Guosong Hong

The inherent opacity of most mammalian tissues limits deep-tissue optical imaging and light delivery. In contrast, the natural transparency of certain species and ocular tissues has been hypothesized to involve proteins with unusually high refractive indices. Here, we systematically analyze the ultraviolet absorption and visible-range refractive index modulation of canonical amino acids to identify key contributors to high-refractive index proteins. We identify arginine as a leading candidate, combining strong ultraviolet absorption, efficient refractive index modulation, physiological pH, and biocompatibility. These properties are validated through successful achievement of optical transparency in both ex vivo and in vivo tissues. Our findings establish a foundation for using abundant endogenous biomolecules to achieve in vivo tissue transparency and suggest a strategy for engineering proteins enriched in high-performing amino acids to enable efficient, biocompatible tissue clearing.

Mechanoluminescent particles, including ZnS, are promising intravenous light sources for biomedical imaging. Here, we report a serendipitous finding that during femtosecond two-photon microscopy, a microsecond-scale prolonged luminescence (“comet tail”) is observed, whereas it is absent or greatly reduced under confocal continuous-wave excitation. We found that the decay time constant of the comet tail is typically 2–5 μs, remains temperature-independent from 0–37 °C, increases with laser power, and decreases with the decay time constant of the particle’s temperature when varying the embedding medium. We further measured the absorption cross section for ZnS microparticles, and the photothermal modeling suggests that while femtosecond pulsed excitation results in instantaneous heating of electrons, it does not lead to a greater increase in bulk temperature in ZnS compared with continuous-wave excitation. Our findings bring new understanding and perspectives to mechanoluminescent particles-based imaging technologies and applications.

Tartrazine, along with many other absorbing molecules, has recently emerged as a promising tissue-clearing agent for achieving transient and reversible optical transparency in live animals. In this mini-review, we describe the fundamental physical origins of tissue opacity, extract engineering principles guiding the design of next-generation dye-based clearing agents, and summarize recent advances in dye-enabled in vivo tissue clearing. We also discuss current challenges and offer forward-looking perspectives to inform future research.

Tissue-clearing techniques have transformed optical imaging of fixed specimens, yet their application to living systems remains limited by toxicity and removal of key tissue components. We recently demonstrated that absorbing molecules such as tartrazine can reversibly render live mouse skin transparent. Subsequently, it was reported that isotonic protein solutions can achieve ex vivo and in vivo cellular clearing. However, discrepancies remain regarding the optimal refractive index (RI) for live-cell clearing and the impact of elevated osmolality on cell viability. Here, using cultured mammalian cells, we systematically examine the dependence of optical contrast on medium RI and the effects of hyperosmolality. We find that, contrary to the recent report of an optimal RI of 1.36 ∼ 1.37 for suspended cells, densely packed adherent cells exhibit a monotonic decrease in phase contrast up to an RI of 1.41 with tartrazine. Moreover, even under highly hyperosmotic conditions (∼1200 mOsm/kg), cultured cells exhibit minimal deformation and negligible loss of viability for up to 30 min in the clearing solution. These results demonstrate that tartrazine enables effective live-cell clearing at RI up to 1.41 while preserving viability under elevated osmolality and motivate future studies to define optimal conditions for in vivo optical clearing.

The editorial highlights articles in a JBO special section, as well as emerging trends in small-animal molecular imaging.

Tissue-clearing techniques have transformed optical imaging of fixed specimens, yet their application to living systems remains limited by toxicity and removal of key tissue components. We recently demonstrated that absorbing molecules such as tartrazine can reversibly render live mouse skin transparent. Subsequently, it was reported that isotonic protein solutions can achieve ex vivo and in vivo cellular clearing. However, discrepancies remain regarding the optimal refractive index (RI) for live-cell clearing and the impact of elevated osmolality on cell viability. Here, using cultured mammalian cells, we systematically examine the dependence of optical contrast on medium RI and the effects of hyperosmolality. We find that, contrary to the recent report of an optimal RI of 1.36∼1.37 for suspended cells, densely-packed adherent cells exhibit a monotonic decrease in phase contrast up to an RI of 1.41 with tartrazine. Moreover, even under highly hyperosmotic conditions (∼1200 mOsm/kg), cultured cells exhibit minimal deformation and negligible loss of viability for up to 30 min in the clearing solution. These results demonstrate that tartrazine enables effective live-cell clearing at RI up to 1.41 while preserving viability under elevated osmolality, and motivate future studies to define optimal conditions for in vivo optical clearing. Graphical

