Stability of gel wax based optical scattering phantoms.

Phantoms with tuneable optical scattering properties are essential in the development and refinement of optical based imaging techniques. Mineral oil based 'gel wax' phantoms are the subject of increasing interest due to their ease and speed of manufacture, non-toxic nature, ability to cast into anatomically realistic shapes, as well as their cost-effective nature of production. The addition of scatterers such as titanium dioxide powder and monodisperse silica microspheres to the gel wax allows for the creation of phantoms with a controllable optical scattering coefficient. To enable repeated use of such phantoms, the stability of the scattering properties must be determined-a property which has yet to be investigated. We present an analysis of the stability of the reduced scattering coefficient ( µ s ' ) of such phantoms over time. We conclude that due to the measurable reduction in scattering coefficient over time, gel wax phantoms embedded with silica spheres may not be suitable for repeated use over time, however gel wax-TiO2 phantoms are much more temporally stable.

Video-rate all-optical ultrasound imaging.

All-optical ultrasound imaging, where ultrasound is generated and detected using light, has recently been demonstrated as a viable modality that is inherently insensitive to electromagnetic interference and exhibits wide bandwidths. High-quality 2D and 3D all-optical ultrasound images of tissues have previously been presented; however, to date, long acquisition times (ranging from minutes to hours) have hindered clinical application. Here, we present the first all-optical ultrasound imaging system capable of video-rate, real-time two-dimensional imaging of biological tissue. This was achieved using a spatially extended nano-composite optical ultrasound generator, a highly sensitive fibre-optic acoustic receiver, and eccentric illumination resulting in an acoustic source exhibiting optimal directivity. This source was scanned across a one-dimensional source aperture using a fast galvo mirror, thus enabling the dynamic synthesis of source arrays comprising spatially overlapping sources at non-uniform source separation distances. The resulting system achieved a sustained frame rate of 15 Hz, a dynamic range of 30 dB, a penetration depth of at least 6 mm, a resolution of 75 µm (axial) by 100 µm (lateral), and enabled the dynamics of a pulsating ex vivo carotid artery to be captured.

Comparing the effective attenuation lengths for long wavelength in vivo imaging of the mouse brain.

Light attenuation in thick biological tissues, caused by a combination of absorption and scattering, limits the penetration depth in multiphoton microscopy (MPM). Both tissue scattering and absorption are dependent on wavelengths, which makes it essential to choose the excitation wavelength with minimum attenuation for deep imaging. Although theoretical models have been established to predict the wavelength dependence of light attenuation in brain tissues, the accuracy of these models in experimental settings needs to be verified. Furthermore, the water absorption contribution to the tissue attenuation, especially at 1450 nm where strong water absorption is predicted to be the dominant contributor in light attenuation, has not been confirmed. Here we performed a systematic study of in vivo three-photon imaging at different excitation wavelengths, 1300 nm, 1450 nm, 1500 nm, 1550 nm, and 1700 nm, and quantified the tissue attenuation by calculating the effective attenuation length at each wavelength. The experimental data show that the effective attenuation length at 1450 nm is significantly shorter than that at 1300 nm or 1700 nm. Our results provide unequivocal validation of the theoretical estimations based on water absorption and tissue scattering in predicting the effective attenuation lengths for long wavelength in vivo imaging.

Real-time cancer detection with an integrated lensless fluorescence contact imager.

Microscopic tumor cell foci left in a patient after surgery significantly increase the chance of cancer recurrence. However, fluorescence microscopes used for intraoperative navigation lack the necessary sensitivity for imaging microscopic disease and are too bulky to maneuver within the resection cavity. We have developed a scalable chip-scale fluorescence contact imager for detecting microscopic cancer in vivo and in real-time. The imager has been characterized under simulated in vivo conditions using ex vivo samples, providing strong evidence that our device can be used in vivo. Angle-selective gratings enhance the resolution of the imager without impacting its physical size. We demonstrate detection of cancer cell clusters containing as few as 25 HCC1569 breast cancer cells and 400 LNCaP prostate cancer cells with integration times of only 50 ms and 70 ms, respectively. A cell cluster recognition algorithm is used to achieve both a sensitivity and specificity of 92 % for HCC1569 cell samples, indicating the reliability of the imager. The signal-to-noise ratio (SNR) degradation with increased separation is only 1.5 dB at 250 µm. Blood scattering and absorption reduce the SNR by less than 2 dB for typical concentrations. Moreover, HER2+ breast cancer tissue taken from a patient is distinguished from normal breast tissue with an integration time of only 75 ms.

