Enhanced-reality video fluorescence: a real-time assessment of intestinal viability.

OBJECTIVE: Our aim was to evaluate a fluorescence-based enhanced-reality system to assess intestinal viability in a laparoscopic mesenteric ischemia model. MATERIALS AND METHODS: A small bowel loop was exposed, and 3 to 4 mesenteric vessels were clipped in 6 pigs. Indocyanine green (ICG) was administered intravenously 15 minutes later. The bowel was illuminated with an incoherent light source laparoscope (D-light-P, KarlStorz). The ICG fluorescence signal was analyzed with Ad Hoc imaging software (VR-RENDER), which provides a digital perfusion cartography that was superimposed to the intraoperative laparoscopic image [augmented reality (AR) synthesis]. Five regions of interest (ROIs) were marked under AR guidance (1, 2a-2b, 3a-3b corresponding to the ischemic, marginal, and vascularized zones, respectively). One hour later, capillary blood samples were obtained by puncturing the bowel serosa at the identified ROIs and lactates were measured using the EDGE analyzer. A surgical biopsy of each intestinal ROI was sent for mitochondrial respiratory rate assessment and for metabolites quantification. RESULTS: Mean capillary lactate levels were 3.98 (SD = 1.91) versus 1.05 (SD = 0.46) versus 0.74 (SD = 0.34) mmol/L at ROI 1 versus 2a-2b (P = 0.0001) versus 3a-3b (P = 0.0001), respectively. Mean maximal mitochondrial respiratory rate was 104.4 (±21.58) pmolO2/second/mg at the ROI 1 versus 191.1 ± 14.48 (2b, P = 0.03) versus 180.4 ± 16.71 (3a, P = 0.02) versus 199.2 ± 25.21 (3b, P = 0.02). Alanine, choline, ethanolamine, glucose, lactate, myoinositol, phosphocholine, sylloinositol, and valine showed statistically significant different concentrations between ischemic and nonischemic segments. CONCLUSIONS: Fluorescence-based AR may effectively detect the boundary between the ischemic and the vascularized zones in this experimental model.

Feasibility of direct mapping of cerebral fluorodeoxy-D-glucose metabolism in situ at subcellular resolution using soft X-ray fluorescence.

Glucose metabolism is difficult to image with cellular resolution in mammalian brain tissue, particularly with (18) fluorodeoxy-D-glucose (FDG) positron emission tomography (PET). To this end, we explored the potential of synchrotron-based low-energy X-ray fluorescence (LEXRF) to image the stable isotope of fluorine (F) in phosphorylated FDG (DG-6P) at 1 μm(2) spatial resolution in 3-μm-thick brain slices. The excitation-dependent fluorescence F signal at 676 eV varied linearly with FDG concentration between 0.5 and 10 mM, whereas the endogenous background F signal was undetectable in brain. To validate LEXRF mapping of fluorine, FDG was administered in vitro and in vivo, and the fluorine LEXRF signal from intracellular trapped FDG-6P over selected brain areas rich in radial glia was spectrally quantitated at 1 μm(2) resolution. The subsequent generation of spatial LEXRF maps of F reproduced the expected localization and gradients of glucose metabolism in retinal Müller glia. In addition, FDG uptake was localized to periventricular hypothalamic tanycytes, whose morphological features were imaged simultaneously by X-ray absorption. We conclude that the high specificity of photon emission from F and its spatial mapping at ≤1 μm resolution demonstrates the ability to identify glucose uptake at subcellular resolution and holds remarkable potential for imaging glucose metabolism in biological tissue. © 2012 Wiley Periodicals, Inc.

Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.

Fluorescence flow cytometer to determine urine particle reference intervals in doping control samples.

