Intravital imaging-based analysis tools for vessel identification and assessment of concurrent dynamic vascular events.

The vasculature undergoes changes in diameter, permeability and blood flow in response to specific stimuli. The dynamics and interdependence of these responses in different vessels are largely unknown. Here we report a non-invasive technique to study dynamic events in different vessel categories by multi-photon microscopy and an image analysis tool, RVDM (relative velocity, direction, and morphology) allowing the identification of vessel categories by their red blood cell (RBC) parameters. Moreover, Claudin5 promoter-driven green fluorescent protein (GFP) expression is used to distinguish capillary subtypes. Intradermal injection of vascular endothelial growth factor A (VEGFA) is shown to induce leakage of circulating dextran, with vessel-type-dependent kinetics, from capillaries and venules devoid of GFP expression. VEGFA-induced leakage in capillaries coincides with vessel dilation and reduced flow velocity. Thus, intravital imaging of non-invasive stimulation combined with RVDM analysis allows for recording and quantification of very rapid events in the vasculature.

Low-cost high sensitivity pulsed endomicroscopy to visualize tricolor optical signatures.

A highly sensitive, modular three-color fluorescence endomicroscopy imaging platform spanning the visible to near-infrared (NIR) range is demonstrated. Light-emitting diodes (LEDs) were sequentially pulsed along with the camera acquisition to provide up to 20 frames per second (fps) three-color imaging performance or 60 fps single color imaging. The system was characterized for bacterial and cellular molecular imaging in ex vivo human lung tissue and for bacterial and indocyanine green imaging in ex vivo perfused sheep lungs. A practical method to reduce background tissue autofluorescence is also proposed. The platform was clinically translated into six patients with pulmonary disease to delineate healthy, cancerous, and fibrotic tissue autofluorescent structures. The instrument is the most broadband clinical endomicroscopy system developed to date (covering visible to the NIR, 500 to 900 nm) and demonstrates significant potential for future clinical utility due to its low cost and modular capability to suit a wide variety of molecular imaging applications.

A high DQE water-equivalent EPID employing an array of plastic-scintillating fibers for simultaneous imaging and dosimetry in radiotherapy.

PURPOSE: First measurements of the imaging performance of a novel prototype water-equivalent electronic portal imaging device (EPID) designed for simultaneous imaging and dose verification in radiotherapy and previously characterized by our group for dosimetry are reported. Experiments were conducted to characterize the prototype's imaging performance relative to a standard commercial EPID and Monte Carlo (MC) simulations were performed to quantify the impact of several detector parameters on image quality and to inform the design of a proposed next-generation prototype. METHODS: The prototype EPID utilizes an array of 3 cm long plastic-scintillating fibers in place of the metal plate/phosphor screen in standard EPIDs. Using a clinical 6 MV photon beam, the prototype's modulation transfer function (MTF), noise power spectrum (NPS), and detective quantum efficiency (DQE) were measured and compared to measurements taken using a standard commercial EPID. A sensitivity analysis was then performed using the MC model by quantifying these metrics while varying the values of several geometrical and optical transport parameters that were unspecified by the prototype manufacturer. Finally, the MC model was used to quantify the imaging performance of a proposed next-generation prototype incorporating 1.5 cm long fibers that is better suited for integration with clinical portal imaging and dosimetry systems. RESULTS: The prototype EPID's zero spatial frequency DQE exceeded 3%, more than doubling that measured with the standard EPID (1.25%). This increased DQE was a consequence of using a prototype array detector with a greater equivalent thickness than the combined copper plate and phosphor screen in a standard EPID. The increased thickness of our prototype decreased spatial resolution relative to the standard EPID; however, the prototype EPID NPS was also lower than that measured with the standard EPID across all spatial frequencies. The sensitivity analysis demonstrated that the NPS was strongly affected by the roughness of the boundaries between fiber core and cladding regions. By comparison, the MTF was most sensitive to beam divergence and the presence of air between the fiber array and underlying photodiode panel. Simulations demonstrated that by optimizing these parameters, DQE(0) >4% may be achievable with the proposed next-generation prototype design. CONCLUSIONS: The first measurements characterizing the imaging performance of a novel water-equivalent EPID for imaging and dosimetry in radiotherapy demonstrated a DQE(0) more than double that of a standard EPID. MC simulations further demonstrated the potential for developing a next-generation prototype better suited for clinical translation with even higher DQE.

A low-cost method for visible fluorescence imaging.

A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins ß-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using ß-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.

Risk assessment of a new acceptance testing procedure for conventional linear accelerators.

