Use and Future Prospects of in Vivo Microdialysis for Epilepsy Studies.

Epilepsy is a common neurological disease characterized by recurrent unpredictable seizures. For the last 30 years, microdialysis sampling has been used to measure changes in excitatory and inhibitory neurotransmitter concentrations before, during, and after seizures. These advances have fostered breakthroughs in epilepsy research by identifying neurochemical changes associated with seizures and correlating them to electrophysiological data. Recent advances in methodology may be useful in further delineating the chemical underpinnings of seizures. A new model of ictogenesis has been developed that allows greater control over the timing of seizures that are similar to spontaneous seizures. This model will facilitate making chemical measurements before and during a seizure. Recent advancements in microdialysis sampling, including the use of segmented flow, "fast" liquid chromatography (LC), and capillary electrophoresis with laser-induced fluorescence (CE-LIF) have significantly improved temporal resolution to better than 1 min, which could be used to measure transient, spontaneous neurochemical changes associated with seizures. Microfabricated sampling probes that are markedly smaller than conventional probes and allow for a much greater spatial resolution have been developed. They may allow the targeting of specific brain regions important to epilepsy studies. Coupling microdialysis sampling to optogenetics and light-stimulated release of neurotransmitters may also prove useful for studying epileptic seizures.

Potentials and pitfalls of inverse fluorescence correlation spectroscopy.

Inverse Fluorescence Correlation Spectroscopy (iFCS) is a variant of FCS where unlabeled particles in solution, or domains in membranes, displace their surrounding, signal-generating molecules and thereby generate fluctuations. iFCS has to date been applied to unlabeled as well as labeled particles and protein molecules, using fluorescence as well as Raman scattering as a signal source, in diffraction-limited detection volumes as well as in nano-wells, and on fixed surfaces as well as in lipid bilayers. This review describes these applications and discusses the potentials and pitfalls when using iFCS.

Measuring embryo metabolism to predict embryo quality.

Measuring the metabolism of early embryos has the potential to be used as a prospective marker for post-transfer development, either alone or in conjunction with other embryo quality assessment tools. This is necessary to maximise the opportunity of couples to have a healthy child from assisted reproduction technology (ART) and for livestock breeders to efficiently improve the genetics of their animals. Nevertheless, although many promising candidate substrates (e.g. glucose uptake) and methods (e.g. metabolomics using different spectroscopic techniques) have been promoted as viability markers, none has yet been widely used clinically or in livestock production. Herein we review the major techniques that have been reported; these are divided into indirect techniques, where measurements are made from the embryo's immediate microenvironment, or direct techniques that measure intracellular metabolic activity. Both have strengths and weaknesses, the latter ruling out some from contention for use in human ART, but not necessarily for use in livestock embryo assessment. We also introduce a new method, namely multi- (or hyper-) spectral analysis, which measures naturally occurring autofluorescence. Several metabolically important molecules have fluorescent properties, which we are pursuing in conjunction with improved image analysis as a viable embryo quality assessment methodology.

Fluorescence correlation spectroscopy: principles and applications.

Fluorescence correlation spectroscopy (FCS) is used to study the movements and the interactions of biomolecules at extremely dilute concentrations, yielding results with good spatial and temporal resolutions. Using a number of technical developments, FCS has become a versatile technique that can be used to study a variety of sample types and can be advantageously combined with other methods. Unlike other fluorescence-based techniques, the analysis of FCS data is not based on the average intensity of the fluorescence emission but examines the minute intensity fluctuations caused by spontaneous deviations from the mean at thermal equilibrium. These fluctuations can result from variations in local concentrations owing to molecular mobility or from characteristic intermolecular or intramolecular reactions of fluorescently labeled biomolecules present at low concentrations. Here, we provide a basic introduction to FCS, including its technical development and theoretical basis, experimental setup of an FCS system, adjustment of a setup, data acquisition, and analysis of FCS measurements. Finally, the application of FCS to the study of lipid bilayer membranes and to living cells is discussed.

