Biology subjects

Ilovitsh, T.

Publications and source records attributed to Ilovitsh, T..

9 recordsLinked to original sources

bioRxiv2026

Nanobubble-Based Ultrasound Localization Microscopy through Interactive Adaptive Processing

This study presents the use of sub-micron nanobubbles (NBs) as contrast agents for ultrasound localization microscopy (ULM), a super-resolution imaging technique that visualizes microvascular structure and flow beyond the acoustic diffraction limit. While ULM has traditionally relied on micron-sized microbubbles (MBs), the reduced dimensions and prolonged circulation times of NBs make them attractive candidates for localization-based imaging. However, their weaker acoustic responses present significant challenges for reliable detection and tracking. To address this challenge, we developed the ULM Master GUI, an interactive framework for optimization of the complete ULM processing pipeline. Using custom ultrasound-compatible wall-less gelatin flow phantoms containing vessel-mimicking channels and bifurcations ranging from 100 to 500 m, we demonstrate that NB-based ULM achieves velocity reconstruction and flow partitioning measurements comparable to conventional MB-based ULM. Across all investigated geometries, NBs faithfully reproduced the underlying flow patterns and hemodynamic behavior despite their substantially reduced acoustic scattering. These findings establish the feasibility of NB-based ULM, expand the range of contrast agents available for localization microscopy, and provide a foundation for future super-resolution ultrasound imaging using nanoscale acoustic contrast agents. The ULM processing GUI is publicly available at https://github.com/grisha1998/ulm-super-resolution-toolbox.

bioengineering↗

bioRxiv2026

Ultrasound-Based Spatiotemporal Monitoring of Coagulation and Thrombolysis via Speed-of-Sound Shift Imaging

Blood clot formation and thrombolysis are dynamic biological processes that play central roles in hemostasis, thrombosis, and thrombolytic therapy. Monitoring clot evolution is challenging, as existing approaches often rely on specialized hardware or complex acquisition protocols. This study presents dense speed-of-sound shift imaging (DSI), a noninvasive ultrasound framework for spatiotemporal monitoring of coagulation and lysis from ultrasound image sequences acquired with a single imaging transducer. DSI estimates interframe displacements using dense optical flow and reconstructs slowness-shift maps by solving a regularized inverse problem, from which relative speed-of-sound (SoS) shifts are derived. Using this approach, we quantified spatially localized acoustic signatures of material solidification, clot formation, and enzymatic clot dissolution across systems of increasing biological complexity, including thermally gelling gelatin, fibrin clots, and porcine and human whole blood. DSI detected composition-dependent clot properties, with fibrinogen primarily affecting SoS shift magnitude and thrombin primarily affecting clotting kinetics. In both porcine and human whole blood, DSI tracked the full transition from rapid clot formation to tPA-mediated thrombolysis, revealing markedly slower lysis kinetics and species-dependent differences in clotting amplitude and stabilization time. Together, these results establish DSI as a simple, ultrasound-based platform for quantitative monitoring of coagulation, thrombolysis, and related biological material transitions.

bioengineering↗

bioRxiv2026

Super-Resolution Macrophage Imaging via Ultrasound Localization Microscopy and Blinking Nanodroplets

Tracking immune cells deep within living tissue remains a fundamental challenge due to the diffraction-limited resolution of ultrasound imaging and the inability to resolve dense cellular populations. Here, we introduce an intracellular super-resolution ultrasound imaging framework based on stochastic phase-changing nanodroplets (NDs) and ultrasound localization microscopy (ULM). We engineer [~]170 nm perfluorocarbon NDs that undergo reversible, stochastic liquid-gas transitions under acoustic excitation, generating temporally sparse "blinking" signals. Leveraging the intrinsic endocytic activity of macrophages, these NDs are internalized, enabling intracellular contrast generation independent of vascular flow. We validate this approach across imaging scales, from controlled phantoms and in vitro systems to in vivo tumor models, demonstrating robust intracellular blinking, high cell viability, and consistent super-resolution reconstruction in dense cellular environments. The stochastic blinking of internalized NDs provides the temporal separation required to localize individual sources, overcoming a central limitation of conventional ULM. Following systemic administration, ND-labeled macrophages are tracked in vivo after homing to the liver, where super-resolution ULM resolves cellular distributions with a spatial resolution of 26.3 {+/-} 3.2 {micro}m, corresponding to a 6.1-fold improvement over diffraction-limited imaging. This work establishes a previously unexplored paradigm for ultrasound-based intracellular super-resolution imaging, enabling non-invasive visualization of immune cell organization in deep tissue. By introducing spatiotemporally programmable intracellular contrast, this approach expands ultrasound beyond vascular imaging toward functional cellular imaging, with broad implications for immunology, diagnostics, and cell-based therapies.

bioengineering↗

bioRxiv2026

Acoustically activatable drug-loaded nanodroplets for mechanochemical therapy in solid tumors