PURPOSE: New techniques and materials have recently been developed to expedite the conventional linac acceptance testing procedure (Med Phys. 2017;22), which use the electronic portal imaging device (EPID) for data collection. This new procedure is designed to be more efficient and robust than the conventional approach. The purpose of this work was to perform a comparative risk assessment of the two acceptance testing procedures (ATPs). MATERIALS AND METHODS: Failure Modes and Effects Analysis was used to assess risks for both ATP approaches. Five domain experts (Medical Physicists) comprised the analysis team. The risk assessment method and ranking scales were adopted from the AAPM TG-100. The number of failure pathways and associated risk priority numbers (RPNs) for the two ATP approaches were compared. RPNs > 100 were considered high-priority failure modes. RESULTS: Fewer failure pathways were determined for the new ATP (ATPEPID ) compared to the conventional ATP (ATPconv ) resulting in a 44% difference (n = 233 vs. n = 534, respectively). There were also 35% fewer RPNs > 100 for the ATPEPID (n = 40) compared to the ATPconv (n = 114). Failure pathways and RPNs > 100 for individual ATP tests were 2.0 and 3.5 times higher, on average, for the ATPconv compared to the ATPEPID , respectively. The EPID pixel sensitivity map was identified as a high risk failure for the ATPEPID . CONCLUSIONS: Potential errors due to human factors were decreased for the ATPEPID compared to ATPconv so it is possible that a largely automated linac ATP can mitigate many error occurrences. Manufacturers should be careful when designing an EPID-based ATP to address errors in the EPID pixel sensitivity map which can potentially lead to a significant impact on patients' treatment.

Low-Cost Charged-Coupled Device (CCD) Based Detectors for Shiga Toxins Activity Analysis.

To improve food safety there is a need to develop simple, low-cost sensitive devices for detection of food-borne pathogens and their toxins. We describe a simple, low-cost webcam-based detector which can be used for various optical detection modalities, including fluorescence, chemiluminescence, densitometry, and colorimetric assays. The portable battery-operated CCD-based detection system consists of four modules: (1) a webcam to measure and record light emission, (2) a sample plate to perform assays, (3) a light emitting diode (LED) for illumination, and (4) a portable computer to acquire and analyze images. To demonstrate the technology, we used a cell based assay for fluorescence detection of the activity of the food borne Shiga toxin type 2 (Stx2), differentiating between biologically active toxin and inactive toxin which is not a risk. The assay is based on Shiga toxin inhibition of cell protein synthesis measured through inhibition of the green fluorescent protein (GFP). In this assay, GFP emits light at 509 nm when excited with a blue LED equipped with a filter at 486 nm. The emitted light is then detected with a green filter at 535 nm. Toxin activity is measured through a reduction in the 509 nm emission. In this system the level of detection (LOD) for Stx2 was 0.1 pg/ml, similar to the LOD of commercial fluorometers. These results demonstrate the utility and potential of low cost detectors for toxin activity. This approach could be readily adapted to the detection of other food-borne toxins.

Instrumentation in molecular imaging.

In vivo molecular imaging is a challenging task and no single type of imaging system provides an ideal solution. Nuclear medicine techniques like SPECT and PET provide excellent sensitivity but have poor spatial resolution. Optical imaging has excellent sensitivity and spatial resolution, but light photons interact strongly with tissues and so only small animals and targets near the surface can be accurately visualized. CT and MRI have exquisite spatial resolution, but greatly reduced sensitivity. To overcome the limitations of individual modalities, molecular imaging systems often combine individual cameras together, for example, merging nuclear medicine cameras with CT or MRI to allow the visualization of molecular processes with both high sensitivity and high spatial resolution.

Versatile Microfluidic Platform for the Assessment of Sialic Acid Expression on Cancer Cells Using Quantum Dots with Phenylboronic Acid Tags.

This work describes a versatile microfluidic platform for evaluation of cell-surface glycan expression at the single-cell level using quantum dots (QDs) tagged with phenylboronic acid. The platform was integrated with dual microwell arrays, allowing the introduction of cells in two states using the same cell culture chamber. The simultaneous analysis of cells in the same environment minimized errors resulting from different culture conditions. As proof-of-concept, the expressions of sialic acid (SA) groups on K562 cells, with or without 3'-azido-3'-deoxythymidine (AZT) treatment, were evaluated in the same chamber. 3-Aminophenylboronic acid functionalized CdSeTe@ZnS-SiO2 QDs (APBA-QDs) were prepared as probes to recognize SA groups on K562 cells with only one-step labeling. The results showed that the expression of SA moieties on K562 cells was increased by 18% and 31% after treatment with 20 and 40 µM AZT, respectively. Performing the drug treatment and control experiments simultaneously in the same chamber significantly improved the robustness and effectiveness of the assay. The strategy presented here provides an alternative tool for glycan analysis in a sensitive, high-throughput, and effective manner.

