Alisha V. DSouza

Review of fluorescence guided surgery systems: identification of key performance capabilities beyond indocyanine green imaging

Abstract. There is growing interest in using fluorescence imaging instruments to guide surgery, and the leading options for open-field imaging are reviewed here. While the clinical fluorescence-guided surgery (FGS) field has been focused predominantly on indocyanine green (ICG) imaging, there is accelerated development of more specific molecular tracers. These agents should help advance new indications for which FGS presents a paradigm shift in how molecular information is provided for resection decisions. There has been a steady growth in commercially marketed FGS systems, each with their own differentiated performance characteristics and specifications. A set of desirable criteria is presented to guide the evaluation of instruments, including: (i) real-time overlay of white-light and fluorescence images, (ii) operation within ambient room lighting, (iii) nanomolar-level sensitivity, (iv) quantitative capabilities, (v) simultaneous multiple fluorophore imaging, and (vi) ergonomic utility for open surgery. In this review, United States Food and Drug Administration 510(k) cleared commercial systems and some leading premarket FGS research systems were evaluated to illustrate the continual increase in this performance feature base. Generally, the systems designed for ICG-only imaging have sufficient sensitivity to ICG, but a fraction of the other desired features listed above, with both lower sensitivity and dynamic range. In comparison, the emerging research systems targeted for use with molecular agents have unique capabilities that will be essential for successful clinical imaging studies with low-concentration agents or where superior rejection of ambient light is needed. There is no perfect imaging system, but the feature differences among them are important differentiators in their utility, as outlined in the data and tables here.

Review of fluorescence guided surgery visualization and overlay techniques.

In fluorescence guided surgery, data visualization represents a critical step between signal capture and display needed for clinical decisions informed by that signal. The diversity of methods for displaying surgical images are reviewed, and a particular focus is placed on electronically detected and visualized signals, as required for near-infrared or low concentration tracers. Factors driving the choices such as human perception, the need for rapid decision making in a surgical environment, and biases induced by display choices are outlined. Five practical suggestions are outlined for optimal display orientation, color map, transparency/alpha function, dynamic range compression, and color perception check.

White light-informed optical properties improve ultrasound-guided fluorescence tomography of photoactive protoporphyrin IX

Abstract. Subsurface fluorescence imaging is desirable for medical applications, including protoporphyrin-IX (PpIX)-based skin tumor diagnosis, surgical guidance, and dosimetry in photodynamic therapy. While tissue optical properties and heterogeneities make true subsurface fluorescence mapping an ill-posed problem, ultrasound-guided fluorescence-tomography (USFT) provides regional fluorescence mapping. Here USFT is implemented with spectroscopic decoupling of fluorescence signals (auto-fluorescence, PpIX, photoproducts), and white light spectroscopy-determined bulk optical properties. Segmented US images provide a priori spatial information for fluorescence reconstruction using region-based, diffuse FT. The method was tested in simulations, tissue homogeneous and inclusion phantoms, and an injected-inclusion animal model. Reconstructed fluorescence yield was linear with PpIX concentration, including the lowest concentration used, 0.025  μg/ml. White light spectroscopy informed optical properties, which improved fluorescence reconstruction accuracy compared to the use of fixed, literature-based optical properties, reduced reconstruction error and reconstructed fluorescence standard deviation by factors of 8.9 and 2.0, respectively. Recovered contrast-to-background error was 25% and 74% for inclusion phantoms without and with a 2-mm skin-like layer, respectively. Preliminary mouse-model imaging demonstrated system feasibility for subsurface fluorescence measurement in vivo. These data suggest that this implementation of USFT is capable of regional PpIX mapping in human skin tumors during photodynamic therapy, to be used in dosimetric evaluations.

Microdose fluorescence imaging of ABY-029 on an operating microscope adapted by custom illumination and imaging modules

Fluorescence guided surgery has the potential to positively impact surgical oncology; current operating microscopes and stand-alone imaging systems are too insensitive or too cumbersome to maximally take advantage of new tumor-specific agents developed through the microdose pathway. To this end, a custom-built illumination and imaging module enabling picomolar-sensitive near-infrared fluorescence imaging on a commercial operating microscope is described. The limits of detection and system specifications are characterized, and in vivo efficacy of the system in detecting ABY-029 is evaluated in a rat orthotopic glioma model following microdose injections, showing the suitability of the device for microdose phase 0 clinical trials.

