Integration of the FGS ETU to the ISIM Structure

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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

Workflow for real-time in-vivo Cherenkov-excited luminescence imaging during radiotherapy

Radiotherapy is a common method for treating tumors, however, radiosensitivity can vary between tumor types or within the tumor microenvironment. The ability to deliver oxygen is crucial for the generation of reactive oxygen species resulting in increased localized cytotoxic effects. Alternatively, hypoxic tumors are thought to indicate a poor prognosis and may benefit from more aggressive treatments, yet identifying tumor hypoxia early in the course of a multi-week fractionated dose regimen is currently impractical. Using a time-gated imaging system and oxygen-sensitive phosphorescent compound (PtG4) we are able to estimate in vivo pO2 distribution at a rate of 2.6 estimates per second, which corresponds to 50+ values during a common 2Gy dose fraction. While our previous work has reported using Cherenkov-excited luminescence to estimate in vivo pO2 during external beam radiotherapy, the dose required was often greater than a standard fraction and camera acquisition parameters required modification during treatments, resulting in interrupted workflows. The current method utilizes custom control software which cycles through camera timing parameters during acquisition. Python code using the web-based user interface JupyterLab allows for interactive analysis of the resulting image stack without the need to pay expensive licensing fees for scientific computing packages. Using open source libraries, the analysis code is able to split the image stack into respective Cherenkov excitation and phosphorescence images, which can then be further automatically segmented to find regions of interest including the subject and phosphorescent region. The intensity of the regions in the phosphorescence images are used to estimate the compound lifetime, which can then be used in the Stern-Volmer relationship to estimate pO2. This entire process does not compromise clinical workflow and is able to provide a pO2 estimate within minutes after delivering the fractionated dose, providing clinicians early feedback about trends in tumor hypoxia. The current method has been validated with both direct injection of 50mM PtG4 in Matrigel in a mouse flank, and 24hrs post IV injection of mouse with MDA-MB-231 tumor implanted in the flank. The mouse with the direct injection was imaged under anesthesia and while awake and mobile to test the ability of the automated segmentation algorithm (Figure below). While the signal from the IV injection was less intense, simultaneous imaging using the previously reported method and current method resulted in similar lifetime estimates. While oxygen-sensitive PtG4 exhibits a lifetime between 16ms under atmospheric oxygen and 47ms when deprived of oxygen, other compounds have also been investigated. Europium chelate nanoparticle (~600ms), Iridium-based small molecules (~5ms), Si nanoparticles (~60ms), and UV-sensitive tattoo inks (~15ms) have all been imaged using Cherenkov-excitation. Camera time-gating can be utilized to discriminate these compound when mixed in the same field, allowing for additional tools in the realm of contrast enhancement during radiotherapy imaging. Ongoing studies with PtG4 and other compounds are being conducted to further improve system sensitivity and refine imaging workflows so they are more clinically translatable. Please click Additional Files below to see the full abstract

Assembly of the SIR Complex and Its Regulation by O-Acetyl-ADP-Ribose, a Product of NAD-Dependent Histone Deacetylation

SummaryAssembly of silent chromatin domains in budding yeast involves the deacetylation of histone tails by Sir2 and the association of the Sir3 and Sir4 proteins with hypoacetylated histone tails. Sir2 couples deacetylation to NAD hydrolysis and the synthesis of a metabolite, O-acetyl-ADP-ribose (AAR), but the functional significance of NAD hydrolysis or AAR, if any, is unknown. Here we examine the association of the Sir2, Sir3, and Sir4 proteins with each other and histone tails. Our analysis reveals that deacetylation of histone H4-lysine 16 (K16), which is critical for silencing in vivo, is also critical for the binding of Sir3 and Sir4 to histone H4 peptides in vitro. Moreover, AAR itself promotes the association of multiple copies of Sir3 with Sir2/Sir4 and induces a dramatic structural rearrangement in the SIR complex. These results suggest that Sir2 activity modulates the assembly of the SIR complex through both histone deacetylation and AAR synthesis

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

Pixel-Based Absorption Correction for Dual-Tracer Fluorescence Imaging of Receptor Binding Potential

