Noninvasive Measurement of Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence Allowing Detection of Murine Glioma In Vivo

Aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence is studied as a contrast agent for noninvasive detection of murine glioma, using the fluorescence-to-transmission ratio measured through the cranium. Signals measured prior to administration of ALA are very similar between control animals, 9L-GFP, and U251 tumor-bearing animals. However, 2 h after ALA administration, the PpIX signal from both tumor-bearing groups is significantly higher than the control group (9L-GFP group p-value=0.016, and U251 group p-value=0.004, relative to the control group). The variance in signal from the 9L-GFP group is much larger than either the control group or the U251 group, which is consistent with higher intrinsic PpIX fluorescence heterogeneity as seen in situ at ex vivo analysis. Decreasing the skin PpIX fluorescence via intentional photobleaching using red light (635 nm) is examined as a tool for increasing PpIX contrast between the tumor-bearing and control groups. The red light bleaching is found to increase the ability to accurately quantify PpIX fluorescence in vivo, but decreases the specificity of detection between tumor-bearing and nontumor-bearing groups

Quantitative Imaging of Scattering Changes Associated with Epithelial Proliferation, Necrosis and Fibrosis in Tumors Using Microsampling Reflectance Spectroscopy

Highly localized reflectance measurements can be used to directly quantify scatter changes in tissues. We present a microsampling approach that is used to raster scan tumors to extract parameters believed to be related to the tissue ultrastructure. A confocal reflectance imager was developed to examine scatter changes across pathologically distinct regions within tumor tissues. Tissue sections from two murine tumors, AsPC-1 pancreas tumor and the Mat-LyLu Dunning prostate tumor, were imaged. After imaging, histopathology-guided region-of-interest studies of the images allowed analysis of the variations in scattering resulting from differences in tissue ultra-structure. On average, the median scatter power of tumor cells with high proliferation index (HPI) was about 26% less compared to tumor cells with low proliferation index (LPI). Necrosis exhibited the lowest scatter power signature across all the tissue types considered, with about 55% lower median scatter power than LPI tumor cells. Additionally, the level and maturity of the tumor\u27s fibroplastic response was found to influence the scatter signal. This approach to scatter visualization of tissue ultrastructure in situ could provide a unique tool for guiding surgical resection, but this kind of interpretation into what the signal means relative to the pathology is required before proceeding to clinical studies

Imaging Targeted-Agent Binding In Vivo with Two Probes

An approach to quantitatively image targeted-agent binding rate in vivo is demonstrated with dual-probe injection of both targeted and nontargeted fluorescent dyes. Images of a binding rate constant are created that reveal lower than expected uptake of epidermal growth factor in an orthotopic xenograft pancreas tumor (2.3×10−5 s−1), as compared to the normal pancreas (3.4×10−5 s−1). This approach allows noninvasive assessment of tumor receptor targeting in vivo to determine the expected contrast, spatial localization, and efficacy in therapeutic agent delivery. Targeting therapeutic drugs to tumors based on their overexpression of cellular receptors is widely researched and has important clinical success.1, 2 Yet there are essentially no good tools to assess the in vivo receptor expression contrast between tumor as compared to normal surrounding tissue.3, 4 In tumors with very high molecular signaling such as in the pancreas,4, 5 it is not obvious when a particular receptor is actually up-regulated as compared to the surrounding normal tissue versus upregulated without biopsy. Imaging of receptor status in vivo is problematic, because the majority of any targeted agent in vivo is often not cell-associated yet. Thus, any single image simply provides a measure of the whole tissue concentration rather than the bound concentration. Delivery from the vascular supply to tumor cells requires transvascular leakage, followed by diffusion through the interstitial space, and binding to the targeted receptor followed by possible internalization.6 As such, imaging concentration values in vivo usually do not provide information about binding,7 since most of the agent is in the interstitial space. In this work, we demonstrate a new methodology for quantitative imaging of effective binding rate in vivo, using the difference in fluorescence signal between a targeted and untargeted agent. We use this to demonstrate that a tumor known to have high EGFR expression in vitro 5 actually has lower EGF activity than the surrounding normal pancreas in vivo. Most contrast agent imaging has been interpreted with a simple pharmacokinetic model that is designed with as few compartments and rate constants as possible to not overinterpret the data. A three compartment model [Fig. 1 ] can be used effectively to model targeted agent delivery in the tumor, which includes compartments for 1. the concentration of drug in the plasma within the vasculature, 2. the concentration in the interstitial space of the tissue, and 3. the cellular-associated fraction of drug.7 The dominant fast rates in this model are transvascular delivery of contrast agent through rate constantK12 role= presentation \u3eK12 , and then cell-associating rate constant due to binding and uptake, K23 role= presentation \u3eK23 . The dominant clearance from the plasma is given by excretion mechanisms, such as those in the liver and kidneys, through rate constant Ke role= presentation \u3eKe . Then the slowest rates tend to be those involved in backflow from the interstitial space to the vasculature K21 role= presentation \u3eK21 , and from the cell-associated space to the interstitial space K32 role= presentation \u3eK32 . Each of these is shown in the illustration of the model in Fig. 1

Protoporphyrin IX Fluorescence Contrast in Invasive Glioblastomas is Linearly Correlated with Gd Enhanced Magnetic Resonance Image Contrast but has Higher Diagnostic Accuracy