Novel analysis of fNIRS acquired dynamic hemoglobin concentrations: application in young children with autism spectrum disorder.

A novel analysis of the spatial complexity of functional connectivity (SCFC) was proposed to investigate the spatial complexity of multiple dynamic functional connectivity series in an fNIRS study, using an approach combining principal component analysis and normalized entropy. The analysis was designed to describe the complex spatial features of phase synchrony based dynamic functional connectivity (dFC), which are unexplained in traditional approaches. The feasibility and validity of this method were verified in a sample of young patients with autism spectrum disorders (ASD). Our results showed that there were information exchange deficits in the right prefrontal cortex (PFC) of children with ASD, with markedly higher interregion SCFCs between the right PFC and other brain regions than those of normal controls. Furthermore, the global SCFC was significantly higher in young patients with ASD, along with considerably higher intraregion SCFCs in the prefrontal and temporal lobes which represents more diverse information exchange in these areas. The study suggests a novel method to analyze the fNIRS required dynamic hemoglobin concentrations by using concepts of SCFC. Moreover, the clinical results extend our understanding of ASD pathology, suggesting the crucial role of the right PFC during the information exchange process.

Wavelet-based method for removing global physiological noise in functional near-infrared spectroscopy.

Functional near-infrared spectroscopy (fNIRS) is a fast-developing non-invasive functional brain imaging technology widely used in cognitive neuroscience, clinical research and neural engineering. However, it is a challenge to effectively remove the global physiological noise in the fNIRS signal. The global physiological noise in fNIRS arises from multiple physiological origins in both superficial tissues and the brain. It has complex temporal, spatial and frequency characteristics, casting significant influence on the results. In the present study, we developed a novel wavelet-based method for fNIRS global physiological noise removal. The method is data-driven and does not rely on any additional hardware or subjective noise component selection procedure. It consists of two steps. Firstly, we use wavelet transform coherence to automatically detect the time-frequency points contaminated by the global physiological noise. Secondly, we decompose the fNIRS signal by using the wavelet transform, and then suppress the wavelet energy of the contaminated time-frequency points. Finally, we transform the signal back to a time series. We validated the method by using simulation and real data at both task- and resting-state. The results showed that our method can effectively remove the global physiological noise from the fNIRS signal and improve the spatial specificity of the task activation and the resting-state functional connectivity pattern.

Full video pulse extraction.

This paper introduces a new method to automate heart-rate detection using remote photoplethysmography (rPPG). The method replaces the commonly used region of interest (RoI) detection and tracking, and does not require initialization. Instead, it combines a number of candidate pulse-signals computed in the parallel, each biased towards differently colored objects in the scene. The method is based on the observation that the temporally averaged colors of video objects (skin and background) are usually quite stable over time in typical application-driven scenarios, such as the monitoring of a subject sleeping in bed, or an infant in an incubator. The resulting system, called full video pulse extraction (FVP), allows the direct use of raw video streams for pulse extraction. Our benchmark set of diverse videos shows that FVP enables long-term sleep monitoring in visible light and in infrared, and works for adults and neonates. Although we only demonstrate the concept for heart-rate monitoring, we foresee the adaptation to a range of vital signs, thus benefiting the larger video health monitoring field.

Voltage-sensitive dye delivery through the blood brain barrier using adenosine receptor agonist regadenoson.

Optical imaging of brain activity has mostly employed genetically manipulated mice, which cannot be translated to clinical human usage. Observation of brain activity directly is challenging due to the difficulty in delivering dyes and other agents through the blood brain barrier (BBB). Using fluorescence imaging, we have demonstrated the feasibility of delivering the near-infrared voltage-sensitive dye (VSD) IR-780 perchlorate to the brain tissue through pharmacological techniques, via an adenosine agonist (regadenoson). Comparison of VSD fluorescence of mouse brains without and with regadenoson showed significantly increased residence time of the fluorescence signal in the latter case, indicative of VSD diffusion into the brain tissue. Dose and timing of regadenoson were varied to optimize BBB permeability for VSD delivery.

