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Although Blood Oxygenation Level Dependent (BOLD) functional MRI (fMRI) is widely used to examine brain function in adults, the need for general anesthesia limits its practical utility in infants and small children. Functional Near-Infrared Spectroscopy - Diffuse Optical Tomography (fNIRS-DOT) imaging promises to be an alternative brain network imaging technique. Yet current versions of continuous-wave fNIRS-DOT systems are restricted to the cortical surface measurements and do not probe deep structures that are frequently injured especially in premature infants. Herein we report a transcranial near infrared optical imaging system, called Cap-based Transcranial Optical Tomography (CTOT) able to image whole brain hemodynamic activity with 3 seconds of data acquisition time. We show the system is capable of whole brain oxygenation mapping in an awake child, and that tomographically reconstructed static CTOT-derived oxy- and deoxygenated blood volumes are spatially correlated with the time-averaged BOLD fMRI volumes. By removing time bottlenecks in the current system, dynamic CTOT mapping should be possible, which would then enable evaluation of functional connectivity in awake infants.

Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viruses. However, its adoption as a safe and effective delivery vector for certain diseases relies on altering its tropism to deliver transgenes to desired cell populations. To this end, we have developed a protease-activatable AAV vector, named provector, that responds to elevated extracellular protease activity commonly found in diseased tissue microenvironments. The AAV9-based provector is initially inactive, but then it can be switched on by matrix metalloproteinases (MMP)-2 and -9. Cryo-electron microscopy and image reconstruction reveal that the provector capsid is structurally similar to that of AAV9, with a flexible peptide insertion at the top of the 3-fold protrusions. In an in vivo model of myocardial infarction (MI), the provector is able to deliver transgenes site specifically to high-MMP-activity regions of the damaged heart, with concomitant decreased delivery to many off-target organs, including the liver. The AAV provector may be useful in the future for enhanced delivery of transgenes to sites of cardiac damage.

Recently, fluorescence imaging using shortwave infrared light (SWIR, 1,000-2,000 nm) has been proposed as having advantage over conventional near-infrared fluorescence (NIRF) imaging due to the reduced tissue scattering, negligible autofluorescence, comparable tissue absorption, and the discovery that indocyanine green (ICG), used clinically as a NIRF contrast agent, also has fluorescence emission in SWIR regime. Images of ICG in small animals acquired by commercial Si-based and InGaAs-based imaging cameras have been qualitatively compared, however the lack of working standards to quantify performance of these imaging systems limits quantitative comparison. Without quantification using a traceable in vitro test, clinical adoption of rapidly evolving advances in both NIRF and SWIR imaging devices will become limited. In this work, we developed an ICG based fluorescent solid working standard calibrated with SI units (mW ${^\centerdot }$ cm $^{-{2}} {^\centerdot }$ sr −1) for quantification of measurement sensitivity of Si, GaAs-intensified Si, and InGaAs based camera systems, their signal-to-noise ratio (SNR), and contrast in non-clinical tests. In addition, we present small animal and large animal imaging with ICG for qualitative comparison of the same SWIR fluorescence and NIRF imaging systems. Results suggest that SWIR fluorescence imaging of ICG may have superior resolution in small animal imaging compared to NIRF imaging, but lack of measurement sensitivity, SNR, contrast, as well as water absorption limits deep penetration in large animals.

Ten head and neck cancer survivors diagnosed with head and neck lymphedema (HNL) were imaged using near-infrared fluorescence lymphatic imaging (NIRFLI) prior to and immediately after an initial advance pneumatic compression device treatment and again after 2 weeks of daily at-home use. Images assessed the impact of pneumatic compression therapy on lymphatic drainage. Facial composite measurement scores assessed reduction/increase in external swelling, and survey results were obtained. After a single pneumatic compression treatment, NIRFLI showed enhanced lymphatic uptake and drainage in all subjects. After 2 weeks of daily treatment, areas of dermal backflow disappeared or were reduced in 6 of 8 subjects presenting with backflow. In general, reductions in facial composite measurement scores tracked with reductions in backflow and subject-reported improvements; however, studies are needed to determine whether longer treatment durations can be impactful and whether advanced pneumatic compression can be used to ameliorate backflow characteristic of HNL.

Cerebrospinal fluid (CSF) outflow from the brain occurs through absorption into the arachnoid villi and, more predominantly, through meningeal and olfactory lymphatics that ultimately drain into the peripheral lymphatics. Impaired CSF outflow has been postulated as a contributing mechanism in Alzheimer's disease (AD). Herein we conducted near-infrared fluorescence imaging of CSF outflow into the peripheral lymph nodes (LNs) and of peripheral lymphatic function in a transgenic mouse model of AD (5XFAD) and wild-type (WT) littermates. CSF outflow was assessed from change in fluorescence intensity in the submandibular LNs as a function of time following bolus, an intrathecal injection of indocyanine green (ICG). Peripheral lymphatic function was measured by assessing lymphangion contractile function in lymphatics draining into the popliteal LN following intradermal ICG injection in the dorsal aspect of the hind paw. The results show 1) significantly impaired CSF outflow into the submandibular LNs of 5XFAD mice and 2) reduced contractile frequency in the peripheral lymphatics as compared to WT mice. Impaired CSF clearance was also evidenced by reduction of fluorescence on ventral surfaces of extracted brains of 5XFAD mice at euthanasia. These results support the hypothesis that lymphatic congestion caused by reduced peripheral lymphatic function could limit CSF outflow and may contribute to the cause and/or progression of AD.

