Sequentially timed all-optical mapping photography boosted by a branched 4f system with a slicing mirror.

We present sequentially timed all-optical mapping photography (STAMP) with a slicing mirror in a branched 4f system for an increased number of frames without sacrificing pixel resolution. The branched 4f system spectrally separates the laser light path into multiple paths by the slicing mirror placed in the Fourier plane. Fabricated by an ultra-precision end milling process, the slicing mirror has 18 mirror facets of differing mirror angles. We used the boosted STAMP to observe dynamics of laser ablation with two image sensors which captured 18 subsequent frames at a frame rate of 126 billion frames per second, demonstrating this technique's potential for imaging unexplored ultrafast non-repetitive phenomena.

Nonlinear photoacoustic waves for light guiding to deep tissue sites.

Light scattering by tissues limits performance in biological sensing or stimulation. Here we present a photoacoustic technique that increases light transmittance by one order of magnitude and enables light localization in deep tissue. Laser-induced nonlinear acoustic waves are utilized to produce a high refractive index contrast in scattering medium without high-intensity pressure. The size of guiding area is around 60 µm, which is equivalent or smaller than the diameter of multimode fibers. To show potential use in biomedical fields, we performed light guiding and imaging of fluorescence, through swine tissues with thickness more than 1 mm.

Dispersive coherent Brillouin scattering spectroscopy.

Frequency- and time-domain Brillouin scattering spectroscopy are powerful tools to read out the mechanical properties of complex systems in material and life sciences. Indeed, coherent acoustic phonons in the time-domain method offer superior depth resolution and a stronger signal than incoherent acoustic phonons in the frequency-domain method. However, it requires scanning of delay time between laser pulses for pumping and probing coherent acoustic phonons. Here, we present Brillouin scattering spectroscopy that spans the time and frequency domains to allow the multichannel detection of Brillouin scattering light from coherent acoustic phonons. Our technique traces the time-evolve Brillouin oscillations at the instantaneous frequency of a chromatic-dispersed laser pulse. The spectroscopic heterodyning of Brillouin scattering light in the frequency domain allows a single-frame readout of gigahertz-frequency oscillations with a spectrometer. As a proof of concept, we imaged heterogeneous thin films and biological cells over a wide bandwidth with nanometer depth resolution.

Cytosolic protein delivery using ultrasound-guided vaporization of perfluorocarbon nano-droplets.

Ultrasound-guided protein delivery is promising for site-specific control of cellular functions in the deep interior of the body in a noninvasive manner. Herein, we propose a method for cytosolic protein delivery based on ultrasound-guided intracellular vaporization of perfluorocarbon nano-droplets. The nano-droplets were conjugated with cargo proteins through a bio-reductively cleavable linker and introduced into living cells via antibody-mediated binding to a cell-surface receptor, which gets internalized through endocytosis. After the cells were exposed to ultrasound for endosomal escape of proteins, the ultrasound-responsive cytosolic release of a cargo enzyme was confirmed by visualizing the hydrolysis of the fluorogenic substrate using confocal microscopy. Moreover, a significant decrease in cell viability was achieved via the release of a cytotoxic protein in response to ultrasound treatment. The results of this study provide the proof of a principle that protein-conjugated nano-droplets can be used as carriers in ultrasound-guided cytosolic delivery of proteins.

Single-shot optical imaging with spectrum circuit bridging timescales in high-speed photography.

Single-shot optical imaging based on ultrashort lasers has revealed nonrepetitive processes in subnanosecond timescales beyond the recording range of conventional high-speed cameras. However, nanosecond photography without sacrificing short exposure time and image quality is still missing because of the gap in recordable timescales between ultrafast optical imaging and high-speed electronic cameras. Here, we demonstrate nanosecond photography and ultrawide time-range high-speed photography using a spectrum circuit that produces interval-tunable pulse trains while keeping short pulse durations. We capture a shock wave propagating through a biological cell with a 1.5-ns frame interval and 44-ps exposure time while suppressing image blur. Furthermore, we observe femtosecond laser processing over multiple timescales (25-ps, 2.0-ns, and 1-ms frame intervals), showing that the plasma generated at the picosecond timescale affects subsequent shock wave formation at the nanosecond timescale. Our technique contributes to accumulating data of various fast processes for analysis and to analyzing multi-timescale phenomena as a series of physical processes.

Cardiac macrophages prevent sudden death during heart stress.

Cardiac arrhythmias are a primary contributor to sudden cardiac death, a major unmet medical need. Because right ventricular (RV) dysfunction increases the risk for sudden cardiac death, we examined responses to RV stress in mice. Among immune cells accumulated in the RV after pressure overload-induced by pulmonary artery banding, interfering with macrophages caused sudden death from severe arrhythmias. We show that cardiac macrophages crucially maintain cardiac impulse conduction by facilitating myocardial intercellular communication through gap junctions. Amphiregulin (AREG) produced by cardiac macrophages is a key mediator that controls connexin 43 phosphorylation and translocation in cardiomyocytes. Deletion of Areg from macrophages led to disorganization of gap junctions and, in turn, lethal arrhythmias during acute stresses, including RV pressure overload and ß-adrenergic receptor stimulation. These results suggest that AREG from cardiac resident macrophages is a critical regulator of cardiac impulse conduction and may be a useful therapeutic target for the prevention of sudden death.

