Logarithmic Intensity Compression in Fluorescence Guided Surgery Applications

The use of fluorescence video imaging to guide surgery is rapidly expanding, and improvements in camera readout dynamic range have not matched display capabilities. Logarithmic intensity compression is a fast, single-step mapping technique that can map the useable dynamic range of high-bit fluorescence images onto the typical 8-bit display and potentially be a variable dynamic contrast enhancement tool. We demonstrate a ∼4.6  times improvement in image quality quantified by image entropy and a dynamic range reduction by a factor of ∼380 by the use of log-compression tools in processing in vivo fluorescence images

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

Review of Fluorescence Guided Surgery Systems: Identification of Key Performance Capabilities Beyond Indocyanine Green Imaging

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

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

White Light-Informed Optical Properties Improve Ultrasound-Guided Fluorescence Tomography of Photoactive Protoporphyrin IX

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

Photodynamic priming with triple-receptor targeted nanoconjugates that trigger T cell-mediated immune responses in a 3D in vitro heterocellular model of pancreatic cancer

Photodynamic priming (PDP), a collateral effect of photodynamic therapy, can transiently alter the tumor microenvironment (TME) beyond the cytotoxic zone. Studies have demonstrated that PDP increases tumor permeability and modulates immune-stimulatory effects by inducing immunogenic cell death, via the release of damage-associated molecular patterns and tumor-associated antigens. Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of cancers with a stubborn immunosuppressive TME and a dense stroma, representing a challenge for current molecular targeted therapies often involving macromolecules. We, therefore, tested the hypothesis that PDP\u27s TME modulation will enable targeted therapy and result in immune stimulation. Using triple-receptor-targeted photoimmuno-nanoconjugate (TR-PINs)-mediated PDP, targeting epidermal growth factor receptor, transferrin receptor, and human epidermal growth factor receptor 2 we show light dose-dependent TR-PINs mediated cytotoxicity in human PDAC cells (MIA PaCa-2), co-cultured with human pancreatic cancer-associated fibroblasts (PCAFs) in spheroids. Furthermore, TR-PINs induced the expression of heat shock proteins (Hsp60, Hsp70), Calreticulin, and high mobility group box 1 in a light dose and time-dependent manner. TR-PINs-mediated T cell activation was observed in co-cultures of immune cells with the MIA PaCa-2-PCAF spheroids. Both CD4+ T and CD8+ T cells showed light dose and time-dependant antitumor reactivity by upregulating degranulation marker CD107a and interferon-gamma post-PDP. Substantial tumor cell death in immune cell-spheroid co-cultures by day 3 shows the augmentation by antitumor T cell activation and their ability to recognize tumors for a light dose-dependent kill. These data confirm enhanced destruction of heterogeneous pancreatic spheroids mediated by PDP-induced phototoxicity, TME modulation and increased immunogenicity with targeted nanoconstructs

Multiple Projection Optical Diffusion Tomography with Plane Wave Illumination

We describe a new data collection scheme for optical diffusion tomography in which plane wave illumination is combined with multiple projections in the slab imaging geometry. Multiple projection measurements are performed by rotating the slab around the sample. The advantage of the proposed method is that the measured data can be much more easily fitted into the dynamic range of most commonly used detectors. At the same time, multiple projections improve image quality by mutually interchanging the depth and transverse directions, and the scanned (detection) and integrated (illumination) surfaces. Inversion methods are derived for image reconstructions with extremely large data sets. Numerical simulations are performed for fixed and rotated slabs

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

Dual-Channel Red/Blue Fluorescence Dosimetry with Broadband Reflectance Spectroscopic Correction Measures Protoporphyrin IX Production during Photodynamic Therapy of Actinic Keratosis

Dosimetry for aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) photodynamic therapy of actinic keratosis was examined with an optimized fluorescence dosimeter to measure PpIX during treatment. While insufficient PpIX generation may be an indicator of incomplete response, there exists no standardized method to quantitate PpIX production at depths in the skin during clinical treatments. In this study, a spectrometer-based point probe dosimeter system was used to sample PpIX fluorescence from superficial (blue wavelength excitation) and deeper (red wavelength excitation) tissue layers. Broadband white light spectroscopy (WLS) was used to monitor aspects of vascular physiology and inform a correction of fluorescence for the background optical properties. Measurements in tissue phantoms showed accurate recovery of blood volume fraction and reduced scattering coefficient from WLS, and a linear response of PpIX fluorescence versus concentration down to 1.95 and 250 nM for blue and red excitations, respectively. A pilot clinical study of 19 patients receiving 1-h ALA incubation before treatment showed high intrinsic variance in PpIX fluorescence with a standard deviation/mean ratio of \u3c0.9 . PpIX fluorescence was significantly higher in patients reporting higher pain levels on a visual analog scale. These pilot data suggest that patient-specific PpIX quantitation may predict outcome response

Scattering phase Function Spectrum Makes Reflectance Spectrum Measured from Intralipid phantoms and Tissue Sensitive to the Device Detection Geometry

Reflectance spectra measured in Intralipid (IL) close to the source are sensitive to wavelength -dependent changes in reduced scattering coefficient (μs′)and scattering phase function (PF). Experiments and simulations were performed using device designs with either single or separate optical fibers for delivery and collection of light in varying concentrations of IL. Spectral reflectance is not consistentl y linear with varying IL concentration, with PF -dependent effects observed for single fiber devices with diameters smaller than ten transport lengths and for separate source- detector devices that collected light at less than half of a transport length from the source. Similar effects are thought to be seen in tissue, limiting the ability to quantitatively compare spectra from different devices without compensation