength differentially tune response kinetics and amplitude, respectively, with amplitude saturating above approximately 8 mT. Magnetic field-effect amplitude is further modulated by cellular culture state. This response generalizes across HEK293T, HeLa, U2OS, and A549 cells and primary mouse cortical neurons, with plateau amplitudes ranging from 1.4% to 2.7%. Together, these results establish that genetically encoded spin-dependent photochemistry can be quantitatively interrogated under standard mammalian live-cell imaging conditions, and generalizes across mammalian cell types.

biophysics↗

bioRxiv2026

Functional ultrasound imaging through a human cranial window for mesoscopic mapping of motor effector encoding within the sensorimotor cortex

Understanding movement encoding within human cortical circuits has been essential for advancing brain computer interfaces (BCIs). However, there are limited minimally invasive, high resolution neurorecording methods sensitive enough to detect single-trial movement-correlated neural activity. Functional ultrasound imaging (fUSI) provides submillimeter spatial resolution of deep cortical tissue with high sensitivity and, when paired with acoustically transparent skull implants, enables transcutaneous recording of human neurovascular changes. Prior studies have used fUSI in participants with acoustically transparent skull implants for on-off task mapping and decoding. Here, we demonstrate fUSIs ability to reliably resolve multi-body-part and single digit movement encoding within the primary sensorimotor cortex in a participant with an acoustically transparent skull implant. We obtained fine-grained mappings of individual effector representation that were consistent with classic somatotopy for both multi-body-part and single digit movement. We were able to resolve single-trial event-related activity, enabling single-trial decoding of both conditions. Analysis of voxels important for decoding suggested differential encoding of single digit movement information across the different Brodmann areas. Finally, we show that these patterns can be approximated across different sessions, allowing for cross session decoding. These results establish that fUSI can reliably delineate somatotopically organized motor representations at submillimeter resolution, bridging a critical gap between invasive electrophysiology and noninvasive hemodynamic imaging in a human subject.

neuroscience↗

bioRxiv2026

Statistical BURST imaging for high-fidelity biomolecular ultrasound

Ultrasound is emerging as a method for molecular and cellular imaging by connecting the versatile physics of sound waves to protein-based contrast agents such as gas vesicles (GVs). BURST is a common imaging mode that leverages the strong, transient echoes generated when GVs collapse under acoustic pressure to enable highly sensitive ultrasound visualization of cells and biomolecules, down to the single cell level. However, BURST is vulnerable to fluctuating background signals, with large-amplitude fluctuations in scattering, as often present in vivo, obscuring genuine GV responses. In this study, we mathematically examine this limitation and show that incorporating statistical metrics such as correlation or temporal contrast-to-noise ratio effectively suppresses unwanted non-GV voxels and quantifies detection confidence, including in image sequences in which GV collapse spans multiple frames. Compared with prior methods, our approach enhances the clarity of BURST images and provides probabilistic interpretations of GV signals, facilitating more reliable analysis of ambiguous in vivo molecular imaging, as we demonstrate in imaging tumor-homing probiotics and gene expression in the brain.

bioengineering↗

bioRxiv2025

Ultrasonic Reporter of Kinase Activity

Protein kinases are an essential class of enzymes that regulate cellular signaling pathways, with their dysregulation implicated in pathologies such as cancer and neurodegenerative diseases. Despite the existence of high-performance fluorescent biosensors of kinase activity, it remains challenging to study the function and regulation pathways of kinases in opaque tissues due to the limited tissue penetration of light. To address this limitation, we introduce the first ultrasonic reporter of kinase activity (UReKA), designed to detect protein kinase A (PKA) activity by altering the ultrasound scattering of gas vesicles, a unique class of air-filled protein nanostructures. We engineer a gas vesicle shell protein to respond to PKA, demonstrate the functionality of UReKA both in purified protein format and expressed in mammalian cells, and showcase its capacity to monitor PKA signaling in response to pharmacological stimulation or genetic mutation. This work makes it possible to visualize cellular functional activity in opaque media, with broad potential for future applications in cancer biology, cellular development, and drug discovery.

bioengineering↗

bioRxiv2025

Rewiring protein function through genetically encoded oxidative chemistry

Oxidative chemistry underlies many endogenous signaling pathways but remains underutilized as a programmable strategy for regulating protein function in living cells. Here we establish genetically encoded oxidative chemistry as a tunable framework for modulating diverse proteins by coupling a photosensitizer to defined intracellular contexts. Using miniSOG to generate reactive oxygen species (ROS), we show that controlled intracellular oxidation increases the fluorescence of the redox reporter HyPerRed and activates redox-sensitive TRP ion channels, with strong responses in TRPA1 and TRPV1 but not TRPV4. Pathway-selective scavengers reveal differential coupling of soluble and membrane targets to distinct oxidative processes, supporting selectivity by context rather than uniform oxidative perturbation. Modulation strength and kinetics are quantitatively tunable through illumination parameters, expression ratios, and subcellular localization, with membrane targeting enhancing coupling to membrane effectors. Finally, fusion targeting of miniSOG to TRPV1 and modulation of endogenous TRPA1 in human fibroblasts extend this approach to protein-proximal and native cellular settings. Together, these results position genetically encoded oxidative chemistry as a versatile and spatially organized modality for engineering protein function in living cells within a defined operating regime.

