Hyuk-Sang Kwon

SignificanceRapid acquisition of high-resolution volumetric images has been critical to effectively monitor dynamic biological processes in vivo, yet it faces tradeoffs between image resolution, penetration depth, and imaging speed. These limitations hinder the ability to study rapid neurophysiological events such as cerebrovascular dynamics and cellular activity, highlighting the need for advanced high-speed 3D imaging system.AimTo address these challenges in volumetric imaging performances, we aimed to develop a high-speed volumetric imaging system capable of resolving fast biological dynamics with minimal compromise in spatial resolution or imaging depth.ApproachWe devised a rapid axially scanned and de-scanned (RASAD) scheme by integrating a TAG lens (tunable acoustic gradient index of refraction lens) into a line-scan confocal microscope. The TAG lens enabled axial (depth) scanning frequency at 70 kHz, allowing 3D projection imaging at rates up to 200 Hz with a detection depth of 135 μm while minimally sacrificing the image quality (i.e., a lateral resolution of ∼2.6 μm).ResultsWe validated its performance through in vitro imaging of spontaneously contracting cardiomyocyte aggregates, capturing real-time calcium transients and synchronized contractions, and through in vivo imaging of the mouse cortical tissue, where volumetric acquisition over a 450×450×100 μm3 region enabled quantification of blood flow velocities up to 3.64 mm/s across various vessel types.ConclusionsThe RASAD system enables high-speed, high-resolution 3D imaging of dynamic biological processes, providing a valuable tool for advancing studies of neurophysiological mechanisms and biomedical applications.

Diabetic neuropathy (DN) is a prevalent and debilitating complication of diabetes, significantly impairing quality of life through chronic pain, sensory deficits, and motor dysfunction. Despite its widespread impact, current rodent behavioral assessments using 2D tracking methods primarily quantify basic locomotion, such as distance and speed, but lack resolution to detect subtle, pattern-based motor impairments characteristic of DN. This study employed MoSeq-based 3D behavioral profiling combined with unsupervised machine learning to identify subtle yet significant alterations in nicotinamide (NA)- and streptozotocin (STZ)-induced DN mouse models. Our analysis identified 22 distinct behavioral syllables, with DN mice exhibiting increased stress-associated behaviors such as head weaving, wall jumping, and nasal hesitancy, while displaying decreased locomotor activities including walking and rearing. These alterations were accompanied by heightened mechanical sensitivity indicative of neuropathic pain and a more predictable, less exploratory behavioral transition pattern, suggesting a restricted behavioral repertoire rather than improved motor coordination. Additionally, MoSeq-based profiling enabled detailed analysis of movement organization and temporal transitions, highlighting stereotyped behavioral sequences and notably decreased exploratory behaviors in DN mice. These behavioral patterns indicate that DN-associated pain is more strongly related to impairments in behavioral adaptability and higher-order motor planning than to simple reductions in movement, suggesting underlying dysfunctions in sensorimotor or cognitive control circuits. These findings indicate that MoSeq can be used as a valuable tool for high-resolution behavioral quantification in diabetic neuropathic animal pain model, enabling refined evaluation of neuropathic phenotypes and therapeutic efficacy in preclinical studies.

Understanding neuronal activity is essential for studying brain function and disease, especially in the context of neurodegenerative disorders such as Alzheimer’s,^{1 – 3} Parkinson’s,^{4 – 6} and Huntington’s,^{7 – 9} as well as conditions such as neuropathic pain^{10 – 12} and chronic pain.^{13 – 15} Advances in neuroimaging, particularly in volumetric imaging techniques, have significantly enhanced our ability to explore these areas. Unlike traditional methods that provide only planar views, volumetric imaging provides a three-dimensional perspective, capturing depth information that has revolutionized our understanding of brain function and pathology. These technologies have enabled unprecedented spatial and temporal resolution, marking a major leap forward in the study of the brain.

In optical imaging of solid tumors, signal contrasts derived from inherent tissue temperature differences have been employed to distinguish tumor masses from surrounding tissue. Moreover, with the advancement of active infrared imaging, dynamic thermal characteristics in response to exogenous thermal modulation (heating and cooling) have been proposed as novel measures of tumor assessment. Contrast factors such as the average rate of temperature changes and thermal recovery time constants have been investigated through an active thermal modulation imaging approach, yielding promising tumor characterization results in a xenograft mouse model. Here, to assess its clinical potential, we developed and deployed an endoscopic infrared thermal modulation imaging system, incorporating anti-reflection germanium lenses. Employing tissue cooling, we evaluated the feasibility of detecting in situ tumors in a syngeneic rectal tumor mouse model. Consequently, early-stage tumors were successfully localized and evaluated based on their heat signatures. Notably, tumors exhibited a higher rate of temperature change induced by thermal modulation compared to adjacent tissues. Through the introduction of this label-free technology, Infrared Thermal Modulation Endoscopy (ITME), our study showcased an effective method for optically delineating and assessing solid tumors. This innovative diagnostic technology holds significant promise for enhancing our ability to detect, classify, and characterize abnormal tissues.