Reflection artifact identification in photoacoustic imaging using multi-wavelength excitation.

Photoacoustic imaging has been a focus of research for clinical applications owing to its ability for deep visualization with optical absorption contrast. However, there are various technical challenges remaining for this technique to find its place in clinics. One of the challenges is the occurrence of reflection artifacts. The reflection artifacts may lead to image misinterpretation. Here we propose a new method using multiple wavelengths for identifying and removing the reflection artifacts. By imaging the sample with multiple wavelengths, the spectral response of the features in the photoacoustic image is obtained. We assume that the spectral response of the reflection artifact is better correlated with the proper image feature of its corresponding absorber than with other features in the image. Based on this, the reflection artifacts can be identified and removed. Here, we experimentally demonstrated the potential of this method for real-time identification and correction of reflection artifacts in photoacoustic images in phantoms as well as in vivo using a handheld photoacoustic imaging probe.

Cellular in vivo 3D imaging of the cornea by confocal laser scanning microscopy.

We present an in vivo confocal laser scanning microscopy based method for large 3D reconstruction of the cornea on a cellular level with cropped volume sizes up to 266 x 286 x 396 µm3. The microscope objective used is equipped with a piezo actuator for automated, fast and precise closed-loop focal plane control. Furthermore, we present a novel concave surface contact cap, which significantly reduces eye movements by up to 87%, hence increasing the overlapping image area of the whole stack. This increases the cuboid volume of the generated 3D reconstruction significantly. The possibility to generate oblique sections using isotropic volume stacks opens the window to slit lamp microscopy on a cellular level.

Erratum: Methods for detecting host genetic modifiers of tumor vascular function using dynamic near-infrared fluorescence imaging: errata.

[This corrects the article on p. 543 in vol. 9, PMID: 29552392.].

Three-dimensional tomographic microscopy technique with multi-frequency combination with partially coherent illuminations.

We demonstrate a three-dimensional (3D) optical diffraction tomographic technique with multi-frequency combination (MFC-ODT) for the 3D quantitative phase imaging of unlabeled specimens. Three sets of through-focus intensity images are captured under an annular aperture and two circular apertures with different coherence parameters. The 3D phase optical transfer functions (POTF) corresponding to different illumination apertures are combined to obtain a synthesized frequency response, achieving high-quality, low-noise 3D reconstructions with imaging resolution up to the incoherent diffraction limit. Besides, the expression of 3D POTF for arbitrary illumination pupils is derived and analyzed, and the 3D imaging performance of annular illumination is explored. It is shown that the phase-contrast washout effect in high-NA circular apertures can be effectively addressed by introducing a complementary annular aperture, which strongly boosts the phase contrast and improves the imaging resolution. By incorporating high-NA illumination as well as high-NA detection, MFC-ODT can achieve a theoretical transverse resolution up to 200 nm and an axial resolution of 645 nm. To test the feasibility of the proposed MFC-ODT technique, the 3D refractive index reconstruction results are based on a simulated 3D resolution target and experimental investigations of micro polystyrene bead and unstained biological samples are presented. Due to its capability for high-resolution 3D phase imaging as well as the compatibility with a widely available commercial microscope, the MFC-ODT is expected to find versatile applications in biological and biomedical research.

Simultaneous monitoring of cerebral perfusion and cytochrome c oxidase by combining broadband near-infrared spectroscopy and diffuse correlation spectroscopy.