Added value of the hybrid tracer indocyanine green- 99mTc-nanocolloid for sentinel node biopsy in a series of patients with different lymphatic drainage patterns

Introducción. El verde de indocianina (ICG)-99mTc-nanocoloide es un novedoso trazador híbrido radioactivo y fluorescente para la biopsia selectiva del ganglio centinela (BSGC). Nuestro objetivo fue demostrar el valor añadido de este trazador en una serie de pacientes con diferentes neoplasias. Material y métodos. Se incluyeron consecutivamente 20 pacientes (con carcinoma de pene, cavidad oral y melanoma) entre marzo y mayo de 2012. A todos se les realizó una gammagrafía planar a los 15 y 120 min tras la inyección de ICG-99mTc-nanocoloide y posteriormente un SPECT/TAC. En quirófano se inyectó 1 ml de colorante vital (blue dye) a 14 pacientes. Durante la cirugía, los ganglios centinelas (GC) fueron localizados utilizando la sonda gammadetectora y visualizados por fluorescencia y colorante vital. Finalmente, se confirmó la extirpación completa de los GC con la gammacámara portátil. Resultados. Mediante una SPECT/TAC se identificó al menos un GC por paciente. Todos los GC (total: 68, 100%) fueron extirpados utilizando la combinación de guía radiofluorescente: 89,7% se localizaron con la sonda gammadetectora. Los ganglios restantes, situados cerca del punto de inyección, fueron ubicados por fluorescencia. Durante la cirugía, un 97% del total de GC fueron fluorescentes y solo el 39,2% azules. ex-vivo, todos los GC fueron radioactivos y fluorescentes. En 5 pacientes el GC fue metastásico. Conclusión. Aplicar una guía radiofluorescente para la BSGC es posible utilizando el ICG-99mTc-nanocoloide. Este enfoque híbrido combina los beneficios de ambas modalidades. La imagen fluorescente mejora la detección visual del GC respecto al colorante vital y demuestra ser especialmente útil cuando el GC está cerca del punto de inyección(AU)

Introduction. Indocyanine green (ICG)-99mTc-nanocolloid is a novel hybrid fluorescent radioactive tracer for sentinel node (SN) biopsy. This study has aimed to evaluate the added value of this novel versatile tracer in a series of patients with different malignancies. Material and methods. Twenty patients (with penile carcinoma, oral cavity tumors, melanoma) were consecutively included between March–May 2012. Planar lymphoscintigraphy was performed 15 min and 2 h after injection of ICG-99mTc-nanocolloid followed by SPECT/CT. Blue dye (1 ml) was injected in 14 patients in surgery room. Intraoperatively, SNs were localized using a gamma probe and visualized by optical SN-detection using blue dye and fluorescence imaging. Finally, a portable gamma camera was used to confirm complete SN removal. Results. At least one SN was identified by SPECT/CT in all patients. All SNs (total 68, 100%) were excised using a combination of radio- and fluorescence guidance: 89.7% were intraoperatively localized with the gamma probe. The remaining SNs, located near the injection site, were localized using fluorescence imaging. During the surgery, 97% of the SNs were fluorescent while only 39.2% were stained blue. Ex vivo, all SNs were both radioactive and fluorescent. The SN was positive in 5 patients. Conclusion. Synchronous radio- and fluorescence guided SN biopsy is feasible using ICG-99mTc-nanocolloid. This hybrid approach combines the beneficial properties of both modalities. Adding fluorescence imaging improves optical SN detection compared to blue dye. It has been shown to be especially useful in the localization of SNs near the injection site(AU)

Autofluorescencia retiniana: aplicaciones y perspectivas / Fundus autofluorescence: applications and perspectives

Objetivos: Describir los hallazgos en el estudio de la autofluorescencia de las distintas enfermedades retinianas incluidas en el estudio. Estudiar en qué enfermedades la autofluorescencia puede ser igual o superior a la angiografía fluoresceínica (AGF) en cuanto a información diagnóstica. Material y métodos: Se estudió la autofluorescencia retiniana de 123 ojos y pacientes, incluyendo diversas enfermedades del fondo de ojo. En todos los casos se exploró el fondo de ojo, la autofluorescencia retiniana, y, en caso de estar indicado, se realizó AGF. La exploración de la autofluorescencia fue llevada a cabo usando el angiógrafo Heidelberg Retina Angiograph 2 (HRA2) de Heidelberg Engeneering (Alemania). Resultados: La autofluorescencia aportó una información igual o mejor (que la AGF) en: 68,18% de casos de edema macular; 50% de desprendimientos del epitelio pigmenario; 100% de atrofias del epitelio pigmentario; 100% de coriorretinopatías centrales serosas; 55,55% de neovascularizaciones coroideas; 100% de las distrofias retinianas con depósito de lipofucsina; 100% de los exudados duros y hemorragias prerretinianas. Conclusiones: La autofluorescencia es un método de exploración rápido, cómodo para el paciente y explorador, no invasivo y con una curva de aprendizaje muy corta, que aporta información para el diagnóstico de múltiples enfermedades con afectación del fondo de ojo. Si bien son precisos más estudios y más experiencia con su uso, su interés radica en la posibilidad de evitar la realización de angiografías en pacientes con estas enfermedades y en la información adicional que nos aporta sobre la situación funcional de las células y pigmentos retinianos(AU)