Anxiety in dental patients has caused inconvenient experiences during their dental visits due to the noise generated by the dental handpiece. High-frequency sounds generated by the handpiece have been challenging to reduce using the active control method that targets low-frequency sounds, as well as the difficulty in applying the noise control method using sound-absorbing materials, because the size of the handpiece is small. As an alternative, a method that can reduce noise and provide stability by playing music to patients is being studied. However, in most studies, there are inconveniences such as the need to turn the music volume higher to cover dental handpiece noise or having to wear headphones to play music. In this study, in order to reduce this inconvenience and optimize the noise reduction effect of music, we propose a technology that converts music into sound masking and a unit chair equipped with a bone conduction speaker that plays music, and through clinical trials with 35 patients, it was confirmed that the proposed system made the patients emotionally stable. In addition, by analyzing the causes of these emotional changes, it suggests that the preferred genre of music by patients should also be considered.

We developed a single-camera-based near-infrared (NIR) fluorescence imaging device using indocyanine green (ICG) NIR fluorescence contrast agents for image-induced surgery. In general, a fluorescent imaging system that simultaneously provides color and NIR images uses two cameras, which is disadvantageous because it increases the imaging head of the system. Recently, a single-camera-based NIR optical imaging device with quantum efficiency partially extended to the NIR region was developed to overcome this drawback. The system used RGB_NIR filters for camera sensors to provide color and NIR images simultaneously; however, the sensitivity and resolution of the infrared images are reduced by 1/4, and the exposure time and gain cannot be set individually when acquiring color and NIR images. Thus, to overcome these shortcomings, this study developed a compact fluorescent imaging system that uses a single camera with two complementary metal–oxide semiconductor (CMOS) image sensors. Sensitivity and signal-to-background ratio were measured according to the concentrations of ICG solution, exposure time, and camera gain to evaluate the performance of the imaging system. Consequently, the clinical applicability of the system was confirmed through the toxicity analysis of the light source and in vivo testing.

Long-term disabilities induced by stroke impose a heavy burden on patients, families, caregivers, and public health systems. Extensive studies have demonstrated the therapeutic value of neuromodulation in enhancing post-stroke recovery. Among them, chemogenetic neuromodulation activated by clozapine-N-oxide (CNO) has been proposed as the potential tool of neuromodulation. However, recent evidence showed that CNO does not cross the blood − brain barrier and may in fact have low binding affinity for chemogenetic tool. Thus, clozapine (CLZ) has been suggested for use in chemogenetic neuromodulation, in place of CNO, because it readily crosses the blood–brain barrier. Previously we reported that low doses of CLZ (0.1 mg/kg) successfully induced neural responses without off-target effects. Here, we show that low-dose clozapine (0.1 mg/kg) can induce prolonged chemogenetic activation while avoiding permeability issues and minimizing off-target effects. In addition, clozapine-induced excitatory chemogenetic neuromodulation (CLZ-ChemoNM) of sensory-parietal cortex with hsyn-hM3Dq-YFP-enhanced motor recovery in a chronic capsular infarct model of stroke in rats, improving post-stroke behavioral scores to 56% of pre-infarct levels. Longitudinal 2-deoxy-2-[18F]-fluoro-D-glucose microPET (FDG-microPET) scans showed that a reduction in diaschisis volume and activation of corticostriatal circuits were both correlated with post-stroke recovery. We also found c-Fos increases in bilateral cortices and BDNF increases in the cortices and striatum after CLZ-ChemoNM, indicating an increase in neural plasticity. These findings suggest the translational feasibility of CLZ-ChemoNM for augmenting recovery in chronic stroke.

In microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and usually suffers from a reduced acquisition rate, which is critical to properly document rapid neurophysiological dynamics. In this study, we implemented an electrically tunable lens (ETL) in the line-scan confocal microscopy (LSCM), enabling the volumetric acquisition at the rate of 20 frames per second with a maximum volume of interest of 315 × 315 × 80 µm³. The axial extent of point-spread-function (PSF) was 17.6 ± 1.6 µm and 90.4 ± 2.1 µm with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth axial penetration by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of both cleared mouse brain ex vivo samples and in vivo brains. The current study showed a successful application of resonant ETL for constructing a high-performance 3D axially scanning LSCM (asLSCM) system. Such advances in rapid volumetric imaging would significantly enhance our understanding of various dynamic biological processes.