Design of a novel 5-camera surface guidance system with multiple imaging isocenters.

PURPOSE/OBJECTIVE(S): Surface-guided radiation therapy (SGRT) can track the patient surface noninvasively to complement radiographic image-guided radiation therapy with a standard 3-camera system and a single radiation/image isocenter. Here we report the commissioning of a novel SGRT system that monitors three imaging isocenters locations in a proton half-gantry room with a unique 5-camera configuration. MATERIALS/METHODS: The proton half-gantry room has three image isocenters, designated ISO-0, ISO-1, and ISO-2, to cover various anatomical sites via a robotic ceiling-mounted cone-beam CT. Although ISO-0 and ISO-1 are used to image the cranium, head and neck, and thoracic regions, ISO-2 is often used to image body and extremity sites and contiguous craniospinal target volumes. The five-camera system was calibrated to the radiographic isocenter by using a stereotactic radiosurgery cube phantom for each image isocenter. RESULTS: The performance of this 5-camera system was evaluated for 6 degrees of freedom in three categories: (1) absolute setup accuracy relative to the radiographic kV image isocenter based on the DICOM reference; (2) relative shift accuracy based on a reference surface capture; and (3) isocenter tracking accuracy from one isocenter to another based on a reference surface capture. The evaluation revealed maximum deviations of 0.8, 0.2, and 0.6 mm in translation and 0.2°, 0.1°, and 0.1° in rotation for the first, second, and third categories, respectively. Comparing the dosimetry and latency with static and gated irradiation revealed a 0.1% dose difference and positional differences of 0.8 mm in X and 0.9 mm in Y with less than 50 ms temporal accuracy. CONCLUSION: The unique 5-camera system configuration provides SGRT at the treatment isocenter (ISO-0) and also imaging isocenter locations (ISO-0, ISO-1, and ISO-2) to ensure correct patient positioning before and after radiographic imaging, especially during transitions from the offset imaging isocenters to the treatment isocenter.

Multi-Field Interference Simultaneously Imaging on Single Image for Dynamic Surface Measurement.

To obtain the dynamic surface of high-frequency vibrating mirrors (VMs), a novel method involving multi-field interference (MFI) pattern imaging on a single image is proposed in this paper. Using multiple reflections and refractions, the proposed method generates three interference patterns at the same time, which improves the traditional time-series methods where a single interference pattern can be obtained at one time. Experimental results show that a series of MFI patterns can be obtained on a single image, with the laser repetition frequency (LRF) ranging from 200 Hz to 10 Hz, and the frame rate of the camera at 10 Hz. Particularly if the LRF (10 Hz) is equal to the frame rate of image, crosstalk is avoided completely, which is particularly desirable in dynamic surface measurement. In summary, the MFI imaging method provides an effective way for VM dynamic surface measurement.

NIR-responsive nanoplatform for pre/intraoperative image-guided carcinoma surgery and photothermal ablation of residual tumor tissue.

Local recurrence is common among patients with advanced cancer who have undergone surgery. Here, we developed a new surgical treatment for cancer based on a nanoparticle that loaded a near-infrared dye (IR780 iodide) and perfluorooctyl bromide into liposomes (NP-IR780). In an orthotopic breast cancer mouse model, NP-IR780 was demonstrated to have excellent tumor-targeting ability due to the selective tumor accumulation of IR780 iodide and the enhanced permeation and retention effect of the nanoparticle. With the excellent targeting ability, concurrent computed tomography and photoacoustic imaging were achieved for preoperative planning. In particular, NP-IR780 could serve as a tumor indicator for near-infrared fluorescence image-guided precise resection of lesions during surgery. Importantly, residual tumors could be ablated through intraoperative photothermal therapy without obvious recurrence. This work provides a theranostic strategy that significantly improved the survival of mice through pre/intraoperative image-guided tumor resection and subsequent photothermal therapy of residual lesions.

Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

Advanced spinning disk-TIRF microscopy for faster imaging of the cell interior and the plasma membrane.

Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high-resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high-resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD-TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD-TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup.

Resolution-matched reflection mode photoacoustic microscopy and optical coherence tomography dual modality system.