Ratiometric approaches to quantifying molecular concentrations have been used for decades in microscopy, but have rarely been exploited in vivo until recently. One dual-tracer approach can utilize an untargeted reference tracer to account for non-specific uptake of a receptor-targeted tracer, and ultimately estimate receptor binding potential quantitatively. However, interpretation of the relative dynamic distribution kinetics is confounded by differences in local tissue absorption at the wavelengths used for each tracer. This study simulated the influence of absorption on fluorescence emission intensity and depth sensitivity at typical near-infrared fluorophore wavelength bands near 700 and 800 nm in mouse skin in order to correct for these tissue optical differences in signal detection. Changes in blood volume [1-3%] and hemoglobin oxygen saturation [0-100%] were demonstrated to introduce substantial distortions to receptor binding estimates (error \u3e 30%), whereas sampled depth was relatively insensitive to wavelength (error \u3c 6%). In response, a pixel-by-pixel normalization of tracer inputs immediately post-injection was found to account for spatial heterogeneities in local absorption properties. Application of the pixel-based normalization method to an in vivo imaging study demonstrated significant improvement, as compared with a reference tissue normalization approach

Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody

Object: Fluorescence imaging has the potential to significantly improve neurosurgical resection of oncologic lesions through improved differentiation between normal and cancerous tissue at the tumor margins. In order to successfully mark glioma tissue a fluorescent tracer must have the ability to penetrate through the blood brain barrier (BBB) and provide delineation in the tumor periphery where heterogeneously intact BBB may exist. In this study it was hypothesized that, due to its smaller size, fluorescently labeled anti-EGFR Affibody protein (~7 kDa) would provide a more clear delineation of the tumor margin than would fluorescently labeled cetuximab, a full antibody (~150 kDa) to the epidermal growth factor receptor (EGFR). Methods: Cetuximab and anti-EGFR targeted Affibody were conjugated to two different fluorescent dyes (both emitting in the near-infrared) and injected intravenously into 6 athymic mice which were inoculated orthotopically with green fluorescent protein (GFP) expressing human U251 glioma cells. Each mouse was sacrificed at 1-h post injection, at which time brains were removed, snap frozen, sectioned and quantitatively analyzed for fluorescence distribution. Results: Ex vivo analysis showed on average, nearly equal concentrations of cetuximab and Affibody within the tumor (on average Affibody made up 49 ± 6% of injected protein), however, the cetuximab was more confined to the center of the tumor with Affibody showing significantly higher concentrations at the tumor periphery (on average Affibody made up 72 ± 15% of injected protein in the outer 50 um of the tumor). Further ex vivo analysis of detection studies showed that the Affibody provided superior discrimination for differentiation of tumor from surrounding normal brain. Conclusions: The present study indicates that fluorescently labeled anti-EGFR Affibody can provide significantly better delineation of tumor margins than a fluorescently labeled anti-EGFR antibody and shows considerable potential for guiding margin detection during neurosurgery

Signal Intensity Analysis and Optimization for in Vivo Imaging of Cherenkov and Excited Luminescence.

During external beam radiotherapy (EBRT), in vivo Cherenkov optical emissions can be used as a dosimetry tool or to excite luminescence, termed Cherenkov-excited luminescence (CEL) with microsecond-level time-gated cameras. The goal of this work was to develop a complete theoretical foundation for the detectable signal strength, in order to provide guidance on optimization of the limits of detection and how to optimize near real time imaging. The key parameters affecting photon production, propagation and detection were considered and experimental validation with both tissue phantoms and a murine model are shown. Both the theoretical analysis and experimental data indicate that the detection level is near a single photon-per-pixel for the detection geometry and frame rates commonly used, with the strongest factor being the signal decrease with the square of distance from tissue to camera. Experimental data demonstrates how the SNR improves with increasing integration time, but only up to the point where the dominance of camera read noise is overcome by stray photon noise that cannot be suppressed. For the current camera in a fixed geometry, the signal to background ratio limits the detection of light signals, and the observed in vivo Cherenkov emission is on the order of 100×  stronger than CEL signals. As a result, imaging signals from depths  \u3c 15 mm is reasonable for Cherenkov light, and depths  \u3c 3 mm is reasonable for CEL imaging. The current investigation modeled Cherenkov and CEL imaging of two oxygen sensing phosphorescent compounds, but the modularity of the code allows for easy comparison of different agents or alternative cameras, geometries or tissues

Contrast Enhanced-Magnetic Resonance Imaging as a Surrogate to Map Verteporfin Delivery in Photodynamic Therapy

The use of in vivo contrast-enhanced magnetic resonance (MR) imaging as a surrogate for photosensitizer (verteporfin) dosimetry in photodynamic therapy of pancreas cancer is demonstrated by correlating MR contrast uptake to ex vivo fluorescence images on excised tissue. An orthotopic pancreatic xenograft mouse model was used for the study. A strong correlation ([i]r=0.57 ) was found for bulk intensity measurements of T1-weighted gadolinium enhancement and verteporfin fluorescence in the tumor region of interest. The use of contrast-enhanced MR imaging shows promise as a method for treatment planning and photosensitizer dosimetry in human photodynamic therapy (PDT) of pancreas cancer