Background: Urine is still the matrix of choice to fight against doping, because it can be collected non-invasively during anti-doping tests. Most of the World Anti-Doping Agency's accredited laboratories have more than 20 years experience in analyzing this biological fluid and the majority of the compounds listed in the 2010 Prohibited List - International Standard are eliminated through the urinary apparatus. Storing and transporting urine samples for doping analyses does not include a specific protocol to prevent microbial and thermal degradation. The use of a rapid and reliable screening method could enable determine reference intervals for urine specimens in doping control samples and evaluate notably the prevalence of microbial contamination known to be responsible for the degradation of chemical substances in urine.Methods: The Sysmex(R) UF-500i is a recent urine flow cytometer analyzer capable of quantifying BACT and other urinary particles such as RBC, WBC, EC, DEBRIS, CAST, PATH. CAST, YLC, SRC as well as measuring urine conductivity. To determine urine anti-doping reference intervals, 501 samples received in our laboratory over a period of two months were submitted to an immediate examination. All samples were collected and then transported at room temperature. Analysis of variance was performed to test the effects of factors such as gender, test type [in-competition, out-of-competition] and delivery time.Results: The data obtained showed that most of the urine samples were highly contaminated with bacteria. The other urine particles were also very different according to the factors.Conclusions: The Sysmex(R) UF-500i was capable of providing a snapshot of urine particles present in the samples at the time of the delivery to the laboratory. These particles, BACT in particular, gave a good idea of the possible microbial degradation which had and/or could have occurred in the sample. This information could be used as the first quality control set up in WADA (World Anti-Doping Agency) accredited laboratories to determine if steroid profiles, endogenous and prohibited substances have possibly been altered. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Direct mapping of 19F in 19FDG-6P in brain tissue at subcellular resolution using soft X-ray fluorescence

Low energy x-ray fluorescence (LEXRF) detection was optimized for imaging cerebral glucose metabolism by mapping the fluorine LEXRF signal of 19 F in 19 FDG, trapped as intracellular 19 F-deoxyglucose-6-phosphate ( 19 FDG-6P) at 1μm spatial resolution from 3μm thick brain slices. 19 FDG metabolism was evaluated in brain structures closely resembling the general cerebral cytoarchitecture following formalin fixation of brain slices and their inclusion in an epon matrix. 2-dimensional distribution maps of 19 FDG-6P were placed in a cytoarchitectural and morphological context by simultaneous LEXRF mapping of N and O, and scanning transmission x-ray (STXM) imaging. A disproportionately high uptake and metabolism of glucose was found in neuropil relative to intracellular domains of the cell body of hypothalamic neurons, showing directly that neurons, like glial cells, also metabolize glucose. As 19 F-deoxyglucose-6P is structurally identical to 18 F-deoxyglucose-6P, LEXRF of subcellular 19 F provides a link to in vivo 18 FDG PET, forming a novel basis for understanding the physiological mechanisms underlying the 18 FDG PET image, and the contribution of neurons and glia to the PET signal.

Determination of the size of quantum dots by fluorescence spectroscopy.

There has been a lack of quick, simple and reliable methods for determination of nanoparticle size. An investigation of the size of hydrophobic (CdSe) and hydrophilic (CdSe/ZnS) quantum dots was performed by using the maximum position of the corresponding fluorescence spectrum. It has been found that fluorescence spectroscopy is a simple and reliable methodology to estimate the size of both quantum dot types. For a given solution, the homogeneity of the size of quantum dots is correlated to the relationship between the fluorescence maximum position (FMP) and the quantum dot size. This methodology can be extended to the other fluorescent nanoparticles. The employment of evolving factor analysis and multivariate curve resolution-alternating least squares for decomposition of the series of quantum dots fluorescence spectra recorded by a specific measuring procedure reveals the number of quantum dot fractions having different diameters. The size of the quantum dots in a particular group is defined by the FMP of the corresponding component in the decomposed spectrum. These results show that a combination of the fluorescence and appropriate statistical method for decomposition of the emission spectra of nanoparticles may be a quick and trusted method for the screening of the inhomogeneity of their solution.