Objective We used near-infrared fluorescence lymphatic imaging in a pilot study to assess the lymphatics in preulcerative (C2-C4) venous insufficiency and determine whether involvement and/or degradation of lymphatic anatomy or function could play a role in the progression of chronic venous insufficiency. We also explored the role of lymphatics in early peripheral arterial disease. Methods After informed consent and intradermal injections of indocyanine green for rapid lymphatic uptake, near-infrared fluorescence lymphatic imaging was used to assess the lymphatic anatomic structure and quantify the lymphatic propulsion rates in subjects with early venous insufficiency. The anatomic observations included interstitial backflow, characterized by the abnormal spreading of indocyanine green from the injection site primarily into the surrounding interstitial tissues; dermal backflow, characterized by the retrograde movement of dye-laden lymph from collecting lymphatics into the lymphatic capillaries; and lymphatic vessel segmentation and dilation. Results Ten subjects with venous insufficiency were enrolled, resulting in two legs with C2 disease, nine legs with C3 disease, eight legs with C4 disease, and one leg with C5 disease. Interstitial and/or dermal backflow were observed in 25%, 33%, and 41% of the injection sites in each limb with C2, C3, and C4 disease, respectively. Distinct vessel segmentation and dilation were observed in limbs with a C3 and higher classification, and dermal backflow proximal to the injection sites was observed in two legs with C4 disease and in the inguinal region of the C5 study subject. The overall average lymph propulsion rates were 1.3 ± 0.4, 1.2 ± 0.7, and 0.8 ± 0.5 contractile events/min for limbs with C2, C3, and C4 disease, respectively. One subject with peripheral arterial disease, who had previously undergone bypass surgery, presented with extensive dermal backflow and lymphatic reflux. Conclusions Near-infrared fluorescence lymphatic imaging demonstrated that, compared with normal health subjects, the lymphatic anatomy and contractile function generally degrade with the severity of venous insufficiency. Lymphatic abnormalities mimic those in early cancer-acquired lymphedema subjects, as previously observed by us and others. Additional studies are needed to decipher the relationship, including any causality, between lymphatic dysfunction and peripheral vascular disease and venous insufficiency.

Background Recent advancements in near-infrared fluorescence lymphatic imaging (NIRFLI) technology provide opportunities for non-invasive, real-time assessment of lymphatic contribution in the etiology and treatment of ulcers. The objective of this study was to assess lymphatics in subjects with venous leg ulcers using NIRFLI and to assess lymphatic impact of a single session of sequential pneumatic compression (SPC). Methods Following intradermal microdoses of indocyanine green (ICG) as a lymphatic contrast agent, NIRFLI was used in a pilot study to image the lymphatics of 12 subjects with active venous leg ulcers (Clinical, Etiologic, Anatomic, and Pathophysiologic [CEAP] C 6 ). The lymphatics were imaged before and after a single session of SPC to assess impact on lymphatic function. Results Baseline imaging showed impaired lymphatic function and bilateral dermal backflow in all subjects with chronic venous insufficiency, even those without ulcer formation in the contralateral limb (C 0 and C 4 disease). SPC therapy caused proximal movement of ICG away from the active wound in 9 of 12 subjects, as indicated by newly recruited functional lymphatic vessels, emptying of distal lymphatic vessels, or proximal movement of extravascular fluid. Subjects with the longest duration of active ulcers had few visible lymphatic vessels, and proximal movement of ICG was not detected after SPC therapy. Conclusions This study provides visible confirmation of lymphatic dysfunction at an early stage in the etiology of venous ulcer formation and demonstrates the potential therapeutic mechanism of SPC therapy in removing excess fluid. The ability of SPC therapy to restore fluid balance through proximal movement of lymph and interstitial fluid may explain its value in hastening venous ulcer healing. Anatomical differences between the lymphatics of longstanding and more recent venous ulcers may have important therapeutic implications.

Head and neck (HN) cancer survivors are particularly susceptible to lymphedema with a reported incidence of 75%. Treatment of HN lymphedema (HNL) consists of manual lymphatic drainage and compression. In this FDA-approved study, we used near-infrared fluorescence lymphatic imaging (NIRFLI) to assess the response of HNL to a single session of sequential, pneumatic compression therapy (PCT) and after two weeks of daily PCT. Following informed consent, 5 HNL subjects were enrolled into this study which consisted of two imaging sessions where each subject received six intradermal injections of 25µg of ICG in 0.1ml saline. Immediately after injection, NIRFLI was performed by illuminating the HN with excitation light and collecting the resultant fluorescent signal using a custom imaging system. After initial imaging, each subject underwent a single PCT session after which NIRFLI was again performed. Subjects were provided a pneumatic compression device for daily use over two weeks, after which imaging was repeated. Abnormal dermal lymphatic backflow was observed in all subjects. Enhanced lymphatic uptake was observed in all subjects following a single PCT session. After two weeks of PCT, NIRFLI revealed the complete resolution of dermal backflow in one subject, the reduction of dermal backflow in two subjects, and no apparent lymphatic change in two subjects though both reported subjective improvements in swallowing or speaking. PCT therapy may be an effective method to treat HNL. Additional studies are needed to assess long term clinical and quality of life impact. This study was funded in part by Tactile Medical.