Purpose: To describe the findings of the study of autofluorescence of the different retinal diseases included in the study. To determine in which diseases autofluorescence may be more, or just as, useful as fluorescein angiography (FAG) in terms of diagnostic information. Material and methods: We studied the retinal autofluorescence of 123 eyes of 93 patients, including various diseases of the eye fundus. In all cases we explored the fundus, retinal autofluorescence, and, if indicated, FAG was performed. Analysis of the autofluorescence was performed using the Heidelberg Retina angiography Angiograph 2 (HRA2) Heidelberg Engineering (Germany) .Results: The autofluorescence information provided was equal or better (than FAG) in: 68.18% of cases of macular edema, 50% of pigment epithelium detachments, 100% of pigment epithelium atrophies, 100% of central serous chorioretinopathy; 55.55% of choroidal neovascularization, 100% of retinal dystrophies with deposition of lipofuscin, 100% of hard exudates and pre-retinal hemorrhages. Conclusions: Autofluorescence is a quick and non-invasive examination method, comfortable for both patient and examiner, and with a very short learning curve. It provides diagnostic information about many eye fundus diseases. While more studies and more experience with its use are needed, its interest lies in the possibility of avoiding the performing of angiography in patients with these diseases, and in the additional information autofluorescence provides about the functional situation of cells and retinal pigments(AU)

Optical methods for single molecule detection and analysis.

As analytical chemists, the highest resolution measurement one can make is at the single molecule level; it just does not get any better than that. To determine the concentration of a molecule in solution, the best way is to count the number of molecules in a given volume. As long as the volume contains a statistically large enough number of molecules and is above the Poisson noise limit, molecular counting is the most accurate way to make a measurement. Molecular counting is the method of the future and is beginning to be performed today.

Single-molecule nanometry for biological physics.

Precision measurement is a hallmark of physics but the small length scale (∼nanometer) of elementary biological components and thermal fluctuations surrounding them challenge our ability to visualize their action. Here, we highlight the recent developments in single-molecule nanometry where the position of a single fluorescent molecule can be determined with nanometer precision, reaching the limit imposed by the shot noise, and the relative motion between two molecules can be determined with ∼0.3 nm precision at ∼1 ms time resolution, as well as how these new tools are providing fundamental insights into how motor proteins move on cellular highways. We will also discuss how interactions between three and four fluorescent molecules can be used to measure three and six coordinates, respectively, allowing us to correlate the movements of multiple components. Finally, we will discuss recent progress in combining angstrom-precision optical tweezers with single-molecule fluorescent detection, opening new windows for multi-dimensional single-molecule nanometry for biological physics.

Análisis de la casuística del Servicio de Atención Toxicológica Veterinaria (SATVe) en el período 2001-2007 / Analysis of the casuistry of the Service of Veterinary Toxicology (SATVe) in the period 2001-2007

El Servicio de Atención Toxicológica Veterinaria (SATVe), localizado en la Facultad de Veterinaria de Lugo (USC), tiene por función resolver problemas relacionados con la toxicología clínica veterinaria remitidos por los profesionales a través de consultas mediante técnicas telemáticas y analíticas. En el presente trabajo se analiza la casuística toxicológica del servicio durante el período 2001-2007 en relación con diversos aspectos: origen geográfico de la demanda, tipo de solicitud efectuada, usuarios solicitantes, especies animales afectadas y tóxicos implicados. Respecto a la casuística, aumentó considerablemente los primeros años, observándose posteriormente una tendencia al mantenimiento de unos 100 casos anuales, siendo esta mayoritariamente procedente de Galicia (92,2%), según orden decreciente: Lugo > Pontevedra > A Coruña > Ourense. La mayoría de las consultas se originaron por profesionales veterinarios (71,6%) frente a la Administración (13,2%) y los particulares (12,3%). Los casos que más se remitieron, procedían de la especie canina (48,7%) seguido de la equina (12,3%), ocupando los félidos la tercera posición (8,4%). Respecto a las analíticas, los tóxicos mayoritariamente detectados (48%) fueron estricnina, metiocarb y fenobarbital (AU)