Photoacoustic microscopy (PAM) and optical coherence tomography (OCT) are sensitive to optical absorption and scattering characteristics, respectively. As such, the integration of these two modalities in order to combine important complementary information has garnered much attention. Due to the relatively low axial resolution of PAM, PAM and OCT dual modality systems generally have a large resolution gap, especially for reflection mode systems. In this study, based on a wide-band transparent pure-optical ultrasonic detector, we developed a dual modality system (PAM-OCT system) in which PAM has a similar spatial resolution (i.e. several micrometers in both the lateral and axial directions) to OCT. In addition, due to the optical transparency advantage, the integrated system works in reflection mode, which is ideal for in vivo biomedical imaging. We successfully imaged the skin of a mouse hindlimb, which cannot be done by a transmission mode dual modality system. Our work demonstrates this dual modality system has potential in biomedical studies with complementary imaging contrasts.

Mesoporous carbon­manganese nanocomposite for multiple imaging guided oxygen-elevated synergetic therapy.

Despite of the extensive application of photodynamic therapy (PDT)nowadays, several restrictions have emerged such as hydrophobility, undesired phototoxicity and low selectivity of photosensitizer as well as the hypoxic tumor microenvironment. To address these challenges, a multifunctional mesoporous carbon­manganese nanocomposite (MC-MnO2) is developed to load Chlorin e6 (Ce6) with a high loading capacity. The MC-MnO2 can prevent Ce6 from being activated by the sunlight to reduce unintentional phototoxicity significantly and realize the hypoxia relief via reacting with the H2O2 overexpressed in tumor tissue, meanwhile, the reduced product Mn2+ ion could act as a T1/T2-weighted MRI contrast. Based on the broad absorption of MC-MnO2 within the range of NIR, the nanoparticle has the potential for serving as a photothermal agent and photoacoustic imaging (PAI) agent. The PEG and iRGD are further decorated on MC-MnO2 (iPMC-MnO2) to improve the biocompatibility, targeting and penetration of the nanoparticle. Taking full advantage of the good photothermal effect of iPMC-MnO2, the photothermal therapy (PTT) and enhanced PDT are subtly integrated into one system, developing an intelligent multimodal diagnostic and therapeutic nanoplatform and realizing our "one nanoparticle fits all" dream.

Multicolor fluorescence microscopy using static light sheets and a single-channel detection.

We present a multicolor fluorescence microscope system, under a selective plane illumination microscopy (SPIM) configuration, using three continuous wave-lasers and a single-channel-detection camera. The laser intensities are modulated with three time-delayed pulse trains that operate synchronously at one third of the camera frame rate, allowing a sequential excitation and an image acquisition of up to three different biomarkers. The feasibility of this imaging acquisition mode is demonstrated by acquiring single-plane multicolor images of living hyphae of Neurospora crassa. This allows visualizing simultaneously the localization and dynamics of different cellular components involved in apical growth in living hyphae. The configuration presented represents a noncommercial, cost-effective alternative microscopy system for the rapid and simultaneous acquisition of multifluorescent images and can be potentially useful for three-dimensional imaging of large biological samples.

Multiple Neuroimaging Measures for Examining Exercise-induced Neuroplasticity in Older Adults: A Quasi-experimental Study.

Physical exercise can improve physical and mental health. A number of imaging studies have examined the role of neuroplasticity in improving cognition with physical exercise; however, such neuroplasticity changes are not consistent across the reports partly due to small sample sizes in some studies. We thought to explore the concept that identifying consistent findings across multi-modality imaging measures would provide relatively reliable results. We designed a 6-week quasi-experiment with Wii-fitness exercise program in 24 healthy adults older than 60, and then examined the changes on neuroimaging measures including brain volume, the amplitude of low-frequency oscillation function (ALFF), regional homogeneity (ReHo), seed-based functional connectivity (FC), and the global efficiency of nodal connectivity during resting state. We focused on whether there were common regions showing changes after exercise across these measures and which measure was closely correlated with cognitive improvement. After the six-week exercise program, participants demonstrated a significant improvement in memory and executive function on neuropsychological tests, and in memory recall on an emotional memory task. The common brain regions that showed significant changes across different measures were the right striatum and the posterior cingulate (PCC). After exercise, the PCC showed decreased ReHo and increased volume, and the striatum did not show volume loss as the control group did and increased its FC with the cingulate, temporal, parietal, and occipital regions. Moreover, the connectivity change between the striatum and the thalamus was correlated with the improvement of executive function. This result implicates the striatum and the PCC associated network in physical exercise. Our work highlights the effectiveness of multi-modality neuroimaging measures in investigating neuroplasticity.

Hemispheric differences in electrical and hemodynamic responses during hemifield visual stimulation with graded contrasts.