Although fMRI technique can be used to scan the whole brain to investigate the brain functional activities in children by using the blood oxygenation level dependent (BOLD) MRI signal, patients often require sedation, which prevents investigation of brain function during the actual disordered movements. Functional near infrared spectroscopy and diffuse optical tomography (fNIRS-DOT) allows a similar assessment to the BOLD MRI signal. However, in a realistic situation there will be a skull, blood vessels, cerebrospinal fluid and a significant variation of distance of the deep hemovascular target from the tissue surface. Transmitted diffuse near-infrared (NIR) light is significantly scattered and attenuated, making it difficult to be detected with sufficient signal-to-noise ratio (SNR) using conventional detectors such as avalanche photodiodes or photomultiplier tubes. In this study, we present an fNIRS-DOT imaging system by use of military based intensification with charge-coupled device (CCD) technology to acquire the transmitted weak diffuse NIR signals with high sensitivity that allows whole brain imaging and the device can be mounted in a comfortable cap on an awake child. The millimeter level spatial resolution of transillumination tomographic reconstructions was demonstrated in studies of phantoms that approximate the size of a child’s brain. In addition, we have been able to obtain preliminary transcranial proof-of-concept data with the reasonable SNR in a 7 month old healthy baby. Further tomographic studies will allow the assessment of the brain network dysfunction in awake children suffering from brain diseases such as brain tumor.

All medical devices for Food and Drug market approval require specifications of performance based upon International System of Units (SI) or units derived from SI for reasons of traceability. Recently, near-infrared fluorescence (NIRF) imaging devices of a variety of designs have emerged on the market and in investigational clinical studies. Yet the design of devices used in the clinical studies vary widely, suggesting variable device performance. Device performance depends upon optimal excitation of NIRF imaging agents, rejection of backscattered excitation and ambient light, and selective collection of fluorescence emanating from the fluorophore. There remains no traceable working standards with SI units of radiance to enable prediction that a given molecular imaging agent can be detected in humans by a given NIRF imaging device. Furthermore, as technologies evolve and as NIRF imaging device components change, there remains no standardized means to track device improvements over time and establish clinical performance without involving clinical trials, often costly. In this study, we deployed a methodology to calibrate luminescent radiance of a stable, solid phantom in SI units of mW/cm 2 /sr for characterizing the measurement performance of ICCD and IsCMOS camera based NIRF imaging devices, such as signal-to-noise ratio (SNR) and contrast. The methodology allowed determination of superior SNR of the ICCD over the IsCMOS system; comparable contrast of ICCD and IsCMOS depending upon binning strategies.

Cardiovascular diseases remain the greatest cause of death in the US and gene therapy has the potential to be an effective therapy. In this study, we demonstrated MMP-9 based protease-activatable virus (PAV) for selective infection of myocardial infarct (MI) that is associated with active MMP-9 expression. To test the specificity of PAV, we used expression of a far-red fluorescence protein (iRFP) delivered by the PAV together with a dual PET/NIRF imaging agent specific for active MMP-9 activity at the site of MI in a murine model. Calibrated fluorescence imaging employed a highly-sensitive intensified camera, laser diode excitation sources, and filtration schemes based upon the spectra of iRFP and the NIRF agent. One to two days after ligation of the left anterior descending artery, the PAV or WT AAV9 virus encoding for iRFP (5x1010 genomic particles) and radiolabeled MMP-9 imaging agent (3 nmol) were injected intravenously (i.v.). PET imaging showed MMP activity was associated with adverse tissue remodeling at the site of the MI. One week after, animals were again injected i.v. with the MMP-9 agent (3 nmol) and 18-24 h later, the animals were euthanized and the hearts were harvested, sliced, and imaged for congruent iRFP transgene expression and NIRF signals associated with MMP-9 tissue activity. The fluorescent margins of iRFP and NIRF contrasted tissues were quantified in terms Standard International units of mW/cm2/sr. The sensitivity, specificity, and accuracy of PAV and WT targeting to sites of MI was determined from these calibrated fluorescence measurements. The PAV demonstrated significantly higher delivery performance than that of the WT AAV9 virus.

Monoclonal antibodies (mAbs) have been shown preclinically as reliable targeting moieties for antigen imaging using near-infrared fluorescence (NIRF) molecular imaging. However, crystallizable fragment-gamma receptor (FcγRs) expressed on immune cells also bind mAbs through defined epitopes on the constant fragment (Fc) of IgG. Herein, we evaluate the potential impact Fc interactions have on mAb agent imaging specificity.Through the removal of conserved glycans within the Fc domain, shown to have Fc/FcγR interactions, we evaluate their impact on non-specific binding/accumulation of a NIRF-labeled mAb-based imaging agent in lymph nodes (LNs) in inflamed animals and in an orthotopic prostate cancer animal model of LN metastasis.Deglycosylation of a murine mAb against the human epithelial cell adhesion marker using endoglycosidase EndoS significantly reduced non-specific binding in the LNs of inflamed animals and in cancer-negative LNs of tumor-bearing animals. Sensitivity remained unchanged while improvement in imaging specificity increased imaging accuracy.The reduction of non-specific binding through deglycosylation of a mAb-based imaging agent shows that reducing Fc/FcγR interactions can improve imaging accuracy.