synthetic biology↗

bioRxiv2025

Trimodal brain-wide ultrasound imaging of brain-tumor interaction

Patients with brain tumors often suffer debilitating neurological dysfunction as their tumors disrupt brain tissue, affecting both local and global neural activity and blood flow. However, studying tumor-brain interactions in animal models is challenging due to a lack of methods that simultaneously capture dynamics of tumor growth, neural activity and vascular alterations over time. Here, we overcome this limitation using a multimodal ultrasound imaging platform, an imaging technique that offers brain-wide coverage in living animals at 10-100 {micro}m resolution. To monitor the co-evolution of tumor growth, neural activity and vascular remodeling, we integrated (1) nonlinear imaging of acoustic reporter gene expressing-tumors, (2) hemodynamic functional imaging of brain activity, and (3) super-resolution microscopy of the vasculature. Integrating these modalities for the first time and applying them to a common model of glioblastoma, we followed tumor-brain interactions in individual animals over their disease lifetimes. Our approach allowed us to precisely map the spatial displacement of functional brain regions, the local and global disruption of functional connectivity, and the remodeling of the blood supply to support tumor growth. This integrated method bridges a critical gap in brain cancer research and therapy development by providing a unified dynamic view of what happens to the brain as tumors grow within it.

bioengineering↗

bioRxiv2025

A modular method for rapidly prototyping targeted gas vesicle protein nanoparticles

Gas vesicles (GVs) are air-filled protein nanoparticles which are proving to be useful in a number of biomedical applications. We hypothesized that it could be possible to develop a modular method for creating rapidly prototyped GVs by modifying their surface chemistry to include targeting peptides in an orientation-specific manner. Here, we describe a modular method to create targeted GVs using His-tagged antibody fragments, ensuring that the antibody fragments are connected to the GV in an orientation-specific manner. This is achieved via the functionalization of the GVs with nickel-nitrilotriacetic acid (Ni-NTA) group. First, we validated that these functionalized GVs can bind His-tagged green fluorescent protein and characterized the particle size and surface charge of functionalized GVs. Then, GVs targeted to prostate-specific membrane antigen (PSMA) using a minibody were validated using a knockout validation in vitro.

bioengineering↗

bioRxiv2025

Ultrasound imaging of in situ transcriptional activity in opaque tissue

Ultrasound imaging and acoustic reporter genes provide unique capabilities for in vivo biological imaging by leveraging ultrasounds ability to visualize opaque tissues with high spatiotemporal resolution. But until now, the expression of acoustic reporter genes - based on gas vesicle (GV) proteins - has been limited to ex vivo-modified cells due to the complexity of the GV gene cluster, precluding valuable in situ applications. Here, we develop a system capable of introducing GV genes directly into native tissues via stoichiometric multi-AAV delivery. We validate this system in the mouse brain, demonstrating well-tolerated in situ gene expression and repeated ultrasound imaging over more than a month in the same animal. Furthermore, by placing GV genes under the control of immediate early gene promoters, we demonstrate the ability to track in vivo gene expression changes arising from elevated neural activity during epileptic seizures. This work connects ultrasound to in situ transcriptional dynamics happening inside the opaque tissues of living creatures.

bioengineering↗

bioRxiv2025

Sono-uncaging for Spatiotemporal Control of Chemical Reactivity

Photo-uncaging - the use of light to reveal the active part of a chemical compound by photolysis of a protecting group - has long been used to study and actuate biochemical processes. However, light scattering limits the applications of photo-uncaging in opaque specimens or tissues. Here, we introduce sono-uncaging, a process in which a chemical functional group becomes exposed upon the application of ultrasound, which can be applied and focused in optically opaque materials. We engineered gas vesicles (GVs), air-filled protein nanostructures sensitive to ultrasound, to contain cysteines on their concealed inner surface, hypothesizing that the application of ultrasound would collapse the GV shell and reveal the cysteines. The resulting SonoCage construct reacted with monobromobimane (mBBr), a fluorogenic, thiol-reactive molecule, only after treatment with ultrasound, establishing the sono-uncaging proof of concept. We then demonstrated the spatial patterning capability of sono-uncaging by embedding the SonoCages in an mBBr-containing hydrogel and creating fluorescent patterns with phased array ultrasound. This patterning could be accomplished using a diagnostic imaging transducer. This work establishes sono-uncaging as a method for spatiotemporal control over chemical reactivity using widely available ultrasound technology.