Stimulus-responsive nanomedicines promise spatiotemporally controlled therapy, yet most systems rely on passive delivery and lack precise, externally programmable activation while maintaining clinical compatibility. Here we engineer sub-200 nm, perfluorocarbon (PFC)-core nanodroplets (NDs) that integrate efficient core drug loading, physiological stability, and acoustically programmable activation within a single nanoscale agent. These NDs are fabricated using microfluidic nanoassembly to achieve controlled size and composition, and are designed to encapsulate fluorinated payloads directly within the liquid core. Upon exposure to a sequential dual-frequency ultrasound (US) paradigm, the NDs undergo acoustic droplet vaporization followed by low-frequency cavitation, enabling spatially confined mechanical disruption and on-demand payload release within clinically relevant acoustic limits. These properties are engineered to overcome physicochemical barriers in solid tumors, including dense extracellular matrix and restricted drug penetration. This approach achieves enhanced payload release and induces potent mechanochemical cytotoxicity in vitro. In vivo, NDs exhibit prolonged circulation and tumor accumulation, while US activation drives substantial tissue fractionation, control drug release, and increases subsequent nanoparticle uptake. When applied to a solid tumor model, this combined mechanochemical strategy improves tumor control and significantly extends survival compared to either modality alone. These acoustically activatable NDs provide a versatile system for stimulus-responsive, site-targeted drug delivery and mechanical tumor disruption, with strong potential for clinical translation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/719550v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@9c07f8org.highwire.dtl.DTLVardef@1cf355aorg.highwire.dtl.DTLVardef@b7afd1org.highwire.dtl.DTLVardef@177fa1a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

Adaptive spatio-temporal phase retrieval for ultra-short pulse wavefront shaping

Beam shaping of ultra-short pulses is essential for medical ultrasound, where single-cycle excitations are required to achieve high axial resolution and improve frame rate. Conventional methods, such as the Gerchberg-Saxton (GS) algorithm or more recent deep learning approaches, are generally effective for continuous-wave excitation but degrade significantly under single-cycle conditions. In diagnostic imaging, high frame rate is critical for applications demanding rapid scanning. In this context, multi-line transmission (MLT) leverages beam shaping to synthesize multiple simultaneous foci, thereby increasing frame rate. In parallel, structured illumination methods for super-resolution and acoustical holography likewise depend on actively shaping single-cycle pulses to produce controlled patterns, highlighting the need for precise short-pulse beam shaping. To address this challenge, we introduce the spatio-temporal adaptive reconstruction (STAR) algorithm, which performs active beam shaping directly in the time domain by integrating the generalized angular spectrum method (GASM) into an iterative optimization scheme. STAR enforces constraints on both the transducer and focal planes, enabling accurate control of single-cycle excitations. Simulations showed that STAR consistently outperformed GS for multi-focus patterns. For example, in a four-foci configuration, STAR achieved a correlation of 0.80 compared to 0.64 for GS, with significantly improved uniformity across focal peaks. Resolution analysis demonstrated that STAR reduced the minimum distinguishable foci spacing from 1.09 mm with GS to 0.87 mm. Experimental hydrophone measurements confirmed these improvements. Across multi-foci patterns, STAR produced more distinct and balanced foci compared to those observed with GS. These results demonstrate that STAR provides robust and efficient active beam shaping of single-cycle pulses, maintaining accuracy across different depths and frequencies for diagnostic applications.

bioengineering↗

bioRxiv2026

Cryoablation temperature monitoring with dense ultrasonic speed-of-sound shift imaging

Accurate temperature monitoring during cryoablation, a minimally invasive technique that destroys tissue locally by forming an ice ball around an inserted cryoprobe, is vital for achieving complete ablation while protecting surrounding tissue. We present a dense slowness-shift imaging method that estimates local speed-of-sound changes from ultrasound B-mode images using optical flow. This single-transducer, image-based approach enables mapping of spatial temperature change without requiring additional hardware. Cryoablation experiments in a tissue-mimicking phantom and ex vivo turkey breast demonstrated that slowness deviation increases with decreasing temperature. In the phantom, the dependence was linear (a = -20.70 s{middle dot}m-1C{degrees}-1), while in turkey breast it presented an exponential relationship (t = 34.04xexp(0.075(-{Delta}T)) s{middle dot}m-1C{degrees}-1). The algorithm detected sub-degree temperature variations and accurately tracked cooling down to -39.4 {+/-} 5.6 {degrees}C. This work demonstrates the feasibility of ultrasound-based, noninvasive temperature monitoring during cryoablation, providing a scalable, real-time alternative to existing invasive or high-cost thermal assessment techniques.

biophysics↗

bioRxiv2025

Nanobubble based sonobiopsy reveals circulating protein signatures of BBB opening and glioblastoma