Assessment of cell lineages and cell death in blastocysts by immunostaining.

During the last decade it has been shown that most mammalian blastocysts consisted of three cell lineages. Immunofluorescence with multiple antibodies enables to identify each cell type allowing an easy detection of eventual defects. It is complementary to RT-PCR experiments as this technique allows to look at cell position and to analyze and count the proportions between the different cell types. Thus after any kind of embryo manipulation such as nuclear transfer (NT), the analysis of the three cell lineages by immunofluorescence will provide criteria for good or poor development.

Non-invasive assessment of cutaneous wound healing using fluorescent imaging.

BACKGROUND/PURPOSE: Optical imaging is a very important technique in the biomedical sciences. The purpose of this study was to develop an in vivo optical system for fluorescent imaging and molecular imaging applications using quantum dots (QDs). METHODS: The in vivo optical system was composed of modular parts, including a light source, light guide, excitation filter wheel, excitation filters, emission filter wheel, emission filters, liquid crystal tunable filter (LCTF), macro lens, dark chamber, and a cooled charged-coupled device (CCD) camera for recording images. Filters were selected based on the excitation and absorption spectra of QDs to allow spectral separation and optimization of the acquired image. In contrast with conventional systems, our system allows selection of the emission bandwidth. RESULTS: The system was tested in an in vivo study using a wound-healing model in nude mice. The healing process was examined after injection of fibroblasts and keratinocytes labeled with two different sets of QDs. The different QD probes were readily detected and distinguished using our system. CONCLUSION: An in vivo optical system is a very useful tool for the detection of genes, proteins, and small-molecule drugs inside living animals, and this imaging modality can also be adopted for real-time visualization of cancer cell metastasis in live animals.

Advances in mid-infrared detection and imaging: a key issues review.

It has been over 200 years since people recognized the presence of infrared radiation, and developed methods to capture this signal. However, current material systems and technologies for infrared detections have not met the increasing demand for high performance infrared detectors/cameras, with each system having intrinsic drawbacks. Type-II InAs/GaSb superlattice has been recently considered as a promising candidate for the next generation of infrared detection and imaging. Type-II superlattice is a man-made crystal structure, consisting of multiple quantum wells placed next to each other in a controlled way such that adjacent quantum wells can interact. The interaction between multiple quantum wells offers an additional degree of freedom in tailoring the material's properties. Another advantage of type-II superlattice is the experimental benefit of inheriting previous research on material synthesis and device fabrication of bulk semiconductors. It is the combination of these two unique strengths of type-II superlattice--novel physics and easy manipulation--that has enabled unprecedented progress in recent years. In this review, we will describe historical development, and current status of type-II InAs/GaSb superlattice for advanced detection and imaging in the mid-infrared regime (λ = 3-5 µm).

Vascular contrast in narrow-band and white light imaging.

Narrow-band imaging (NBI) is a spectrally selective reflectance imaging technique that is used clinically for enhancing visualization of superficial vasculature and has shown promise for applications such as early endoscopic detection of gastrointestinal neoplasia. We have studied the effect of vessel geometry and illumination wavelength on vascular contrast using idealized geometries in order to more quantitatively understand NBI and broadband or white light imaging of mucosal tissue. Simulations were performed using a three-dimensional, voxel-based Monte Carlo model incorporating discrete vessels. In all cases, either 415 or 540 nm illumination produced higher contrast than white light, yet white light did not always produce the lowest contrast. White light produced the lowest contrast for small vessels and intermediate contrast for large vessels (diameter≥100 µm) at deep regions (vessel depth≥200 µm). The results show that 415 nm illuminations provided superior contrast for smaller vessels at shallow depths while 540 nm provided superior contrast for larger vessels in deep regions. Besides 540 nm, our studies also indicate the potential of other wavelengths to achieve high contrast of large vessels at deep regions. Simulation results indicate the importance of three key mechanisms in determining spectral variations in contrast: intravascular hemoglobin (Hb) absorption in the vessel of interest, diffuse Hb absorption from collateral vasculature, and bulk tissue scattering. Measurements of NBI contrast in turbid phantoms incorporating 0.1-mm-diameter hemoglobin-filled capillary tubes indicated good agreement with modeling results. These results provide quantitative insights into light-tissue interactions and the effect of device and tissue properties on NBI performance.