Biological systems operate across distributed regions with fast, localized dynamics, yet existing biointerfaces fall short of providing both high spatiotemporal precision and the ability to dynamically target any region without disturbing surrounding tissue. Here we present an in vivo deep-tissue light source based on focused ultrasound scanning of mechanoluminescent nanotransducers circulating through the vasculature. We demonstrate the programmability of this approach in tissue-mimicking phantoms and the endogenous circulatory system of animals, where tunable spatial resolution and dynamic light patterning are achieved. We validate the functionality of the ultrasound-scanning light source in opsin-expressing neurons through electrophysiological recordings and immunostaining in both the brain and the spinal cord. We showcase dynamic three-dimensional brain targeting and temporally resolved behavioural control in freely moving animals via the ultrasound-scanning in vivo light source. This non-invasive deep-tissue light source offers a versatile strategy for body-wide optical interfacing.

Fabricating materials within optically opaque structures, such as biological tissue, is a considerable challenge. Recently, ultrasound-based printing (“sonoprinting”) approaches have emerged as a promising strategy to address this challenge. However, an approach to sonoprint conductive materials has yet to be realized, limiting potential bioelectronic applications. Here, we extend sonoprinting to conductive materials by designing temperature-based and pressure-based methods to polymerize conductive polymers with focused ultrasound (FUS). Our temperature-based approach relies on the acoustic attenuation of the surrounding medium to generate heat under FUS, whereas our pressure-based approach leverages the acoustic vaporization of perfluorohexane double emulsions to trigger polymerization. We demonstrate that both approaches can be used to print the conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) through optically opaque hydrogels and biological tissue with high spatial resolution. Taken together, our results establish complementary temperature- and pressure-based methods for sonoprinting conductive polymers, paving the way for future efforts to fabricate bioelectronic interfaces in tissue.

Temporal interference (TI) electrical stimulation is promising for noninvasive neuromodulation with the potential advantages of deep brain targeting. This technique uses high-frequency electric fields applied transcranially, which intersects within the brain to generate a low-frequency modulation field. This intersection enables precise, noninvasive targeting of deep brain regions, addressing a major limitation of typical noninvasive neuromodulation methods, which often yield scattered effects and reduced precision in deep tissue. Recent advances in TI stimulation, supported by computational models and behavioral studies, have demonstrated efficacy in targeting the hippocampus and modulating neuronal activity without notably affecting cortical regions. Its noninvasive nature, coupled with high energy efficiency and precision, positions TI stimulation as a potential tool for activating deep brain regions crucial for behavior and cognition. This review explores the recent developments in TI stimulation, highlighting its mechanisms and pivotal role in precise, noninvasive neuromodulation. The potential of TI stimulation in treating neurological and psychiatric disorders is emphasized, setting the stage for future advances in neurotherapy.

Light scattering in biological tissue presents a significant challenge for deep in vivo imaging. Our previous work demonstrated the ability to achieve optical transparency in live mice using intensely absorbing dye molecules, which created transparency in the red spectrum while blocking shorter-wavelength photons. In this paper, we extend this capability to achieve optical transparency across the entire visible spectrum by employing molecules with strong absorption in the ultraviolet spectrum and sharp absorption edges that rapidly decline upon entering the visible spectrum. This color-neutral and reversible tissue transparency method enables optical transparency for imaging commonly used fluorophores in the green and yellow spectra. Notably, this approach facilitates tissue transparency for structural and functional imaging of the live mouse brain labeled with yellow fluorescent protein and GCaMP through the scalp and skull. We show that this method enables longitudinal imaging of the same brain regions in awake mice over multiple days during development. Histological analyses of the skin and systemic toxicology studies indicate minimal acute or chronic damage to the skin or body using this approach. This color-neutral and reversible tissue transparency technique opens opportunities for noninvasive deep-tissue optical imaging, enabling long-term visualization of cellular structures and dynamic activity with high spatiotemporal resolution and chronic tracking capabilities.