A multimodal neuroimaging technique based on electroencephalography (EEG) and functional near-infrared spectroscopy (fNIRS) was used with horizontal hemifield visual stimuli with graded contrasts to investigate the retinotopic mapping more fully as well as to explore hemispheric differences in neuronal activity, the hemodynamic response, and the neurovascular coupling relationship in the visual cortex. The fNIRS results showed the expected activation over the contralateral hemisphere for both the left and right hemifield visual stimulations. However, the EEG results presented a paradoxical lateralization, with the maximal response located over the ipsilateral hemisphere but with the polarity inversed components located over the contralateral hemisphere. Our results suggest that the polarity inversion as well as the latency advantage over the contralateral hemisphere cause the amplitude of the VEP over the contralateral hemisphere to be smaller than that over the ipsilateral hemisphere. Both the neuronal and hemodynamic responses changed logarithmically with the level of contrast in the hemifield visual stimulations. Moreover, the amplitudes and latencies of the visual evoked potentials (VEPs) were linearly correlated with the hemodynamic responses despite differences in the slopes.

Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP.

The pH-sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EGFP or EYFP is good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is an urgent need. Here a pH-sensitive red fluorescent protein, pHuji, is selected and verified to remain pH-sensitive in HM20 resin. We observe 183% fluorescence intensity of pHuji in resin-embeded mouse brain and 29.08-fold fluorescence intensity of reactivated pHuji compared to the quenched state. pHuji and EGFP can be quenched and chemically reactivated simultaneously in resin, thus enabling simultaneous two-color micro-optical sectioning tomography of resin-embedded mouse brain. This method may greatly facilitate the visualization of neuronal morphology and neural circuits to promote understanding of the structure and function of the brain.

Three-dimensional reconstruction and measurements of zebrafish larvae from high-throughput axial-view in vivo imaging.

High-throughput imaging is applied to provide observations for accurate statements on phenomena in biology and this has been successfully applied in the domain of cells, i.e. cytomics. In the domain of whole organisms, we need to take the hurdles to ensure that the imaging can be accomplished with a sufficient throughput and reproducibility. For vertebrate biology, zebrafish is a popular model system for High-throughput applications. The development of the Vertebrate Automated Screening Technology (VAST BioImager), a microscope mounted system, enables the application of zebrafish high-throughput screening. The VAST BioImager contains a capillary that holds a zebrafish for imaging. Through the rotation of the capillary, multiple axial-views of a specimen can be acquired. For the VAST BioImager, fluorescence and/or confocal microscopes are used. Quantitation of a specific signal as derived from a label in one fluorescent channel requires insight in the zebrafish volume to be able to normalize quantitation to volume units. However, from the setup of the VAST BioImager, a specimen volume cannot be straightforwardly derived. We present a high-throughput axial-view imaging architecture based on the VAST BioImager. We propose profile-based 3D reconstruction to produce 3D volumetric representations for zebrafish larvae using the axial-views. Volume and surface area can then be derived from the 3D reconstruction to obtain the shape characteristics in high-throughput measurements. In addition, we develop a calibration and a validation of our methodology. From our measurements we show that with a limited amount of views, accurate measurements of volume and surface area for zebrafish larvae can be obtained. We have applied the proposed method on a range of developmental stages in zebrafish and produced metrical references for the volume and surface area for each stage.

Fully integrated optical coherence tomography, ultrasound, and indocyanine green-based fluorescence tri-modality system for intravascular imaging.

We present a tri-modality imaging system and fully integrated tri-modality probe for intravascular imaging. The tri-modality imaging system is able to simultaneously acquire optical coherence tomography (OCT), ultrasound (US), and fluorescence imaging. Moreover, for fluorescence imaging, we used the FDA-approved indocyanine green (ICG) dye as the contrast agent to target lipid-loaded macrophages. We conducted imaging from a male New Zealand white rabbit to evaluate the performance of the tri-modality system. In addition, tri-modality images of rabbit aortas were correlated with hematoxylin and eosin (H&E) histology to check the measurement accuracy. The fully integrated miniature tri-modality probe, together with the use of ICG dye suggest that the system is of great potential for providing a more accurate assessment of vulnerable plaques in clinical applications.

Registration of free-hand OCT daughter endoscopy to 3D organ reconstruction.