Objective Investigational, near-infrared fluorescence (NIRF) lymphatic imaging was used to assess lymphatic architecture and contractile function in participants diagnosed with Dercum’s disease, a rare, poorly understood disorder characterized by painful lipomas in subcutaneous adipose tissues.

Wide-field surgical excision reduces the chance of residual disease, but can also lead to disfigurement and devastating morbidities when resection is close to critical structures. We hypothesize that near-infrared fluorescence (NIRF) imaging can enable accurate detection of tumor margins for image-guided resection. An orthotopic model of human prostate cancer (PCa) was used to assess primary tumor margins using a NIRF-labeled antibody against epithelial cell adhesion molecule (EpCAM). PCa cells stably expressing far red fluorescent gene reporter, iRFP, enabled colocalization with NIRF signals for direct assessment of tumor margins. Using receiver operating characteristic analysis, far red fluorescence was validated against standard pathology of primary and metastatic lesions with >96 % accuracy. Primary tumor margins were more accurately detected by quantitative NIRF imaging using the EpCAM-targeting antibody as compared to a NIRF-labeled isotype control antibody. NIRF molecular imaging may enable real-time and accurate assessment of tumor margins.

The use of tissue phantoms as calibrators to transfer SI-referenced scale to an imager offers convenience, compared to other methods of calibration. The tissue phantoms are calibrated separately for radiance at emission wavelength per irradiance at excitation wavelength. This calibration is only performed at a single geometric configuration, typically with the detector normal to the sample. In the clinic however, the imager can be moved around, resulting in a geometric configuration different from the calibration configuration. In this study, radiometric measurements are made at different sample-imager angles to test whether the tissue phantoms are Lambertian and the angular limits to which the calibration values hold true.

The success of optical surgical navigation depends upon being able to intraoperatively employ a contrast agent and an imaging device to successfully guide surgery. Development of devices and contrast agents typically occur separately even though it is their combined performance that ultimately determines success and clinical adoption. Herein, we review critical issues and summarize our strategies and approaches for validating molecularly-targeted, near-infrared fluorescent contrast agents and the devices sufficiently sensitive enough for their detection in order to guide lymph node dissection.

The brain connectivity in joint attention skills using NIRS and imaging was studied, indicating differences in the neuronal pathways. To further improve the spatial resolution, an intensified CCD based NIRS and imaging system was developed.

The work presented herein describes the system design and performance evaluation of a miniaturized near-infrared fluorescence (NIRF) frequency-domain photon migration (FDPM) system with non-contact excitation and homodyne detection capability for small animal fluorescence tomography. The FDPM system was developed specifically for incorporation into a Siemens micro positron emission tomography/computed tomography (microPET/CT) commercial scanner for hybrid small animal imaging, but could be adapted to other systems. Operating at 100 MHz, the system noise was minimized and the associated amplitude and phase errors were characterized to be ±0.7% and ±0.3°, respectively. To demonstrate the tomographic ability, a commercial mouse-shaped phantom with 50 µM IRDye800CW and ⁶⁸Ga containing inclusion was used to associate PET and NIRF tomography. Three-dimensional mesh generation and anatomical referencing was accomplished through CT. A third-order simplified spherical harmonics approximation (SP₃) algorithm, for efficient prediction of light propagation in small animals, was tailored to incorporate the FDPM approach. Finally, the PET-NIRF target co-localization accuracy was analyzed in vivo with a dual-labeled imaging agent targeting orthotopic growth of human prostate cancer. The obtained results validate the integration of time-dependent fluorescence tomography system within a commercial microPET/CT scanner for multimodality small animal imaging.

With the aid of indocyanine green (ICG), lymphatic architecture and function in both mice and humans has been successfully imaged non-invasively using near-infrared (NIR) fluorescence imaging devices. Maximal measurement sensitivity of NIR fluorescence imaging devices is needed for “first-in-humans” molecularly targeting NIR fluorescence agents that are brighter than non-specific ICG. In this study, we developed a solid phantom and measurement approach for the quantification of excitation light leakage and measurement sensitivity of NIR fluorescence imaging devices. The constructed solid phantom, consisting of quantum dots impregnated onto specularly reflective surface, shows long-term stability and can be used as a traceable fluorescence standard. With the constructed solid phantom, the intensified CCD (ICCD)-based device demonstrated more than 300% higher measurement sensitivity compared to the Electron Multiplying CCD (EMCCD) based device when integration time was maintained less than 1.0 s.