The sensitivity and specificity of in vivo magnetic resonance (MR) imaging is compared with production of protoporphyrin IX (PpIX), determined ex vivo, in a diffusely infiltrating glioma. A human glioma transfected with green fluorescent protein, displaying diffuse, infiltrative growth, was implanted intracranially in athymic nude mice. Image contrast from corresponding regions of interest (ROIs) in in vivo MR and ex vivo fluorescence images was quantified. It was found that all tumor groups had statistically significant PpIX fluorescence contrast and that PpIX contrast demonstrated the best predictive power for tumor presence. Contrast from gadolinium enhanced T1-weighted (T1W+Gd) and absolute T2 images positively predicted the presence of a tumor, confirmed by the GFP positive (GFP+) and hematoxylin and eosin positive (H&E+) ROIs. However, only the absolute T2 images had predictive power from controls in ROIs that were GFP+ but H&E negative. Additionally, PpIX fluorescence and T1W+Gd image contrast were linearly correlated in both the GFP+ (r = 0.79, p\u3c1×10−8) and H&E+ (r = 0.74, p\u3c0.003) ROIs. The trace diffusion images did not have predictive power or significance from controls. This study indicates that gadolinium contrast enhanced MR images can predict the presence of diffuse tumors, but PpIX fluorescence is a better predictor regardless of tumor vascularity

Imaging of Glioma Tumor with Endogenous Fluorescence Tomography

Tomographic imaging of a glioma tumor with endogenous fluorescence is demonstrated using a noncontact single-photon counting fan-beam acquisition system interfaced with microCT imaging. The fluorescence from protoporphyrin IX (PpIX) was found to be detectable, and allowed imaging of the tumor from within the cranium, even though the tumor presence was not visible in the microCT image. The combination of single-photon counting detection and normalized fluorescence to transmission detection at each channel allowed robust imaging of the signal. This demonstrated use of endogenous fluorescence stimulation from aminolevulinic acid (ALA) and provides the first in vivo demonstration of deep tissue tomographic imaging with protoporphyrin IX. Fluorescence tomography provides a tool for preclinical molecular contrast agent assessment in oncology.1, 2, 3, 4 Systems have advanced in complexity to where noncontact imaging,5 automated boundary recovery,6 and sophisticated internal tissue shapes can be included in the recovered images. The translation of this work to humans will require molecular contrast agents that are amenable to regulatory approval and maintain tumor specificity in humans, where often nonspecific uptake of molecular imaging agents can decrease their utility. In this study, a new fluorescence tomography system coupled to microCT7 was used to illustrate diagnostic detection of orthotopic glioma tumors that were not apparent in the microCT images, using endogenous fluorescent contrast from protoporphyrin IX (PpIX). Glioma tumors provide significant endogenous fluorescence from PpIX,8, 9, 10, 11 and this is enhanced when the subject imaged has been administered aminolevulinic acid (ALA). The endogenous production process of PpIX is known to stem from the administered, ALA bypassing the regulatory inhibition of ALA synthase, allowing the heme synthesis pathway to proceed uninhibited. Since there is a limited supply of iron in the body, this process produces overabundance of PpIX rather than heme, and many tumors have been shown to have high yields of PpIX. Clinical trials with PpIX fluorescence guided resection of tumors have shown significant promise,12 and yet deep tissue imaging with PpIX fluorescence has not been exploited in clinical use. Early studies have shown that detection of these tumors with PpIX is feasible,13, 14 but no tomographic imaging has been used. This limitation in development has largely been caused by problems in wavelength filtering and low signal intensity, as well as background fluorescence from the skin limiting sensitivity to deeper structures. In the system developed and used here, this feasibility is demonstrated by imaging a human xenograft glioma model. To solve the sensitivity problem and study the ability to diagnostically image PpIX in vivo, time-correlated single-photon counting was used in the fluorescence tomography system, which provides maximum sensitivity. Figure 1a shows the system designed to match up with a microCT, allowing both x-ray structural and optical functional imaging sequentially. Lens-coupled detection of signals is acquired from the mouse using five time-resolved photomultiplier tubes (H7422P-50, Hamamatsu, Japan) with single-photon counting electronics (SPC-134 modules, Becker and Hickl GmbH, Germany). The system has fan-beam transmission geometry similar to a standard CT scanner, with single source delivery of a1-mW role= presentation \u3e1-mW pulsed diode laser light at 635nm role= presentation \u3e635nm , collimated to a 1-mm role= presentation \u3e1-mm effective area on the animal. The five detection lenses were arranged in an arc, each with 22.5-deg role= presentation \u3e22.5-deg angular separation, centered directly on the opposite side of the animal with long working distance pickup,7 allowing noncontact measurement of the diffuse light through the animal. The diffuse intensity signals collected at each of the five channels were then translated via 400-μm role= presentation \u3e400-μm fibers and split using beamsplitters to be directed toward the fluorescence (95%) and transmission (5%) channel detectors. A 650-nm role= presentation \u3e650-nm long-pass filter was used in the fluorescence channels to isolate the signal, and in the transmitted intensity signals, a neutral density filter (2 OD) was used to attenuate the signals. This latter filtering was necessary to ensure that the fluorescence and transmission. Intensity signals fell within the same dynamic range, allowing a single 1s role= presentation \u3e1s acquisition for each detector. Scans were then performed by rotating the fan-beam around the specimen to 32 locations. A GE eXplore Locus SP scanner (GE Healthcare, London, Ontario, Canada) that incorporated a detector with 94-micronpixel role= presentation \u3e94-micronpixel resolution, a 80-kV role= presentation \u3e80-kV peak voltage, and a tube current of 450μAs role= presentation \u3e450μAs , was used in acquiring the microCT data, as displayed in Fig. 2 . In this example, since soft tissue was being imaged, the CT data was largely used to image the exterior of the animal, although in future studies, it could be used to isolate the cranium region as well

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives