Robotic transcranial magnetic stimulation in the treatment of depression: a pilot study.

There has been an increasing demand for robotic coil positioning during repetitive transcranial magnetic stimulation (rTMS) treatment. Accurate coil positioning is crucial because rTMS generally targets specific brain regions for both research and clinical application with other reasons such as safety, consistency and reliability and individual variablity. Some previous studies have employed industrial robots or co-robots and showed they can more precisely stimulate the target cortical regions than traditional manual methods. In this study, we not only developed a custom-TMS robot for better TMS coil placement but also analyzed the therapeutic effects on depression. Treatment effects were evaluated by measuring regional cerebral blood flow (rCBF) using single-photon emission computed tomography and depression severity before and after rTMS for the two positioning methods. The rTMS preparation time with our robotic coil placement was reduced by 53% compared with that of the manual method. The position and orientation errors were also significantly reduced from 11.17 mm and 4.06° to 0.94 mm and 0.11°, respectively, confirming the superiority of robotic positioning. The results from clinical and neuroimaging assessments indicated comparable improvements in depression severity and rCBF in the left dorsolateral prefrontal cortex between the robotic and manual rTMS groups. A questionnaire was used to determine the patients' feelings about the robotic system, including the safety and preparation time. A high safety score indicated good acceptability of robotic rTMS at the clinical site.

A phased array ultrasound system with a robotic arm for neuromodulation.

BACKGROUND: Ultrasonic neuromodulation (UNMOD) provides a non-invasive brain stimulation. However, the high-resolution region-specificity of UNMOD with a single element transducer combined with a mechanical positioning system could have limits due to the intrinsic positioning error from mechanical systems. OBJECTIVE/HYPOTHESIS: A phased array system could lead to highly selective neuromodulation with electronic control. METHODS: A specialized phased-array system with a robotic arm is implemented for a rhesus monkey model. Various primary motor cortex areas related to tail, hand, and mouth were stimulated with a 200 µm step size. The ultrasonic parameters were ISPTA of 840 mW/cm2, pulse repetition frequency of 100 Hz, and a 5% duty factor at 600 kHz. The induced movement were recorded and analyzed. RESULTS: Separate digits, mouth, and tongue motions were successfully induced by electronically controlling the focus. The identical body part movement could be induced when the focus was moved back to the identical primary motor cortex with electronic control. Accordingly, the reproducibility of UNMOD could be partially validated with rhesus monkey model. CONCLUSION: A phased-array system appears to have a potential for the non-invasive and region-selective neuromodulation method.

Development of a Wide Area 3D Scanning System with a Rotating Line Laser.

In a 3D scanning system, using a camera and a line laser, it is critical to obtain the exact geometrical relationship between the camera and laser for precise 3D reconstruction. With existing depth cameras, it is difficult to scan a large object or multiple objects in a wide area because only a limited area can be scanned at a time. We developed a 3D scanning system with a rotating line laser and wide-angle camera for large-area reconstruction. To obtain 3D information of an object using a rotating line laser, we must be aware of the plane of the line laser with respect to the camera coordinates at every rotating angle. This is done by estimating the rotation axis during calibration and then by rotating the laser at a predefined angle. Therefore, accurate calibration is crucial for 3D reconstruction. In this study, we propose a calibration method to estimate the geometrical relationship between the rotation axis of the line laser and the camera. Using the proposed method, we could accurately estimate the center of a cone or cylinder shape generated while the line laser was rotating. A simulation study was conducted to evaluate the accuracy of the calibration. In the experiment, we compared the results of the 3D reconstruction using our system and a commercial depth camera. The results show that the precision of our system is approximately 65% higher for plane reconstruction, and the scanning quality is also much better than that of the depth camera.

Fluorescence microscopy tensor imaging representations for large-scale dataset analysis.

Understanding complex biological systems requires the system-wide characterization of cellular and molecular features. Recent advances in optical imaging technologies and chemical tissue clearing have facilitated the acquisition of whole-organ imaging datasets, but automated tools for their quantitative analysis and visualization are still lacking. We have here developed a visualization technique capable of providing whole-organ tensor imaging representations of local regional descriptors based on fluorescence data acquisition. This method enables rapid, multiscale, analysis and virtualization of large-volume, high-resolution complex biological data while generating 3D tractographic representations. Using the murine heart as a model, our method allowed us to analyze and interrogate the cardiac microvasculature and the tissue resident macrophage distribution and better infer and delineate the underlying structural network in unprecedented detail.