Acquired lymphedema is thought to arise from the damage of the lymphatic vasculature that transports excess fluid and macromolecules away from tissues for return to the blood vasculature. The onset of the cancer acquired disease can occur months to years after lymph node dissection and manifests itself as an accumulation of fluid and macromolecules in tissues that leads to edema and irresolvable swelling. The rare disease of primary lymphedema is identical to cancer acquired lymphedema, with the exception that there is no trauma or cancer treatment that can be attributed as its cause. Primary lymphedema has been attributed to genetic causes since the late nineteenth century. Although there are five known genetic causes of hereditary or primary lymphedema, the majority of patients with lymphedema do not possess mutations in these genes. More recently, it has been proposed that a genetic link between cancer acquired and primary lymphedema exists. If a genetic susceptibility for cancer acquired lymphedema could be found, then we could predict which survivors will encounter the disease and could develop new therapies which are more effective than the current treatments that have remained unchanged for the past 80 years. In an FDA approved investigational study, we used near-infrared (NIR) fluorescence imaging to phenotype the lymphatic architecture of subjects with both acquired and primary lymphedema, as well as their unaffected family members. We collected blood for DNA analyses. NIR fluorescence provided the phenotype of abnormal lymphatic function while whole exome sequencing provided the genotype. Bioinformatics analyses were then used to identify causative genes using cosegregation of familial genotypes using the phentotypes found through NIR fluorescence imaging. The first family analyzed had members with primary and acquired lymphedema in which mutations encoding for proteins that participate in the HGF/c-MET and PI3K pathways could potentially explain the inheritance of lymphedema in this family. The father and affected daughters were heterozygous for a de novo SNP HGF in the kringle binding domain that interacts with tyrosine kinase receptor c-MET. The father had a normal lymphatic phenotype. On the other hand, the mother and daughters were heterozygous for the de novo mutation of INPPL1 (SHIP-2), adjacent to the SH2 domain of the protein that is known to bind to the multifunctional docking site of c-MET and associates with proteins in the Rho pathway for cytoskeletal reorganization. The daughters possessed both HGF and INPPL1 mutations and were diagnosed with primary lymphedema while the mother, who possessed the INPPL1 mutation, was diagnosed at the time of NIR imaging with acquired lymphedema. Analyses of remaining families as well as breast cancer related lymphedema patients are underway to confirm whether INPPL1 may be a candidate susceptibility gene for acquired lymphedema. Supported in parts by R01 HL092923 and CA128919, The Texas Star Award, and the Cullen Foundation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-12-04.

Near-infrared fluorescence (NIRF) imaging involves the separation of weak fluorescence signals from backscattered excitation light. The measurement sensitivity of current NIRF imaging systems is limited by the excitation light leakage through rejection filters. In this contribution, the authors demonstrate that the excitation light leakage can be suppressed upon using appropriate filter combination and permutations. The excitation light leakage and measurement sensitivity were assessed and compared in this study by computing the transmission ratios of excitation to emission light collected and the signal-to-noise ratios in well-controlled phantom studies with different filter combinations and permutations. Using appropriate filter combinations and permutations, we observe as much as two orders of magnitude reduction in the transmission ratio and higher signal-to-noise ratio.

Autism is a socio-communication brain development disorder. It is marked by degeneration in the ability to respond to joint attention skill tasks, from as early as 12 to 18 months of age. This trait is used to distinguish autistic from typically developing populations. In this study, near infrared (NIR) spectroscopy was used for the first time to study brain connectivity and lateralisation in response to joint attention experience in normal adults. The pre- and anterior frontal regions of the brain were non-invasively imaged using a frequency-domains based NIR system. The imaging studies were performed on 11 normal right-handed adults and optical measurements were acquired in response to joint-attention based video clips. Functional connectivity analysis was performed using intensity and relative phase shift data of the frequency-domain optical signals. Functional connectivity was computed using zero-order correlations and the cortical lateralisation analyses was also performed. Strong functional connectivity (in terms of HbO (oxy-haemoglobin), HbT (total haemoglobin) and relative phase shift data) in the pre-frontal cortex, was observed, irrespective of the stimuli (joint-attention (J), non-joint attention (NJ), and baseline rest (R); J, NJ or R). However, in the anterior frontal cortex, although the connectivity (based on HbO and HbT) demonstrated a strong correlation between the left and right regions, the connectivity (based on relative phase shift data) demonstrated a weaker correlation. The cortical lateralisation study demonstrated a right pre-frontal dominance during NJ and R stimuli, and not so in the J stimulus. The lateralisation study complemented the connectivity study by revealing that, although both the volume and pathlengths of the left and right pre-frontal cortex are similar, the dominance can vary with respect to stimuli. The high temporal resolution of NIR spectroscopy enabled study of the neural pathway associated with the socio-communicative skills, and hence the results have potential to elucidate the neural pathways associated with autism. Future NIR studies on autistic and typically developing children in response to the socio-communicative tasks (e.g. joint attention experience), may have a significant impact in early stage diagnostic intervention of autism in much younger children (< three years of age).

Purpose: Fluorescence-enhanced optical imaging using near-infrared (NIR) light developed for in vivo molecular targeting and reporting of various diseases provides promising opportunities for diagnostic imaging. However, the measurement sensitivity of NIR fluorescence (NIRF) optical imaging systems is limited by the leakage of the strong backscattered excitation light through rejection filters. In this article, the authors present a systematic method for improving sensitivity and validating the NIRF optical imager currently used for clinical imaging of human lymphatic function. Methods: The proposed systemic method consists of an appropriate filter combination and a collimation optics adapted to an NIRF optical imager. The spectral contributions were first assessed due to the excitation light backscattered from the tissue and from non-normal-incidence of the excitation light on the optical filters used in the authors’ NIRF clinical imaging system. Then two tests were conducted to assess the system with and without the components of appropriate filters combination and collimation optics using: (1) a phantom to evaluate excitation light leakage as a function of target depth and (2) deployment in an actual human study. Results: The phantom studies demonstrate as much as two to three orders of magnitude reduction in the transmission ratio, indicating that the excitation light leakage can be reduced upon using the appropriate filter combination and collimation optics while an in vivo investigatory human study confirms improved imaging. Conclusions: The method for reducing the excitation light leakage is presented for validating collected signals for fluorescence imaging.