[Development of cloud chamber having thin-film entrance windows and proposal of practical training for beginners using X-ray equipment and unsealed radioactive material].

A cloud chamber is a detector that can visualize the tracks of charged particles. Hayashi, et al. suggested a visualization experiment in which X-rays generated by diagnostic X-ray equipment were directed into a cloud chamber; however, there was a problem in that the wall of the cloud chamber scattered the incoming X-rays. In this study, we developed a new cloud chamber with entrance windows. Because these windows are made of thin film, we were able to direct the X-rays through them without contamination by scattered X-rays from the cloud chamber wall. We have newly proposed an experiment in which beta-particles emitted from radioisotopes are directed into a cloud chamber. We place shielding material in the cloud chamber and visualize the various shielding effects seen with the material positioned in different ways. During the experiment, electrons scattered in the air were measured quantitatively using GM counters. We explained the physical phenomena in the cloud chamber using Monte Carlo simulation code EGS5. Because electrons follow a tortuous path in air, the shielding material must be placed appropriately to be able to effectively block their emissions. Visualization of the tracks of charged particles in this experiment proved effective for instructing not only trainee radiological technologists but also different types of healthcare professionals.

Co-registered pulse-echo/photoacoustic transvaginal probe for real time imaging of ovarian tissue.

We present the design and construction of a prototype imaging probe capable of co-registered pulse-echo ultrasound and photoacoustic (optoacoustic) imaging in real time. The probe consists of 36 fibers of 200 micron core diameter each that are distributed around a commercial transvaginal ultrasound transducer, and housed in a protective shield. Its performance was demonstrated by two sets of experiments. The first set involved imaging of blood flowing through a tube mimicking a blood vessel, the second set involved imaging of human ovaries ex vivo. The results suggest that the system along with the probe has great potential for imaging and characterizing of ovarian tissue in vivo.

Experimental validation of an inverse fluorescence Monte Carlo model to extract concentrations of metabolically relevant fluorophores from turbid phantoms and a murine tumor model.

An inverse Monte Carlo based model has been developed to extract intrinsic fluorescence from turbid media. The goal of this work was to experimentally validate the model to extract intrinsic fluorescence of three biologically meaningful fluorophores related to metabolism from turbid media containing absorbers and scatterers. Experimental studies were first carried out on tissue-mimicking phantoms that contained individual fluorophores and their combinations, across multiple absorption, scattering, and fluorophore concentrations. The model was then tested in a murine tumor model to determine both the kinetics of fluorophore uptake as well as overall tissue fluorophore concentration through extraction of the intrinsic fluorescence of an exogenous contrast agent that reports on glucose uptake. Results show the model can be used to recover the true intrinsic fluorescence spectrum with high accuracy (R(2)=0.988) as well as accurately compute fluorophore concentration in both single and multiple fluorophores phantoms when appropriate calibration standards are available. In the murine tumor, the model-corrected intrinsic fluorescence could be used to differentiate drug dose injections between different groups. A strong linear correlation was observed between the extracted intrinsic fluorescence intensity and injected drug dose, compared with the distorted turbid tissue fluorescence.

Investigation of X-ray fluorescence computed tomography (XFCT) and K-edge imaging.

This work provides a comprehensive Monte Carlo study of X-ray fluorescence computed tomography (XFCT) and K-edge imaging system, including the system design, the influence of various imaging components, the sensitivity and resolution under various conditions. We modified the widely used EGSnrc/DOSXYZnrc code to simulate XFCT images of two acrylic phantoms loaded with various concentrations of gold nanoparticles and Cisplatin for a number of XFCT geometries. In particular, reconstructed signal as a function of the width of the detector ring, its angular coverage and energy resolution were studied. We found that XFCT imaging sensitivity of the modeled systems consisting of a conventional X-ray tube and a full 2-cm-wide energy-resolving detector ring was 0.061% and 0.042% for gold nanoparticles and Cisplatin, respectively, for a dose of ∼ 10 cGy. Contrast-to-noise ratio (CNR) of XFCT images of the simulated acrylic phantoms was higher than that of transmission K-edge images for contrast concentrations below 0.4%.

Molecular imaging of small animals with fluorescent proteins: from projection to multimodality.