bioengineering↗

bioRxiv2025

Thermally controlled state switches for macrophage immunotherapy

Advances in cellular immunotherapy promise new treatments for conditions such as cancer, autoimmune disease, and heart disease. While engineered cells have the ability to recognize clinically relevant signals, traffic to disease sites and interface with the host immune system, their activity must be tightly controlled to minimize undesirable effects in healthy tissues. One approach to obtaining specificity is to activate the cells spatially using externally applied energy, such as ultrasound-delivered heating. To facilitate such control, we designed and characterized a genetic circuit that enables stable transcriptional activation of macrophages after a brief thermal stimulus, resulting in the expression of reporters or secretion of the cytokine IL-12. We demonstrate that in vivo activation of a mouse macrophage cell line containing this bioswitch results in spatially localized gene expression for at least 14 days after ultrasound heating. This thermal bioswitch provides a precise control element for cell-therapeutic agents.

bioengineering↗

bioRxiv2025

VARS-fUSI: Variable Sampling for Fast and Efficient Functional Ultrasound Imaging using Neural Operators

Functional ultrasound imaging (fUSI) is a promising neuroimaging method that infers neural activity by detecting cerebral blood volume changes. It offers high sensitivity and spatial resolution relative to fMRI and is an epidural alternative to electrophysiology for medical and neuroscience applications, including brain-computer interfaces. However, current fUSI methods require hundreds of compounded images and ultrasound pulse emissions, leading to high computational costs, memory demands, and potential probe heating. We propose VARiable Sampling fUSI (VARS-fUSI), the first deep learning fUSI method to allow for different sampling durations and rates during training and inference by using neural operators. VARS-fUSI reconstructs high-quality fUSI images using 10 - 15% of the time or sampling rate needed per image while preserving decodable behavior-correlated signals. Additionally, VARS-fUSI offers efficient finetuning for generalization to new animals and humans. Demonstrated across mouse, monkey, and human data, VARS-fUSI achieves state-of-the-art performance, enhancing imaging efficiency by significantly reducing storage and processing needs.

bioengineering↗

bioRxiv2024

Acoustic tumor paint for real-time imaging, surgical guidance and recurrence monitoring of brain tumors with ultrasound

The rapid growth, invasiveness, and resistance to treatment of glioblastoma multiforme (GBM) underscore the urgent need for improved diagnostics and therapies. Current surgical practice is limited by challenges with intraoperative imaging, while recurrence monitoring requires expensive magnetic resonance or nuclear imaging scans. Here we introduce "acoustic tumor paint", an approach to labeling brain tumors for ultrasound imaging - a widely accessible imaging modality. We show that gas vesicles (GVs), natural air-filled protein nanostructures, preferentially accumulate in brain tumors following systemic administration in syngeneic and xenograft mouse models of GBM. This enables real-time tumor visualization during surgery and postoperative monitoring of recurrence. We characterize GV uptake and breakdown by tumors and their resident cells and support clinical translatability by documenting non-toxic repeated administration. We also demonstrate the potential for post-operative monitoring in humans by imaging GVs through a human skull and an FDA-approved skull prosthesis. Acoustic tumor paint has the potential to enhance diagnostic accuracy, improve surgical outcomes, make monitoring more accessible, and extend survival in GBM patients.

bioengineering↗

bioRxiv2024

Real-time volumetric imaging of cells and molecules in deep tissues with Takoyaki ultrasound

Acoustic contrast agents and reporter genes play a critical role in allowing ultrasound to visualize blood flow, map molecules and track cellular function in opaque living organisms. However, existing ultrasound methods to image acoustic contrast agents predominantly focus on 2D planar imaging, while the biological phenomena of interest unfurl in three dimensions. Here, we introduce a method for efficient, dynamic imaging of contrast agents and reporter genes in 3D using multiplexed matrix array transducers. Our "Takoyaki" pulse sequence uses the simultaneous scanning of multiple focal points to excite contrast agents with sufficient acoustic pressure for nonlinear imaging while efficiently covering 3D space. Through in vitro experiments, we first show that the Takoyaki sequence produces highly sensitive volume images of gas vesicle contrast agents and compare its performance with alternative imaging schemes. We then establish its utility in cellular imaging in vivo by visualizing acoustic reporter gene-expressing tumors in a mouse model of glioblastoma. Finally, we demonstrate real-time volumetric imaging by tracking the dynamics of fluid motion in brain ventricles after intraventricular contrast injection. Takoyaki imaging enables a more comprehensive understanding of biological processes by providing spatiotemporal information in 3D within the constraints of accessible multiplexed matrix array systems.