Fluorescence molecular imaging/tomography may play an important future role in preclinical research and clinical diagnostics. Time- and frequency-domain fluorescence imaging can acquire more measurement information than the continuous wave (CW) counterpart, improving the image quality of fluorescence molecular tomography. Although diffusion approximation (DA) theory has been extensively applied in optical molecular imaging, high-order photon migration models need to be further investigated to match quantitation provided by nuclear imaging. In this paper, a frequency-domain parallel adaptive finite element solver is developed with simplified spherical harmonics (SP(N)) approximations. To fully evaluate the performance of the SP(N) approximations, a fast time-resolved tetrahedron-based Monte Carlo fluorescence simulator suitable for complex heterogeneous geometries is developed using a convolution strategy to realize the simulation of the fluorescence excitation and emission. The validation results show that high-order SP(N) can effectively correct the modeling errors of the diffusion equation, especially when the tissues have high absorption characteristics or when high modulation frequency measurements are used. Furthermore, the parallel adaptive mesh evolution strategy improves the modeling precision and the simulation speed significantly on a realistic digital mouse phantom. This solver is a promising platform for fluorescence molecular tomography using high-order approximations to the radiative transfer equation.

Noisy measurements may impact the quality of image reconstructions in fluorescence-enhanced optical tomography studies. In this contribution, different noise pre-filtration techniques were developed using fluorescence measurements of homogeneous breast phantoms. The noise pre-filtration techniques were determined based on correlation studies among various measurement parameters. We demonstrated that noise pre-filtration techniques can improve the target localization in image reconstructions when the target is deeply located (>2 cm) in imperfect uptake case (i.e. target: background fluorescence absoroption contrast ratio of 100 : 1). Moreover, filtration of measurement noise using different criteria can possibly differentiate the target from artifacts, potentially minimizing the false-positive cases in a clinical environment.

Hand-held based optical imaging systems are a recent development towards diagnostic imaging of breast cancer. To date, all the hand-held based optical imagers are used to perform only surface mapping and target localization, but are not capable of demonstrating tomographic imaging. Herein, a novel hand-held probe based optical imager is developed towards three-dimensional (3-D) optical tomography studies. The unique features of this optical imager, which primarily consists of a hand-held probe and an intensified charge coupled device detector, are its ability to; (i) image large tissue areas ( 5 × 10 sq. cm ) in a single scan, (ii) perform simultaneous multiple point illumination and collection, thus reducing the overall imaging time; and (iii) adapt to varying tissue curvatures, from a flexible probe head design. Experimental studies are performed in the frequency domain on large slab phantoms ( ∼ 650 ml ) using fluorescence target(s) under perfect uptake (1:0) contrast ratios, and varying target depths ( 1 – 2 cm ) and X - Y locations. The effect of implementing simultaneous over sequential multiple point illumination towards 3-D tomography is experimentally demonstrated. The feasibility of 3-D optical tomography studies has been demonstrated for the first time using a hand-held based optical imager. Preliminary fluorescence-enhanced optical tomography studies are able to reconstruct 0.45 ml target(s) located at different target depths ( 1 – 2 cm ) . However, the depth recovery was limited as the actual target depth increased, since only reflectance measurements were acquired. Extensive tomography studies are currently carried out to determine the resolution and performance limits of the imager on flat and curved phantoms.

The performance evaluation of a variety of small animal tomography measurement approaches and algorithms for recovery of fluorescent absorption cross section has not been conducted. Herein, we employed an intensified CCD system installed in a commercial small animal CT (Computed Tomography) scanner to compare image reconstructions from time-independent, continuous wave (CW) measurements and from time-dependent, frequency domain (FD) measurements in a series of physical phantoms specifically designed for evaluation. Comparisons were performed as a function of 1) number of projections, 2) the level of preprocessing filters used to improve the signal-to-noise ratio (SNR), 3) endogenous heterogeneity of optical properties, as well as in the cases of 4) two fluorescent targets and 5) a mouse-shaped phantom. Assessment of quantitative recovery of fluorescence absorption cross section was performed using a fully parallel, regularization-free, linear reconstruction algorithm with diffusion approximation (DA) and high order simplified spherical harmonics ( ${\rm SP}_{\rm N}$ ) approximation to the radiative transport equation (RTE). The results show that while FD measurements may result in superior image reconstructions over CW measurements, data acquisition times are significantly longer, necessitating further development of multiple detector/source configurations, improved data read-out rates, and detector technology. FD measurements with ${\rm SP}_3$ reconstructions enabled better quantitative recovery of fluorescent target strength, but required increased computational expense. Despite the developed parallel reconstruction framework being able to achieve more than 60 times speed increase over sequential implementation, further development in faster parallel acceleration strategies for near-real time and real-time image recovery and more precise forward solution is necessary.