Preterm infants born with very low birth weights are at a high risk of brain injury, in part because the premature brain is believed to be prone to periods of low cerebral blood flow (CBF). Tissue damage is likely to occur if reduction in CBF is sufficient to impair cerebral energy metabolism for extended periods. Therefore, a neuromonitoring method that can detect reductions in CBF, large enough to affect metabolism, could alert the neonatal intensive care team before injury occurs. In this report, we present the development of an optical system that combines diffuse correlation spectroscopy (DCS) for monitoring CBF and broadband near-infrared spectroscopy (B-NIRS) for monitoring the oxidation state of cytochrome c oxidase (oxCCO) - a key biomarker of oxidative metabolism. The hybrid instrument includes a multiplexing system to enable concomitant DCS and B-NIRS measurements while avoiding crosstalk between the two subsystems. The ability of the instrument to monitor dynamic changes in CBF and oxCCO was demonstrated in a piglet model of neonatal hypoxia-ischemia (HI). Experiments conducted in eight animals, including two controls, showed that oxCCO exhibited a delayed response to ischemia while CBF and tissue oxygenation (StO2) responses were instantaneous. These findings suggest that simultaneous neuromonitoring of perfusion and metabolism could provide critical information regarding clinically significant hemodynamic events prior to the onset of brain injury.

A spread spectrum approach to time-domain near-infrared diffuse optical imaging using inexpensive optical transceiver modules.

We introduce a compact time-domain system for near-infrared spectroscopy using a spread spectrum technique. The proof-of-concept single channel instrument utilises a low-cost commercially available optical transceiver module as a light source, controlled by a Kintex 7 field programmable gate array (FPGA). The FPGA modulates the optical transceiver with maximum-length sequences at line rates up to 10Gb/s, allowing us to achieve an instrument response function with full width at half maximum under 600ps. The instrument is characterised through a set of detailed phantom measurements as well as proof-of-concept in vivo measurements, demonstrating performance comparable with conventional pulsed time-domain near-infrared spectroscopy systems.

Improving mesoscopic fluorescence molecular tomography via preconditioning and regularization.

Mesoscopic fluorescence molecular tomography (MFMT) is a novel imaging technique capable of obtaining 3-D distribution of molecular probes inside biological tissues at depths of a few millimeters with a resolution up to ~100 µm. However, the ill-conditioned nature of the MFMT inverse problem severely deteriorates its reconstruction performances. Furthermore, dense spatial sampling and fine discretization of the imaging volume required for high resolution reconstructions make the sensitivity matrix (Jacobian) highly correlated, which prevents even advanced algorithms from achieving optimal solutions. In this work, we propose two computational methods to respectively increase the incoherence of the sensitivity matrix and improve the convergence rate of the inverse solver. We first apply a compressed sensing (CS) based preconditioner on either the whole sensitivity matrix or sub sensitivity matrices to reduce the coherence between columns of the sensitivity matrix. Then we employed a regularization method based on the weight iterative improvement method (WIIM) to mitigate the ill-condition of the sensitivity matrix and to drive the iterative optimization process towards convergence at a faster rate. We performed numerical simulations and phantom experiments to validate the effectiveness of the proposed strategies. In both in silico and in vitro cases, we were able to improve the quality of MFMT reconstructions significantly.

Spectral-resolved cone-beam X-ray luminescence computed tomography with principle component analysis.

Cone-beam X-ray luminescence computed tomography (CB-XLCT) has become a promising technique for its higher utilization of X-ray and shorter scanning time compared to the narrow-beam XLCT, but it suffers from the low-spatial resolution that results in the insufficiency to resolve the adjacent multiple probes. In multispectral CB-XLCT, multiple probes show different emission behaviors in the dimension of the spectrum. In this work, a spectral-resolved CB-XLCT method combining multispectral CB-XLCT with principle component analysis (PCA) was proposed to improve the imaging resolution. Results of digital simulation and the phantom experiment illustrated that the proposed method was capable of resolving adjacent multiple probes accurately and had better performance than the common multispectral CB-XLCT with spectrum information priori.

Distinction of directional coupling in sensorimotor networks between active and passive finger movements using fNIRS.