bioengineering↗

bioRxiv2024

Multiplexed ultrasound imaging of gene expression

Acoustic reporter genes (ARGs) have enabled the imaging of gene expression with ultrasound, which provides high-resolution access to deep, optically opaque living tissues. However, unlike their fluorescent counterparts, ARGs have so far been limited to a single "color," preventing multiplexed imaging of cellular states or populations. Here, we use rational protein design and directed evolution to develop two novel ARGs that can be distinguished from each other based on their acoustic pressure response profiles, enabling "two-color" ultrasound imaging of gene expression. We demonstrate the utility of multiplexed ARGs for delineating bacterial cell species and cell states in vitro, and then apply them towards imaging distinct subpopulations of probiotics in the mouse gastrointestinal tract and in tumor-colonizing bacterial agents in vivo. Just as the first wavelength-shifted derivatives of fluorescent proteins opened a vivid world for optical microscopy, our next-generation acoustic proteins set the stage for a richer symphony of ultrasound signals from living subjects.

bioengineering↗

bioRxiv2024

Non-invasive imaging of cell-based therapies using acoustic reporter genes

Cell-based therapies are a major emerging category of medicine. The ability of engineered cells to traffic to and function at specific anatomical locations is a major aspect of their performance. However, there is a lack of non-invasive, non-ionizing, cost-accessible methods to track these therapies inside the body and ensure proper function. Here, we establish a platform for in vivo imaging of primary cell therapies using ultrasound - a ubiquitously accessible technology for high-resolution non-invasive imaging. We introduce and optimize a lentiviral delivery system to express acoustic reporter genes based on gas vesicles in primary mammalian cells such as T cells, showing that this results in robust ultrasound contrast. Additionally, we develop genetic circuits making it possible to monitor T cell activation via activity-dependent promoters. We apply this technology to primary human T cells, using it to non-invasively track their accumulation and proliferation as a targeted therapy in a mouse tumor xenograft model and compare it to invasive, terminal measures such as immunohistology. By making it possible to visualize cell-based therapies and their function inside opaque living organs with unprecedented resolution and accessibility, this technology has the potential to significantly accelerate their development and effective use.

bioengineering↗

bioRxiv2024

Characterisation of Gas Vesicles as Cavitation Nuclei for Ultrasound Therapy using Passive Acoustic Mapping

Genetically encodable gas filled particles known as gas vesicles (GVs) have shown promise as a biomolecular contrast agent for ultrasound imaging and have the potential to be used as cavitation nuclei for ultrasound therapy. In this study, we used passive acoustic mapping techniques to characterize GV-seeded cavitation, utilizing 0.5 and 1.6 MHz ultrasound over peak rarefactional pressures ranging from 100 to 2200 kPa. We found that GVs produce cavitation for the duration of the first applied pulse, up to at least 5000 cycles, but that bubble activity diminishes rapidly over subsequent pulses. At 0.5 MHz the frequency content of cavitation emissions was predominantly broadband in nature, whilst at 1.6 MHz narrowband content at harmonics of the main excitation frequency dominated. Simulations and high-speed camera imaging suggest that the received cavitation emissions come not from individual GVs but instead from the coalescence of GV-released gas into larger bubbles during the applied ultrasound pulse. These results will aid the future development of GVs as cavitation nuclei in ultrasound therapy.

bioengineering↗

bioRxiv2024

Probiotic acoustic biosensors for noninvasive imaging of gut inflammation

Inflammatory bowel diseases (IBD) affect millions of people globally, result in severe symptoms, and are difficult to diagnose and monitor - often necessitating the use of invasive and costly methods such as colonoscopies or endoscopies. Engineered gut bacteria offer a promising alternative due to their ability to persist in the gastrointestinal (GI) tract and sense and respond to specific environmental signals. However, probiotics that have previously been engineered to report on inflammatory and other disease biomarkers in the Gl tract rely on fluorescent or bioluminescent reporters, whose signals cannot be resolved in situ due to the poor penetration of light in tissue. To overcome this limitation, we introduce probiotic biosensors that can be imaged in situ using ultrasound - a widely available, inexpensive imaging modality providing sub-mm spatial resolution deep inside the body. These biosensors are based on the clinically approved probiotic bacterium E. coli Nissle, which we engineered to transiently colonize the GI tract, sense inflammatory biomarkers, and respond by expressing air-filled sound-scattering protein nanostructures called gas vesicles. After optimizing biomolecular signaling circuits to respond sensitively to the biomarkers thiosulfate and tetrathionate and produce strong and stable ultrasound contrast, we validated our living biosensors in vivo by noninvasively imaging antibiotic-induced inflammation in mice. By connecting cell-based diagnostic agents to ultrasound, this "diagnostic yogurt" will make it easier, cheaper, and less painful to diagnose and monitor IBD or other GI conditions.

bioengineering↗