Investigating the optimum size of nanoparticles for their delivery into the brain assisted by focused ultrasound-induced blood-brain barrier opening.

The blood-brain barrier (BBB) has hampered the efficiency of nanoparticle delivery into the brain via conventional strategies. The widening of BBB tight junctions via focused ultrasound (FUS) offers a promising approach for enhancing the delivery of nanoparticles into the brain. However, there is currently an insufficient understanding of how nanoparticles pass through the opened BBB gaps. Here we investigated the size-dependence of nanoparticle delivery into the brain assisted by FUS-induced BBB opening, using gold nanoparticles (AuNPs) of 3, 15, and 120 nm diameter. For 3- and 15-nm AuNPs, FUS exposure significantly increased permeation across an in vitro BBB model by up to 9.5 times, and the permeability was higher with smaller diameter. However, in vivo transcranial FUS exposure in mice demonstrated that smaller particles were not necessarily better for delivery into the brain. Medium-sized (15 nm) AuNPs showed the highest delivery efficiency (0.22% ID), compared with 3- and 120-nm particles. A computational model suggested that this optimum size was determined by the competition between their permeation through opened BBB gaps and their excretion from blood. Our results would greatly contribute to designing nanoparticles for their delivery into the brain for the treatment of central nervous system diseases.

Selective intracellular delivery of perfluorocarbon nanodroplets for cytotoxicity threshold reduction on ultrasound-induced vaporization.

BACKGROUND: Phase-change nanodroplets (PCNDs), which are liquid perfluorocarbon nanoparticles, have garnered much attention as ultrasound-responsive nanomedicines. The vaporization phenomenon has been employed to treat tumors mechanically. However, the ultrasound pressure applied to induce vaporization must be low to avoid damage to nontarget tissues. AIMS: Here, we report that the pressure threshold for vaporization to induce cytotoxicity can be significantly reduced by selective intracellular delivery of PCNDs into targeted tumors. METHODS AND RESULTS: In vitro experiments revealed that selective intracellular delivery of PCNDs induced PCND aggregation specifically inside the targeted cells. This close-packed configuration decreased the pressure threshold for vaporization to induce cytotoxicity. Moreover, following ultrasound exposure, significant decrease was observed in the viability of cells that incorporated PCNDs (35%) but not in the viability of cells that did not incorporate PCNDs (88%). CONCLUSIONS: Intracellular delivery of PCNDs reduced ultrasound pressure applied for vaporization to induce cytotoxicity. Confocal laser scanning microscopy and flow cytometry revealed that prolonged PCND-cell incubation increased PCND uptake and aggregation. This aggregation effect might have contributed to the cytotoxicity threshold reduction effect.

The lifetime evaluation of vapourised phase-change nano-droplets.

Phase-change nano-droplets (PCNDs) are sub-micron particles that are coated with phospholipid and contain liquid-state perfluorocarbons such as perfluoropentane (boiling point=29°C) and perfluorohexane (boiling point=57°C), which can vapourise upon application of ultrasound. The bubbles generated by such reactions can serve as ultrasound contrast agents or HIFU sensitisers. However, the lifetime of bubbles generated from PCNDs on µs-order is not well known. Knowledge of the condition of PCND-derived bubbles on µs-order is essential for producing bubbles customised for specific purposes. In this study, we use an optical measurement system to measure the vapourisation and stability of the bubbles (bubble-lifetime) as well as the stability-controlling method of the nucleated bubbles on µs-order while changing the internal composition of PCNDs and the ambient temperature. PCND-derived bubbles remain in a bubble state when the boiling point of the internal composition is lower than the ambient temperature, but lose their optical contrast after approximately 10µs by re-condensation or dissolution when the boiling point of the internal composition is higher than the ambient temperature. We reveal that the superheating condition significantly affects the fate of vapourised PCNDs and that the bubble-lifetime can be controlled by changing both the ambient temperature conditions and the internal composition of PCNDs.

Automated frame selection process for high-resolution microendoscopy.

We developed an automated frame selection algorithm for high-resolution microendoscopy video sequences. The algorithm rapidly selects a representative frame with minimal motion artifact from a short video sequence, enabling fully automated image analysis at the point-of-care. The algorithm was evaluated by quantitative comparison of diagnostically relevant image features and diagnostic classification results obtained using automated frame selection versus manual frame selection. A data set consisting of video sequences collected in vivo from 100 oral sites and 167 esophageal sites was used in the analysis. The area under the receiver operating characteristic curve was 0.78 (automated selection) versus 0.82 (manual selection) for oral sites, and 0.93 (automated selection) versus 0.92 (manual selection) for esophageal sites. The implementation of fully automated high-resolution microendoscopy at the point-of-care has the potential to reduce the number of biopsies needed for accurate diagnosis of precancer and cancer in low-resource settings where there may be limited infrastructure and personnel for standard histologic analysis.