Despite the trend to pair white light endoscopy with secondary image modalities for in vivo characterization of suspicious lesions, challenges remain to co-register such data. We present an algorithm to co-register two different optical imaging modalities as a mother-daughter endoscopy pair. Using white light cystoscopy (mother) and optical coherence tomography (OCT) (daughter) as an example, we developed the first forward-viewing OCT endoscope that fits in the working channel of flexible cystoscopes and demonstrated our algorithm's performance with optical phantom and clinical imaging data. The ability to register multimodal data opens opportunities for advanced analysis in cancer imaging applications.

Depth-resolved imaging of colon tumor using optical coherence tomography and fluorescence laminar optical tomography.

Early detection of neoplastic changes remains a critical challenge in clinical cancer diagnosis and treatment. Many cancers arise from epithelial layers such as those of the gastrointestinal (GI) tract. Current standard endoscopic technology is difficult to detect the subsurface lesions. In this research, we investigated the feasibility of a novel multi-modal optical imaging approach including high-resolution optical coherence tomography (OCT) and high-sensitivity fluorescence laminar optical tomography (FLOT) for structural and molecular imaging. The C57BL/6J-ApcMin/J mice were imaged using OCT and FLOT, and the correlated histopathological diagnosis was obtained. Quantitative structural (scattering coefficient) and molecular (relative enzyme activity) parameters were obtained from OCT and FLOT images for multi-parametric analysis. This multi-modal imaging method has demonstrated the feasibility for more accurate diagnosis with 88.23% (82.35%) for sensitivity (specificity) compared to either modality alone. This study suggested that combining OCT and FLOT is promising for subsurface cancer detection, diagnosis, and characterization.

Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.

Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a "precise color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans.

Human retinal imaging using visible-light optical coherence tomography guided by scanning laser ophthalmoscopy.

We achieved human retinal imaging using visible-light optical coherence tomography (vis-OCT) guided by an integrated scanning laser ophthalmoscopy (SLO). We adapted a spectral domain OCT configuration and used a supercontinuum laser as the illumating source. The center wavelength was 564 nm and the bandwidth was 115 nm, which provided a 0.97 µm axial resolution measured in air. We characterized the sensitivity to be 86 dB with 226 µW incidence power on the pupil. We also integrated an SLO that shared the same optical path of the vis-OCT sample arm for alignment purposes. We demonstrated the retinal imaging from both systems centered at the fovea and optic nerve head with 20° × 20° and 10° × 10° field of view. We observed similar anatomical structures in vis-OCT and NIR-OCT. The contrast appeared different from vis-OCT to NIR-OCT, including slightly weaker signal from intra-retinal layers, and increased visibility and contrast of anatomical layers in the outer retina.

Multimodal optical imaging system for in vivo investigation of cerebral oxygen delivery and energy metabolism.

Improving our understanding of brain function requires novel tools to observe multiple physiological parameters with high resolution in vivo. We have developed a multimodal imaging system for investigating multiple facets of cerebral blood flow and metabolism in small animals. The system was custom designed and features multiple optical imaging capabilities, including 2-photon and confocal lifetime microscopy, optical coherence tomography, laser speckle imaging, and optical intrinsic signal imaging. Here, we provide details of the system's design and present in vivo observations of multiple metrics of cerebral oxygen delivery and energy metabolism, including oxygen partial pressure, microvascular blood flow, and NADH autofluorescence.

Diagnosis and treatment of SAPHO syndrome: A case report.

The present study reports a rare case of synovitis, acne, pustulosis, hyperostosis and osteitis (SAPHO) syndrome in an adult male. The 42-year-old man complained of skin lesions, chest pain and lumbago. Laboratory evaluations demonstrated an elevated erythrocyte sedimentation rate and increased levels of C-reactive protein. Computerized tomography, bone scintigraphy and magnetic resonance imaging revealed multiple bone lesions. A diagnosis of SAPHO syndrome was made. Non-steroidal anti-inflammatory drugs, alendronate sodium and steroids were administered, which resulted in clinical improvement. The current case study demonstrates that skin manifestation and multiple imaging modalities are important in generating a definite diagnosis of SAPHO syndrome, and that early treatment is vital for a positive outcome.

Multi-modal hard x-ray imaging with a laboratory source using selective reflection from a mirror.

Multi-modal hard x-ray imaging sensitive to absorption, refraction, phase and scattering contrast is demonstrated using a simple setup implemented with a laboratory source. The method is based on selective reflection at the edge of a mirror, aligned to partially reflect a pencil x-ray beam after its interaction with a sample. Quantitative scattering contrast from a test sample is experimentally demonstrated using this method. Multi-modal imaging of a house fly (Musca domestica) is shown as proof of principle of the technique for biological samples.