The purpose of this study is to investigate cerebral cortex activation during active movement and passive movement by using a functional near-infrared spectroscopy (fNIRS). Tasks were the flexion/extension of the right hand finger by active movement and passive movement. Oxy-hemoglobin concentration changes calculated from fNIRS and analyzed the activation and connectivity so as to understand dynamical brain relationship. The results demonstrated that the brain activation in passive movements is similar to motor execution. During active movement, the estimated causality patterns showed significant causality value from the supplementary motor area (SMA) to the primary motor cortex (M1). During the passive movement, the causality from the primary somatosensory cortex (S1) to the primary motor cortex (M1) was stronger than active movement. These results demonstrated that active and passive movements had a direct effect on the cerebral cortex but the stimulus pathway of active and passive movement is different. This study may contribute to better understanding how active and passive movements can be expressed into cortical activation by means of fNIRS.

Quantitative diagnosis of tissue microstructure with wide-field high spatial frequency domain imaging.

Non-contact and minimally invasive endoscopic optical imaging is an invaluable diagnostic tool for tissue examination and cancer screening. The point sampling techniques with high sensitivity to the tissue microenvironment are time consuming and often not affordable in clinics. There is a major clinical need for a large field-of-view (FOV) rapid screening method to highlight subtle tissue microstructural alterations. To address this unmet need, we have developed High Spatial Frequency Domain Imaging (HSFDI)-a non-contact imaging modality that spatially maps the tissue microscopic scattering structures over a large field of view (>1cm2). Based on an analytical reflectance model of sub-diffusive light from forward-peaked highly scattering media, HSFDI quantifies the spatially-resolved parameters of the light scattering phase function (i.e., the backscattering probability and the light spreading length) from the reflectance of structured light modulated at high spatial frequencies. Enhanced signal to noise ratio (SNR) is achieved at even ultra-high modulation frequencies with single snapshot multiple frequency demodulation (SSMD). The variations in tissue microstructures, including the strength of the background (pudding) refractive index fluctuation and the prominent scattering structure (plum) morphology, can then be inferred. After validation with optical phantoms, measurements of fresh ex vivo tissue samples revealed significant contrast and differentiation of the phase function parameters between different types and disease states (normal, inflammatory, and cancerous) of tissue whereas tissue absorption and reduced scattering coefficients only show modest changes. HSFDI may provide wide-field images of microscopic structural biomarkers unobtainable with either diffuse light imaging or point-based optical sampling. Potential clinical applications include the rapid screening of excised tissue and the noninvasive examination of suspicious lesions during operation.

Pilot study of freshly excised breast tissue response in the 300-600 GHz range.

The failure to accurately define tumor margins during breast conserving surgery (BCS) results in a 20% re-excision rate. The present paper reports the investigation to evaluate the potential of terahertz imaging for breast tissue recognition within the under-explored 300 - 600 GHz range. Such a frequency window matches new BiCMOS technology capabilities and thus opens up the opportunity for near-field terahertz imaging using these devices. To assess the efficacy of this frequency band, data from 16 freshly excised breast tissue samples were collected and analyzed directly after excision. Complex refractive indices have been extracted over the as-mentioned frequency band, and amplitude frequency images show some contrast between tissue types. Principal component analysis (PCA) has also been applied to the data in an attempt to automate tissue classification. Our observations suggest that the dielectric response could potentially provide contrast for breast tissue recognition within the 300 - 600 GHz range. These results open the way for silicon-based terahertz subwavelength near field imager design, efficient up to 600 GHz to address ex vivo life-science applications.

Decoding cortical brain states from widefield calcium imaging data using visibility graph.

Widefield optical imaging of neuronal populations over large portions of the cerebral cortex in awake behaving animals provides a unique opportunity for investigating the relationship between brain function and behavior. In this paper, we demonstrate that the temporal characteristics of calcium dynamics obtained through widefield imaging can be utilized to infer the corresponding behavior. Cortical activity in transgenic calcium reporter mice (n=6) expressing GCaMP6f in neocortical pyramidal neurons is recorded during active whisking (AW) and no whisking (NW). To extract features related to the temporal characteristics of calcium recordings, a method based on visibility graph (VG) is introduced. An extensive study considering different choices of features and classifiers is conducted to find the best model capable of predicting AW and NW from calcium recordings. Our experimental results show that temporal characteristics of calcium recordings identified by the proposed method carry discriminatory information that are powerful enough for decoding behavior.