Geometric correction of deformed chromosomes for automatic Karyotyping

Automatic Karyotyping is the process of classifying chromosomes from an unordered karyogram into their respective classes to create an ordered karyogram. Automatic karyotyping algorithms typically perform geometrical correction of deformed chromosomes for feature extraction; these features are used by classifier algorithms for classifying the chromosomes. Karyograms of bone marrow cells are known to have poor image quality. An example of such karyograms is the Lisbon-K1 (LK1) dataset that is used in our work. Thus, to correct the geometrical deformation of chromosomes from LK1, a robust method to obtain the medial axis of the chromosome was necessary. To address this problem, we developed an algorithm that uses the seed points to make a primary prediction. Subsequently, the algorithm computes the distance of boundary from the predicted point, and the gradients at algorithm-specified points on the boundary to compute two auxiliary predictions. Primary prediction is then corrected using auxiliary predictions, and a final prediction is obtained to be included in the seed region. A medial axis is obtained this way, which is further used for geometrical correction of the chromosomes. This algorithm was found capable of correcting geometrical deformations in even highly distorted chromosomes with forked ends.

Nodal lymph flow quantified with afferent vessel input function allows differentiation between normal and cancer-bearing nodes.

Morbidity and complexity involved in lymph node staging via surgical resection and biopsy could ideally be improved using node assay techniques that are non-invasive. While visible blue dyes are often used to locate the sentinel lymph nodes from draining lymphatic vessels near a tumor, they do not provide an in situ metric to evaluate presence of cancer. In this study, the transport kinetics of methylene blue were analyzed to determine the potential for better in situ information about metastatic involvement in the nodes. A rat model with cancer cells in the axillary lymph nodes was used, with methylene blue injection to image the fluorescence kinetics. The lymphatic flow from injection sites to nodes was imaged and the relative kinetics from feeding lymphatic ducts relative to lymph nodes was quantified. Large variability existed in raw fluorescence and transport patterns within each cohort resulting in no systematic difference between average nodal uptake in normal, sham control and cancer-bearing nodes. However, when the signal from the afferent lymph vessel fluorescence was used to normalize the signal of the lymph nodes, the high signal heterogeneity was reduced. Using a model, the lymph flow through the nodes [Formula: see text] was estimated to be 1.49 ± 0.64 ml/g/min in normal nodes, 1.53 ± 0.45 ml/g/min in sham control nodes, and reduced to 0.50 ± 0.24 ml/g/min in cancer-cell injected nodes. This summarizes the significant difference (p = 0.0002) between cancer-free and cancer-bearing nodes in normalized flow. This process of normalized flow imaging could be used as an in situ tool to detect metastatic involvement in nodes.

Cherenkov-excited Multi-Fluorophore Sensing in Tissue-Simulating Phantoms and In Vivo from External Beam Radiotherapy

In this work, Cherenkov-excited molecular sensing was used to assess the potential for simultaneous quantitative sensing of two NIR fluorophores within tissue-simulating phantoms through spectral separation of signals. Cherenkov emissions induced by external beam gamma photon radiation treatment to tissues/tissue-simulating phantoms were detectable over the 500–900-nm wavelength range. The presence of blood was demonstrated to reduce the integrated intensity of detected Cherenkov emissions by nearly 50%, predominantly at wavelengths below 620 nm. The molecular dyes, IRDye 680RD and IRDye 800CW, have excitation and emission spectra at longer wavelengths than the strongest blood absorption peaks, and also where the intensity of Cherenkov light is at its lowest, so that the emission signal relative to background signal is maximized. Tissue phantoms composed of 1% intralipid and 1% blood were used to simulate human breast tissue, and vials containing fluorophore were embedded in the media, and irradiated with gamma photons for Cherenkov excitation. We observed that fluorescence emissions excited by the Cherenkov signal produced within the phantom could be detected at 5-mm depth into the media within a 0.1–25 μM fluorophore concentration range. The detected fluorescence signals from these dyes showed linear relationships with radiation doses down to the cGy level. In vivo tests were successful only within the range near a μM, suggesting that these could be used for metabolic probes in vivo where the local concentrations are near this range.