We present a one-dimensional optical fibre-based imaging catheter specifically developed for the atherosclerotic plaque detection of emerging novel near infrared fluorescence imaging agents. We show that femtomole amounts of fluorochromes can be detected, especially in the presence of a blood-free medium. We further studied the catheter responses for a wide range of laser powers and biologically relevant concentrations of fluorochrome. In vitro tissue-like phantoms and human carotid plaque specimen measurements further demonstrate the feasibility of atherosclerotic plaque detection.

We propose a new concept for wavefront curvature sensing, which is based on diffraction grating and fractional Fourier transforms systems. An explicit formula is derived expressing the wavefront curvature, and the wavefront shape can also be retrieved rapidly. The experimental setup is presented and numerical simulations show the validity of the proposed method.

We design a novel interferometer for the real-time wavefront measurement, which is based on the diffraction grating holography technique, where the diffraction gratings act as a beam splitter and a phase retarder, thus four interference patterns can be simultaneously obtained. Computer simulations show the validity of the approach and experimental setup is also presented.

Summary We propose a novel image encryption method that combines the pure intensity random encoding and the digital holography technique. A phase-shifting interferometer records both phase and amplitude information of a complex object with a CCD sensor array. The encryption is performed by placing two pure intensity random masks between the image to be encrypted and an intensity recording device. Electronic decryption can be performed with fast Fresnel reconstruction procedure. Numerical simulation results show the validity of the algorithm and an optoelectronic implementation setup is also presented.

Summary A novel three-dimensional (3-D) transparent object-recognition method is proposed. It combines the digital holographic technique with the fractional Fourier transform. The complex amplitude distribution of the 3-D transparent object can be recorded by use of the phase-shifting interferometry, and its bidimensional (2-D) Fresnel transform contents the entire information of the 3-D object, such as shape, orientation and location. By use of the fractional Fourier transform, the 3-D transparent objects can be recognized efficiently with the obtained 2-D information. An opto-electronic hybrid implementation has been also suggested.

Although there has been a plethora of devices advanced for clinical translation, there has been no standards to compare and determine the optical device for fluorescence molecular imaging. In this work, we compare different CCD configurations using a solid phantom developed to mimic pM - fM concentrations of near-infrared fluorescent dyes in tissues. Our results show that intensified CCD systems (ICCDs) offer greater contrast at larger signal-tonoise ratios (SNRs) in comparison to their un-intensified CCD systems operated at clinically reasonable, sub-second acquisition times. Furthermore, we compared our investigational ICCD device to the commercial NOVADAQ SPY system, demonstrating different performance in both SNR and contrast.

A tri-modal (PET/CT/Optical) small animal tomographic imaging system was developed by integrating our advanced non-contact intensified CCD (ICCD) frequency-domain fluorescence imaging components into a Siemens Inveon scanner. We performed a performance evaluation of the developed imaging system by using the developed regularization-free high-order radiative-transfer-based reconstruction algorithm and custom solid phantoms. Our results show that frequency-domain photon migration (FDPM) fluorescence tomography can achieve better tomographic images with less artifacts and more precise fluorescent source localization compared to the continuous-wave counterpart. The developed multimodal tomographic imaging system provides a powerful tool for translational biomedical research.

In this proof-of-concept study we seek to demonstrate the delivery of fluorescent contrast agent to the tumor-draining lymph node basin following intraparenchymal breast injections and intradermal arm injection of micrograms of indocyanine green in 20 breast cancer patients undergoing complete axillary lymph node dissection. Individual lymph nodes were assessed ex vivo for presence of fluorescent signal. In all, 88% of tumor-negative lymph nodes and 81% of tumor-positive lymph nodes were fluorescent. These results indicate that future studies utilizing targeted fluorescent contrast agents may demonstrate improved surgical and therapeutic intervention.

Although fluorescence molecular imaging is rapidly evolving as a new combinational drug/device technology platform for molecularly guided surgery and noninvasive imaging, there remains no performance standards for efficient translation of "first-in-humans" fluorescent imaging agents using these devices.The authors employed a stable, solid phantom designed to exaggerate the confounding effects of tissue light scattering and to mimic low concentrations (nM-pM) of near-infrared fluorescent dyes expected clinically for molecular imaging in order to evaluate and compare the commonly used charge coupled device (CCD) camera systems employed in preclinical studies and in human investigational studies.The results show that intensified CCD systems offer greater contrast with larger signal-to-noise ratios in comparison to their unintensified CCD systems operated at clinically reasonable, subsecond acquisition times.Camera imaging performance could impact the success of future "first-in-humans" near-infrared fluorescence imaging agent studies.

A new kind of generalized fractional convolution operation is introduced, which contains six degrees of freedom provided by the fractional orders. We apply such a fractional convolution operation to optical image encryption. The algorithm convolves the primary image with the randomly encoded mask in the fractional Fourier domain with the fractional orders as additional keys. Numerical simulation results have been given to verify its flexibility and the performance of security. The optical implementation setup is also suggested.

