John C. Rasmussen

Imaging of Lymph Flow in Breast Cancer Patients after Microdose Administration of a Near-Infrared Fluorophore: Feasibility Study

PURPOSEnTo prospectively demonstrate the feasibility of using indocyanine green, a near-infrared (NIR) fluorophore at the minimum dose needed for noninvasive optical imaging of lymph nodes (LNs) in breast cancer patients undergoing sentinel lymph node mapping (SLNM).nnnMATERIALS AND METHODSnInformed consent was obtained from 24 women (age range, 30-85 years) who received intradermal subcutaneous injections of 0.31-100 microg indocyanine green in the breast in this IRB-approved, HIPAA-compliant, dose escalation study to find the minimum microdose for imaging. The breast, axilla, and sternum were illuminated with NIR light and the fluorescence generated in the tissue was collected with an NIR-sensitive intensified charged-coupled device. Lymphoscintigraphy was also performed. Resected LNs were evaluated for the presence of radioactivity, blue dye accumulation, and fluorescence. The associations between the resected LNs that were fluorescent and (a) the time elapsed between NIR fluorophore administration and resection and (b) the dosage of NIR fluorophores were tested with the Spearman rank and Pearson product moment correlation tests, respectively.nnnRESULTSnLymph imaging consistently failed with indocyanine green microdosages between 0.31 and 0.77 microg. When indocyanine green dosages were 10 microg or higher, lymph drainage pathways from the injection site to LNs were imaged in eight of nine women; lymph propulsion was observed in seven of those eight. When propulsion in the breast and axilla regions was present, the mean apparent velocities ranged from 0.08 to 0.32 cm/sec, the time elapsed between packets of propelled fluid varied from 14 to 92 seconds. In patients who received 10 microg of indocyanine green or more, a weak negative correlation between the fluorescence status of resected LNs and the time between NIR fluorophore administration and LN resection was found. No statistical association was found between the fluorescence status of resected LNs and the dose of NIR fluorophore.nnnCONCLUSIONnNIR fluorescence imaging of lymph function and LNs is feasible in humans at microdoses that would be needed for future molecular imaging of cancer-positive LNs.

Near-Infrared Fluorescence Imaging in Humans with Indocyanine Green: A Review and Update.

Near-infrared (NIR) fluorescence imaging clinical studies have been reported in the literature with six different devices that employ various doses of indocyanine green (ICG) as a non-specific contrast agent. To date, clinical applications range from (i) angiography, intraoperative assessment of vessel patency, and tumor/metastasis delineation following intravenous administration of ICG, and (ii) imaging lymphatic architecture and function following subcutaneous and intradermal ICG administration. In the latter case, NIR fluorescence imaging may enable new discoveries associated with lymphatic function due to (i) a unique niche that is not met by any other conventional imaging technology and (ii) its exquisite sensitivity enabling high spatial and temporal resolution. Herein, we (i) review the basics of clinical NIR fluorescence imaging, (ii) survey the literature on clinical application of investigational devices using ICG fluorescent contrast, (iii) provide an update of non-invasive dynamic lymphatic imaging conducted with our FDPM device, and finally, (iv) comment on the future NIR fluorescence imaging for non-invasive and intraoperative use given recent demonstrations showing capabilities for imaging following microdose administration of contrast agent.

New Horizons for Imaging Lymphatic Function

In this review, we provide a comprehensive summary of noninvasive imaging modalities used clinically for the diagnosis of lymphatic diseases, new imaging agents for assessing lymphatic architecture and cancer status of lymph nodes, and emerging near‐infrared (NIR) fluorescent optical imaging technologies and agents for functional lymphatic imaging. Given the promise of NIR optical imaging, we provide example results of functional lymphatic imaging in mice, swine, and humans, showing the ability of this technology to quantify lymph velocity and frequencies of propulsion resulting from the contractility of lymphatic structures.

Molecular imaging with optics: primer and case for near-infrared fluorescence techniques in personalized medicine

We compare and contrast the development of optical molecular imaging techniques with nuclear medicine with a didactic emphasis for initiating readers into the field of molecular imaging. The nuclear imaging techniques of gamma scintigraphy, single-photon emission computed tomography, and positron emission tomography are first briefly reviewed. The molecular optical imaging techniques of bioluminescence and fluorescence using gene reporter/probes and gene reporters are described prior to introducing the governing factors of autofluorescence and excitation light leakage. The use of dual-labeled, near-infrared excitable and radio-labeled agents are described with comparative measurements between planar fluorescence and nuclear molecular imaging. The concept of time-independent and -dependent measurements is described with emphasis on integrating time-dependent measurements made in the frequency domain for 3-D tomography. Finally, we comment on the challenges and progress for translating near-infrared (NIR) molecular imaging agents for personalized medicine.

Assessment of Lymphatic Contractile Function After Manual Lymphatic Drainage Using Near-Infrared Fluorescence Imaging

OBJECTIVEnTo investigate the feasibility of assessing the efficacy of manual lymphatic drainage (MLD), a method for lymphedema (LE) management, by using near-infrared (NIR) fluorescence imaging.nnnDESIGNnExploratory pilot study.nnnSETTINGnPrimary care unit.nnnPARTICIPANTSnSubjects (N=10; age, 18-68y) with a diagnosis of grade I or II LE and 12 healthy control subjects (age, 22-59y).nnnINTERVENTIONnIndocyanine green (25 μg in 0.1 mL each) was injected intradermally in bilateral arms or legs of subjects. Diffused excitation light illuminated the limbs, and NIR fluorescence images were collected by using custom-built imaging systems. Subjects received MLD therapy, and imaging was performed pre- and posttherapy.nnnMAIN OUTCOME MEASURESnApparent lymph velocities and periods between lymphatic propulsion events were computed from fluorescence images. Data collected pre- and post-MLD were compared and evaluated for differences.nnnRESULTSnBy comparing pre-MLD lymphatic contractile function against post-MLD lymphatic function, results showed that average apparent lymph velocity increased in both the symptomatic (+23%) and asymptomatic (+25%) limbs of subjects with LE and control limbs (+28%) of healthy subjects. The average lymphatic propulsion period decreased in symptomatic (-9%) and asymptomatic (-20%) limbs of subjects with LE, as well as in control limbs (-23%).nnnCONCLUSIONSnWe showed that NIR fluorescence imaging could be used to quantify immediate improvement of lymphatic contractile function after MLD.