Development and evaluation of a connective tissue phantom model for subsurface visualization of cancers requiring wide local excision

Abstract. Wide local excision (WLE) of tumors with negative margins remains a challenge because surgeons cannot directly visualize the mass. Fluorescence-guided surgery (FGS) may improve surgical accuracy; however, conventional methods with direct surface tumor visualization are not immediately applicable, and properties of tissues surrounding the cancer must be considered. We developed a phantom model for sarcoma resection with the near-infrared fluorophore IRDye 800CW and used it to iteratively define the properties of connective tissues that typically surround sarcoma tumors. We then tested the ability of a blinded surgeon to resect fluorescent tumor-simulating inclusions with ∼1-cm margins using predetermined target fluorescence intensities and a Solaris open-air fluorescence imaging system. In connective tissue-simulating phantoms, fluorescence intensity decreased with increasing blood concentration and increased with increasing intralipid concentrations. Fluorescent inclusions could be resolved at ≥1-cm depth in all inclusion concentrations and sizes tested. When inclusion depth was held constant, fluorescence intensity decreased with decreasing volume. Using targeted fluorescence intensities, a blinded surgeon was able to successfully excise inclusions with ∼1-cm margins from fat- and muscle-simulating phantoms with inclusion-to-background contrast ratios as low as 2∶1. Indirect, subsurface FGS is a promising tool for surgical resection of cancers requiring WLE.

Optical tracer size differences allow quantitation of active pumping rate versus Stokes–Einstein diffusion in lymphatic transport

Lymphatic uptake of interstitially administered agents occurs by passive convective–diffusive inflow driven by interstitial concentration and pressure, while the downstream lymphatic transport is facilitated by active propulsive contractions of lymphatic vessel walls. Near-infrared fluorescence imaging in mice was used to measure these central components of lymphatic transport for the first time, using two different-sized molecules––methylene blue (MB) and fluorescence-labeled antibody immunoglobulin G (IgG)-IRDye 680RD. This work confirms the hypothesis that lymphatic passive inflow and active propulsion rates can be separated based upon the relative differences in Stokes–Einstein diffusion coefficient. This coefficient specifically affects the passive-diffusive uptake when the interstitial volume and pressure are constant. Parameters such as mean time-to-peak signal, overall fluorescence signal intensities, and number of active peristaltic pulses, were estimated from temporal imaging data. While the mean time to attain peak signal representative of diffusion-dominated flow in the lymph vessels was 0.6±0.2??min for MB and 8±6??min for IgG, showing a size dependence, the active propulsion rates were 3.4±0.8??pulses/min and 3.3±0.5??pulses/min, respectively, appearing size independent. The propulsion rates for both dyes decreased with clearance from the interstitial injection-site, indicating intrinsic control of the smooth muscles in response to interstitial pressure. This approach to size-comparative agent flow imaging of lymphatic function can enable noninvasive characterization of diseases related to uptake and flow in lymph networks.

Subsurface PpIX imaging in vivo with ultrasound-guided tomographic spectroscopy: reconstruction vs. born-normalized data

Aminolevulinic acid (ALA)-induced Protoporphyrin IX (PpIX)-based photodynamic therapy (PDT) is an effective treatment for skin cancers including basal cell carcinoma (BCC). Topically applied ALA promotes PpIX production preferentially in tumors, and many strategies have been developed to increase PpIX distribution and PDT treatment efficacy at depths > 1mm is not fully understood. While surface imaging techniques provide useful diagnosis, dosimetry, and efficacy information for superficial tumors, these methods cannot interrogate deeper tumors to provide in situ insight into spatial PpIX distributions. We have developed an ultrasound-guided, white-light-informed, tomographics spectroscopy system for the spatial measurement of subsurface PpIX. Detailed imaging system specifications, methodology, and optical-phantom-based characterization will be presented separately. Here we evaluate preliminary in vivo results using both full tomographic reconstruction and by plotting individual tomographic source-detector pair data against US images.