Ultrasonic Neuromodulation via Astrocytic TRPA1.

Low-intensity, low-frequency ultrasound (LILFU) is the next-generation, non-invasive brain stimulation technology for treating various neurological and psychiatric disorders. However, the underlying cellular and molecular mechanism of LILFU-induced neuromodulation has remained unknown. Here, we report that LILFU-induced neuromodulation is initiated by opening of TRPA1 channels in astrocytes. The Ca2+ entry through TRPA1 causes a release of gliotransmitters including glutamate through Best1 channels in astrocytes. The released glutamate activates NMDA receptors in neighboring neurons to elicit action potential firing. Our results reveal an unprecedented mechanism of LILFU-induced neuromodulation, involving TRPA1 as a unique sensor for LILFU and glutamate-releasing Best1 as a mediator of glia-neuron interaction. These discoveries should prove to be useful for optimization of human brain stimulation and ultrasonogenetic manipulations of TRPA1.

Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

Motion characterization scheme to minimize motion artifacts in intravital microscopy.

Respiratory- and cardiac-induced motion artifacts pose a major challenge for in vivo optical imaging, limiting the temporal and spatial imaging resolution in fluorescence laser scanning microscopy. Here, we present an imaging platform developed for in vivo characterization of physiologically induced axial motion. The motion characterization system can be straightforwardly implemented on any conventional laser scanning microscope and can be used to evaluate the effectiveness of different motion stabilization schemes. This method is particularly useful to improve the design of novel tissue stabilizers and to facilitate stabilizer positioning in real time, therefore facilitating optimal tissue immobilization and minimizing motion induced artifacts.

Prolonged stimulation with low-intensity ultrasound induces delayed increases in spontaneous hippocampal culture spiking activity.

Ultrasound is a promising neural stimulation modality, but an incomplete understanding of its range and mechanism of effect limits its therapeutic application. We investigated the modulation of spontaneous hippocampal spike activity by ultrasound at a lower acoustic intensity and longer time scale than has been previously attempted, hypothesizing that spiking would change conditionally upon the availability of glutamate receptors. Using a 60-channel multielectrode array (MEA), we measured spontaneous spiking across organotypic rat hippocampal slice cultures (N = 28) for 3 min each before, during, and after stimulation with low-intensity unfocused pulsed or sham ultrasound (spatial-peak pulse average intensity 780 µW/cm2 ) preperfused with artificial cerebrospinal fluid, 300 µM kynurenic acid (KA), or 0.5 µM tetrodotoxin (TTX) at 3 ml/min. Spike rates were normalized and compared across stimulation type and period, subregion, threshold level, and/or perfusion condition using repeated-measures ANOVA and generalized linear mixed models. Normalized 3-min spike counts for large but not midsized, small, or total spikes increased after but not during ultrasound relative to sham stimulation. This result was recapitulated in subregions CA1 and dentate gyrus and replicated in a separate experiment for all spike size groups in slices pretreated with aCSF but not KA or TTX. Increases in normalized 18-sec total, midsized, and large spike counts peaked predominantly 1.5 min following ultrasound stimulation. Our low-intensity ultrasound setup exerted delayed glutamate receptor-dependent, amplitude- and possibly region-specific influences on spontaneous spike rates across the hippocampus, expanding the range of known parameters at which ultrasound may be used for neural activity modulation. © 2016 Wiley Periodicals, Inc.

Imaging the beating heart in the mouse using intravital microscopy techniques.

Real-time microscopic imaging of moving organs at single-cell resolution represents a major challenge in studying complex biology in living systems. Motion of the tissue from the cardiac and respiratory cycles severely limits intravital microscopy by compromising ultimate spatial and temporal imaging resolution. However, significant recent advances have enabled single-cell resolution imaging to be achieved in vivo. In this protocol, we describe experimental procedures for intravital microscopy based on a combination of thoracic surgery, tissue stabilizers and acquisition gating methods, which enable imaging at the single-cell level in the beating heart in the mouse. Setup of the model is typically completed in 1 h, which allows 2 h or more of continuous cardiac imaging. This protocol can be readily adapted for the imaging of other moving organs, and it will therefore broadly facilitate in vivo high-resolution microscopy studies.

New techniques for motion-artifact-free in vivo cardiac microscopy.