Radiative transport-based frequency-domain fluorescence tomography

We report the development of radiative transport model-based fluorescence optical tomography from frequency-domain boundary measurements. The coupled radiative transport model for describing NIR fluorescence propagation in tissue is solved by a novel software based on the established Attila particle transport simulation platform. The proposed scheme enables the prediction of fluorescence measurements with non-contact sources and detectors at a minimal computational cost. An adjoint transport solution-based fluorescence tomography algorithm is implemented on dual grids to efficiently assemble the measurement sensitivity Jacobian matrix. Finally, we demonstrate fluorescence tomography on a realistic computational mouse model to locate nM to microM fluorophore concentration distributions in simulated mouse organs.

Fully adaptive FEM based fluorescence optical tomography from time-dependent measurements with area illumination and detection

Using an area-illumination and area-detection scheme, we acquire fluorescence frequency domain measurements from a tissue phantom with an embedded fluorescent target and obtain tomographic reconstructions of the interior fluorescence absorption map with an adaptive finite element based scheme. The tissue phantom consisted of a clear acrylic cubic box (512 ml) filled with 1% Liposyn solution, while the fluorescent targets were 5 mm diameter glass bulbs filled with 1 microM Indocyanine Green dye solution in 1% Liposyn. Frequency domain area illumination and detection employed a planar excitation source using an expanded intensity modulated (100 MHz) 785 nm diode laser light and a gain modulated image intensified charge coupled device camera, respectively. The excitation pattern was characterized by isolating the singly scattered component with cross polarizers and was input into a dual adaptive finite element-based scheme for three dimensional reconstructions of fluorescent targets embedded beneath the phantom surface. Adaptive mesh refinement techniques allowed efficient simulation of the incident excitation light and the reconstruction of fluorescent targets buried at the depths of 1 and 2 cm. The results demonstrate the first clinically relevant noncontact fluorescence tomography with adaptive finite element methods.

Radiative transport in fluorescence-enhanced frequency domain photon migration

Small animal optical tomography has significant, but potential application for streamlining drug discovery and pre-clinical investigation of drug candidates. However, accurate modeling of photon propagation in small animal volumes is critical to quantitatively obtain accurate tomographic images. Herein we present solutions from a robust fluorescence-enhanced, frequency domain radiative transport equation (RTE) solver with unique attributes that facilitate its deployment within tomographic algorithms. Specifically, the coupled equations describing time-dependent excitation and emission light transport are solved using discrete ordinates (SN) angular differencing along with linear discontinuous finite-element spatial differencing on unstructured tetrahedral grids. Source iteration in conjunction with diffusion synthetic acceleration is used to iteratively solve the resulting system of equations. This RTE solver can accurately and efficiently predict ballistic as well as diffusion limited transport regimes which could simultaneously exist in small animals. Furthermore, the solver provides accurate solutions on unstructured, tetrahedral grids with relatively large element sizes as compared to commonly employed solvers that use step differencing. The predictions of the solver are validated by a series of frequency-domain, phantom measurements with optical properties ranging from diffusion limited to transport limited propagation. Our results demonstrate that the RTE solution consistently matches measurements made under both diffusion and transport-limited conditions. This work demonstrates the use of an appropriate RTE solver for deployment in small animal optical tomography.

Plane-wave fluorescence tomography with adaptive finite elements

We present three-dimensional fluorescence yield tomography of a tissue phantom in a noncontact reflectance imaging setup. The method employs planar illumination with modulated light and frequency domain fluorescence measurements made on the illumination plane. An adaptive finite-element algorithm is used to handle the ill-posed and computationally demanding inverse image reconstruction problem. Tomographic images of fluorescent targets buried at 1-2 cm depths from the illumination surface demonstrate the feasibility of fluorescence tomography from reflectance tomography in clinically relevant tissue volumes.

Improved Excitation Light Rejection Enhances Small-Animal Fluorescent Optical Imaging

Small-animal fluorescence-enhanced imaging involves the detection of weak fluorescent signals emanating from nanomolar to picomolar concentrations of exogenous or endogenously produced fluorophore concurrent with the rejection of an overwhelmingly large component of backscattered excitation light. The elimination of the back-reflected excitation light of the collected signal remains a major and often unrecognized challenge for further reducing the noise floor and increasing sensitivity of small-animal fluorescence imaging. Herein, we show that the combination of three-cavity interference and holographic super notch filters with appropriate imaging lenses to collimate light improves rejection of excitation light, enabling more accurate imaging. To assess excitation leakage, the “out-of-band (S(Λ x ))” to “in-band (S(Λ m )–S(Λ x ))” signal ratio from phantom studies and the target-to-background ratio (TBR) from in vivo animal imaging was acquired with and without collimating optics. The addition of collimating optics resulted in a 51% to 75% reduction in the ratio of (S(Λ x ))/(S(Λ m )–S(Λ x )) for the phantom studies and an improvement of TBR from 11% to 31% and of signal-to-noise ratio from 11% to 142% for an integrin-targeting conjugate in human glioma xenografts.