Fluorescent proteins (FPs) have been widely adopted in cell research for protein trafficking and reporter gene expression studies, as well as to study other biological processes. However, biological tissue has high light scattering and high absorption coefficients of visible light; hence, using FPs in small animal imaging remains a challenge, especially when the FPs are located deep in the tissue. In small animals, fluorescence molecular imaging could potentially address this difficulty. We constructed fluorescence molecular imaging systems that have two modes: a planner mode (projection imaging) and a multimodality mode (fluorescence molecular tomography and micro-CT). The planner mode can provide projection images of a fluorophore in the whole body of a small animal, whereas three-dimensional information can be offered by multimodality mode. The planner imaging system works in the reflection mode and is designed to provide fast imaging. The multimodality imaging system is designed to allow quantification and three-dimensional localization of fluorophores. A nude mouse with a tumour targeted with a far-red FP, which is appropriate for in vivo imaging, was adopted to validate the two systems. The results indicate that the planner imaging system is probably suitable for high throughput molecular imaging, whereas the multimodality imaging system is fit for quantitative research.

Quantitative determination of dynamical properties using coherent spatial frequency domain imaging.

Laser speckle imaging (LSI) is a fast, noninvasive method to obtain relative particle dynamics in highly light scattering media, such as biological tissue. To make quantitative measurements, we combine LSI with spatial frequency domain imaging, a technique where samples are illuminated with sinusoidal intensity patterns of light that control the characteristic path lengths of photons in the sample. We use both diffusion and radiative transport to predict the speckle contrast of coherent light remitted from turbid media. We validate our technique by measuring known Brownian diffusion coefficients (D(b)) of scattering liquid phantoms. Monte Carlo (MC) simulations of radiative transport were found to provide the most accurate contrast predictions. For polystyrene microspheres of radius 800 nm in water, the expected and fit D(b) using radiative transport were 6.10E-07 and 7.10E-07 mm²/s, respectively. For polystyrene microspheres of radius 1026 nm in water, the expected and fit D(b) were 4.7E-07 and 5.35 mm²/s, respectively. For scattering particles in water-glycerin solutions, the fit fractional changes in D(b) with changes in viscosity were all found to be within 3% of the expected value.

In vivo multispectral photoacoustic and photothermal flow cytometry with multicolor dyes: a potential for real-time assessment of circulation, dye-cell interaction, and blood volume.

Recently, photoacoustic (PA) flow cytometry (PAFC) has been developed for in vivo detection of circulating tumor cells and bacteria targeted by nanoparticles. Here, we propose multispectral PAFC with multiple dyes having distinctive absorption spectra as multicolor PA contrast agents. As a first step of our proof-of-concept, we characterized high-speed PAFC capability to monitor the clearance of three dyes (Indocyanine Green [ICG], Methylene Blue [MB], and Trypan Blue [TB]) in an animal model in vivo and in real time. We observed strong dynamic PA signal fluctuations, which can be associated with interactions of dyes with circulating blood cells and plasma proteins. PAFC demonstrated enumeration of circulating red and white blood cells labeled with ICG and MB, respectively, and detection of rare dead cells uptaking TB directly in bloodstream. The possibility for accurate measurements of various dye concentrations including Crystal Violet and Brilliant Green were verified in vitro using complementary to PAFC photothermal (PT) technique and spectrophotometry under batch and flow conditions. We further analyze the potential of integrated PAFC/PT spectroscopy with multiple dyes for rapid and accurate measurements of circulating blood volume without a priori information on hemoglobin content, which is impossible with existing optical techniques. This is important in many medical conditions including surgery and trauma with extensive blood loss, rapid fluid administration, and transfusion of red blood cells. The potential for developing a robust clinical PAFC prototype that is safe for human, and its applications for studying the liver function are further highlighted.

Characterization of a rotating slat collimator system dedicated to small animal imaging.

Some current investigations based on small animal models are dedicated to functional cerebral imaging. They represent a fundamental tool to understand the mechanisms involved in neurodegenerative diseases. In the radiopharmaceutical development approach, the main challenge is to measure the radioactivity distribution in the brain of a subject with good temporal and spatial resolutions. Classical SPECT systems mainly use parallel hole or pinhole collimators. In this paper we investigate the use of a rotating slat collimator system for small animal brain imaging. The proposed prototype consists of a 64-channel multi-anode photomultiplier tube (H8804, Hamamatsu Corp.) coupled to a YAP:Ce crystal highly segmented into 32 strips of 0.575 × 18.4 × 10 mm(3). The parameters of the rotating slat collimator are optimized using GATE Monte Carlo simulations. The performance of the proposed prototype in terms of spatial resolution, detection efficiency and signal-to-noise ratio is compared to that obtained with a gamma camera equipped with a parallel hole collimator. Preliminary experimental results demonstrate that a spatial resolution of 1.54 mm can be achieved with a detection efficiency of 0.012% for a source located at 20 mm, corresponding to the position of the brain in the prototype field of view.