Intravital imaging microscopy (i.e., imaging in live animals at microscopic resolution) has become an indispensable tool for studying the cellular micro-dynamics in cancer, immunology and neurobiology. High spatial and temporal resolution, combined with large penetration depth and multi-reporter visualization capability make fluorescence intravital microscopy compelling for heart imaging. However, tissue motion caused by cardiac contraction and respiration critically limits its use. As a result, in vitro cell preparations or non-contracting explanted heart models are more commonly employed. Unfortunately, these approaches fall short of understanding the more complex host physiology that may be dynamic and occur over longer periods of time. In this review, we report on novel technologies, which have been recently developed by our group and others, aimed at overcoming motion-induced artifacts and capable of providing in vivo subcellular resolution imaging in the beating mouse heart. The methods are based on mechanical stabilization, image processing algorithms, gated/triggered acquisition schemes or a combination of both. We expect that in the immediate future all these methodologies will have considerable applications in expanding our understanding of the cardiac biology, elucidating cardiomyocyte function and interactions within the organism in vivo, and ultimately improving the treatment of cardiac diseases.

Automated motion artifact removal for intravital microscopy, without a priori information.

Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.

Advanced Motion Compensation Methods for Intravital Optical Microscopy.

Intravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the micro-dynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions.

Sequential average segmented microscopy for high signal-to-noise ratio motion-artifact-free in vivo heart imaging.

In vivo imaging is often severely compromised by cardiovascular and respiratory motion. Highly successful motion compensation techniques have been developed for clinical imaging (e.g. magnetic resonance imaging) but the use of more advanced techniques for intravital microscopy is largely unexplored. Here, we implement a sequential cardiorespiratory gating scheme (SCG) for averaged microscopy. We show that SCG is very efficient in eliminating motion artifacts, is highly practical, enables high signal-to-noise ratio (SNR) in vivo imaging, and yields large field of views. The technique is particularly useful for high-speed data acquisition or for imaging scenarios where the fluorescence signal is not significantly above noise or background levels.

Improved intravital microscopy via synchronization of respiration and holder stabilization.

A major challenge in high-resolution intravital confocal and multiphoton microscopy is physiologic tissue movement during image acquisition. Of the various physiological sources of movement, respiration has arguably the largest and most wide-ranging effect. We describe a technique for achieving stabilized microscopy imaging using a dual strategy. First, we designed a mechanical stabilizer for constraining physical motion; this served to simultaneously increase the in-focus range over which data can be acquired as well as increase the reproducibility of imaging a certain position within each confocal imaging plane. Second, by implementing a retrospective breathing-gated imaging modality, we performed selective image extraction gated to a particular phase of the respiratory cycle. Thanks to the high reproducibility in position, all gated images presented a high degree of correlation over time. The images obtained using this technique not only showed significant improvements over images acquired without the stabilizer, but also demonstrated accurate in vivo imaging during longitudinal studies. The described methodology is easy to implement with any commercial imaging system, as are used by most biological imaging laboratories, and can be used for both confocal and multiphoton laser scanning microscopy.

Real-time in vivo imaging of the beating mouse heart at microscopic resolution.

Real-time imaging of moving organs and tissues at microscopic resolutions represents a major challenge in studying the complex biology of live animals. Here we present a technique based on a novel stabilizer setup combined with a gating acquisition algorithm for the imaging of a beating murine heart at the single-cell level. The method allows serial in vivo fluorescence imaging of the beating heart in live mice in both confocal and nonlinear modes over the course of several hours. We demonstrate the utility of this technique for in vivo optical sectioning and dual-channel time-lapse fluorescence imaging of cardiac ischaemia. The generic method could be adapted to other moving organs and thus broadly facilitate in vivo microscopic investigations.

Motion compensation using a suctioning stabilizer for intravital microscopy.

Motion artifacts continue to present a major challenge to single cell imaging in cardiothoracic organs such as the beating heart, blood vessels, or lung. In this study, we present a new water-immersion suctioning stabilizer that enables minimally invasive intravital fluorescence microscopy using water-based stick objectives. The stabilizer works by reducing major motion excursions and can be used in conjunction with both prospective or retrospective gating approaches. We show that the new approach offers cellular resolution in the beating murine heart without perturbing normal physiology. In addition, because this technique allows multiple areas to be easily probed, it offers the opportunity for wide area coverage at high resolution.