Focused ultrasound (FUS) and microbubbles can transiently increase blood brain barrier (BBB) permeability, yet verification of BBB opening (BBBO) relies mainly on contrast-enhanced MRI, offering limited insight into the molecular consequences of barrier modulation. Sonobiopsy, which uses FUS induced BBBO to release brain derived molecules into the bloodstream, provides a molecular readout from blood samples. Nanobubbles (NBs) are smaller agents that circulate more effectively in the brain microvasculature and have shown enhanced BBBO in capillaries. Here, NB-mediated proteomic sonobiopsy is used to improve biomarker efflux and increase molecular sensitivity, with the goal of defining the molecular signature produced by BBBO in healthy and glioblastoma (GBM) models, and distinguishing biomarkers associated with tumor pathology from BBBO biomarkers. Plasma collected before and after NB-mediated FUS underwent data-independent acquisition-based mass spectrometry, revealing post-BBBO changes in healthy and tumor bearing mice. In healthy cohorts, 77 proteins were reproducibly altered after BBBO. Six proteins (Dpysl3, Myl1, Mybpc1, Vsig4, Krt33a, Krtap6-5) were detectable only after BBBO in healthy mice. In 005 glioma bearing mice, the BBBO signature identified in healthy animals was preserved, and comparison with matched shams isolated tumor specific effects. Three proteins, Itih4, Lrg1, and Hp, rose significantly after BBBO only in GBM and reached higher post treatment levels than in shams, nominating candidate GBM-associated markers accessible via blood. These findings establish NB-mediated proteomic sonobiopsy as a promising method for BBBO verification and for detecting GBM associated protein signals, supporting the development of scalable BBBO confirmation and protein-based diagnostics in neuro oncology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/692317v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@d0186aorg.highwire.dtl.DTLVardef@1f2fe8borg.highwire.dtl.DTLVardef@25921forg.highwire.dtl.DTLVardef@9c3566_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Low-frequency ultrasound-mediated blood-brain barrier opening enables non-invasive lipid nanoparticle RNA delivery to glioblastoma

Ionizable Lipid Nanoparticles (LNPs) are an FDA-approved non-viral RNA delivery system, though their use for brain therapy is restricted by the blood-brain barrier (BBB). Focused ultrasound combined with microbubbles can disrupt the BBB, but delivering large particles requires balancing increased peak negative pressures while maintaining microvascular integrity. Herein, we optimized low-frequency ultrasound parameters to induce high-amplitude microbubble oscillations, enabling the safe delivery of LNPs across the BBB. First, BBB opening was assessed at different frequencies (850, 250, and 80 kHz) and pressures by monitoring the extravasation of Evans blue (~1 kDa). Next, the delivery of 4, 70, and 150 kDa Dextrans, LNPs entrapping Cy5-siRNAs (~70 nm in diameter), and LNPs entrapping mRNA (~100 nm in diameter) was evaluated via microscopy and bioluminescence. In a glioblastoma syngeneic mouse model, siRNA-Cy5-LNP was successfully delivered. A frequency of 850 kHz and 125 kPa pressure induced safe BBB opening, enabling delivery of both small molecules and LNPs. In healthy brains, LNP entrapping siRNAs delivery increased 10-fold compared to controls, and LNPs with mRNAs showed a 12-fold increase in bioluminescence after 24 hours. In glioblastoma tumors, LNPs with siRNAs delivery resulted in a 6.7-fold increase in fluorescence. This study paves the way for non-invasive LNP delivery to the brain, offering a versatile platform for brain therapies. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/634427v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@cad2bdorg.highwire.dtl.DTLVardef@1d1936corg.highwire.dtl.DTLVardef@259d83org.highwire.dtl.DTLVardef@1883aed_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Enhanced capillary delivery with nanobubble-mediated blood-brain barrier opening and advanced high resolution vascular segmentation

Overcoming the blood-brain barrier (BBB) is essential to enhance brain therapy. Here, we utilized nanoscale nanobubbles with focused ultrasound for targeted and improved BBB opening in mice. A microscopy method assessed BBB opening at a single blood vessel resolution employing a dual-dye labeling technique using green fluorescent molecules to label blood vessels and Evans blue brain-permeable dye for quantifying BBB extravasation. A UNET-based deep learning architecture enabled blood vessels segmentation, delivering comparable accuracy to manual segmentation with a significant time reduction. Segmentation outcomes were applied to the Evans blue channel to quantify extravasation of each blood vessel. Results were compared to microbubble-mediated BBB opening, where reduced extravasation was observed in capillaries with 2-6m diameter. In comparison, nanobubbles yield an improved opening in these capillaries, and equivalent efficacy to that of microbubbles in larger vessels. These results indicate the potential of nanobubbles to serve as enhanced agents for BBB opening, amplifying bioeffects in capillaries while preserving comparable opening in larger vessels.

bioengineering↗