We propose a novel architecture of optical image encryption that uses cascaded multi-stages of fractional Fourier transforms with a random phase mask at each stage. Without increasing the complexity of the optical hardware, such algorithm can result in a more secure image encryption with the addition of the extra degrees of freedom provided by the fractional Fourier transforms, i.e. the fractional orders. Numerical simulations have verified its validity.

Investigational, near-infrared fluorescence (NIRF) lymphatic imaging was used to assess lymphatic architecture and contractile function in participants diagnosed with Dercum's disease, a rare, poorly understood disorder characterized by painful lipomas in subcutaneous adipose tissues.After informed consent and as part of an FDA-approved feasibility study to evaluate lymphatics in diseases in which their contribution has been implicated, three women diagnosed with Dercum's disease and four control subjects were imaged. Each participant received multiple intradermal and subcutaneous injections of indocyanine green (ICG, total dose ≤400 µg) in arms, legs, and/or trunk. Immediately after injection, ICG was taken up by the lymphatics and NIRF imaging was conducted.The lymphatics in the participants with Dercum's disease were intact and dilated, yet sluggishly propelled lymph when compared to control lymphatics. Palpation of regions containing fluorescent lymphatic pathways revealed tender, fibrotic, tubular structures within the subcutaneous adipose tissue that were associated with painful nodules, and, in some cases, masses of fluorescent tissue indicating that some lipomas may represent tertiary lymphoid tissues.These data support the hypothesis that Dercum's disease may be a lymphovascular disorder and suggest a possible association between abnormal adipose tissue deposition and abnormal lymphatic structure and function.

One limitation of fluorescence molecular imaging that can limit clinical implementation and hamper small animal imaging is the inability to eliminate ambient light. Herein, we demonstrate the ability to conduct rapid non-invasive, far-red and near-infrared fluorescence imaging in living animals and a phantom under ambient light conditions using a modulated image intensified CCD (ICCD) and a laser diode operated in homodyne detection. By mapping AC amplitude from three planar images at varying phase delays, we show improvement in target-to-background ratios (TBR) and reasonable signal-to-noise ratios (SNR) over continuous wave measurements. The rapid approach can be used to accurately collect fluorescence in situations where ambient light cannot be spectrally conditioned or controlled, such as in the case of fluorescent molecular image-guided surgery.

As part of a proof-of-concept study for future delivery of targeted near-infrared fluorescent (NIRF) tracers, we sought to assess the delivery of micrograms of indocyanine green to all the axillary lymph nodes following intraparenchymal breast injections and intradermal arm injections in 20 subjects with advanced breast carcinoma and undergoing complete axillary lymph node dissection. Lymphatic vessels and nodes were assessed in vivo. Ex vivo images demonstrated that 87% of excised lymph nodes, including 81% of tumor-positive lymph nodes, were fluorescent. Future clinical studies using microdose amounts of tumor-targeting NIRF contrast agents may demonstrate improved surgical intervention with reduced morbidity.

Fluorescence gene reporters have recently become available for excitation at far-red wavelengths, enabling opportunities for small animal in vivo gene reporter fluorescence tomography (GRFT). We employed multiple projections of the far-red fluorescence gene reporters IFP1.4 and iRFP, excited by a point source in transillumination geometry in order to reconstruct the location of orthotopically implanted human prostate cancer (PC3), which stably expresses the reporter. Reconstruction was performed using a linear radiative-transfer-based regularization-free tomographic method. Positron emission tomography (PET) imaging of a radiolabeled antibody-based agent that targeted epithelial cell adhesion molecule overexpressed on PC3 cells was used to confirm in vivo GRFT results. Validation of GRFT results was also conducted from ex vivo fluorescence imaging of resected prostate tumor. In addition, in mice with large primary prostate tumors, a combination of GRFT and PET showed that the radiolabeled antibody did not penetrate the tumor, consistent with known tumor transport limitations of large (∼150 kDa ) molecules. These results represent the first tomography of a living animal using far-red gene reporters.

Over the past decade, we developed near-infrared fluorescence (NIRF) devices for non-invasive lymphatic imaging using microdosages of ICG in humans and for detection of lymph node metastasis in animal models mimicking metastatic human prostate cancer. To validate imaging, a NIST traceable phantom is needed so that developed “first-inhumans” drugs may be used with different luorescent imaging platforms. In this work, we developed a QDots 800 based fluorescent solid phantom for installation and operational qualification of clinical and preclinical, NIRF imaging devices. Due to its optical clearance, polyurethane was chosen as the base material. Titanium dioxide was used as the scattering agent because of its miscibility in polyurethane. QDots 800 was chosen owing to its stability and NIR emission spectra. A first phantom was constructed for evaluation of the noise floor arising from excitation light leakage, a phenomenon that can be minimized during engineering and design of fluorescent imaging systems. A second set of phantoms were constructed to enable quantification of device sensitivity associated with our preclinical and clinical devices. The phantoms have been successfully applied for installation and operational qualification of our preclinical and clinical devices. Assessment of excitation light leakage provides a figure of merit for “noise floor” and imaging sensitivity can be used to benchmark devices for specific imaging agents.

Solid fluorescent phantoms were constructed to assess detection limitations of investigational NIRF imaging devices. The phantoms enabled the quantitative assessment of the fluorescent status of lymph nodes following resection from breast cancer patients.