The function of the Veterinary Toxicology Attention Service (SATVe), located at the Veterinary School of Lugo (USC), is to solve questions of veterinary clinical and analytical toxicology submitted by health professionals through telematics consultations and to provides analytical techniques. This work analyzes the toxicological casuistry of the period 2001-2007 on different aspects: geographical origin of consultations, type of request, requesting users, animal species affected and toxic compounds involved. Regarding the casuistry, significantly increased the first years, later observed an evolution to the maintenance of about 100 cases a year, this being mainly from Galicia (92.2%), according to the decreasing order: Lugo > Pontevedra > A Coruña > Ourense. Most queries were originated from veterinary practitioners (71.6%) compared to government agencies (13.2%) and private users (12.3%). Most of the cases referreed, came from canines (48.7%), followed by equines (12.3%), occupying the third place felines (8.4%). Regarding toxicological analysis, the main toxic compounds detected (48%) were strychnine, methiocarb and phenobarbital (AU)

Fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure concentrations, mobilities, and interactions of fluorescent biomolecules. It can be applied to various biological systems such as simple homogeneous solutions, cells, artificial, or cellular membranes and whole organisms. Here, we introduce the basic principle of FCS, discuss its application to biological questions as well as its limitations and challenges, present an overview of novel technical developments to overcome those challenges, and conclude with speculations about the future applications of fluorescence fluctuation spectroscopy.

The design and application of fluorophore-gold nanoparticle activatable probes.

Fluorescence-based assays and detection techniques are among the most highly sensitive and popular biological tests for researchers. To match the needs of research and the clinic, detection limits and specificities need to improve, however. One mechanism is to decrease non-specific background signals, which is most efficiently done by increasing fluorescence quenching abilities. Reports in the literature of theoretical and experimental work have shown that metallic gold surfaces and nanoparticles are ultra-efficient fluorescence quenchers. Based on these findings, subsequent reports have described gold nanoparticle fluorescence-based activatable probes that were designed to increase fluorescence intensity based on a range of stimuli. In this way, these probes can detect and signify assorted biomarkers and changes in environmental conditions. In this review, we explore the various factors and theoretical models that affect gold nanoparticle fluorescence quenching, explore current uses of activatable probes, and propose an engineering approach for future development of fluorescence based gold nanoparticle activatable probes.

Single-walled carbon nanotubes as optical materials for biosensing.

In this review, we summarize recent progress in the development of single-walled carbon nanotubes (SWNTs) as optical materials for biosensing applications. First, as optical labels, we discuss the use of SWNTs in Raman-based protein detection. Strong and simple resonance Raman spectroscopy of SWNTs opens up a method of protein microarray with detection sensitivity down to femtomolar range. Also, tunable isotopic SWNT-Raman signature enables the simultaneous detection of multiple analytes in complex fluids. Second, the photoluminescence properties of SWNTs are also explored. We examine fluorescence biosensors that integrate the quenching property of SWNTs and the recognition property of functional nucleic acids. Particularly, SWNTs are established as an efficient signal transduction substrate in different biosensing systems, including the detection of specific proteins and DNA sequences, regulation of singlet oxygen generation and label-free fluorescence assays, and all have exhibited very high selectivity and sensitivity.

New developments in fluorescence diagnostics.

In the last decade, significant advances have been achieved in the direct viewing of the skin. Non-invasive analysis of various skin diseases in vivo has become possible by special skin display devices, allowing the physician to view the structure and properties of the skin in greater detail than can be achieved by simple visual examination. We review the last 100 years of fluorescence imaging development from clinical observation to advanced spectral imaging, addressing the role of fluorescence diagnostics (FD) in modern dermatology as well as the detection of autofluorescence.

Fluorescence correlation spectroscopy: past, present, future.

In recent years fluorescence correlation spectroscopy (FCS) has become a routine method for determining diffusion coefficients, chemical rate constants, molecular concentrations, fluorescence brightness, triplet state lifetimes, and other molecular parameters. FCS measures the spatial and temporal correlation of individual molecules with themselves and so provides a bridge between classical ensemble and contemporary single-molecule measurements. It also provides information on concentration and molecular number fluctuations for nonlinear reaction systems that complement single-molecule measurements. Typically implemented on a fluorescence microscope, FCS samples femtoliter volumes and so is especially useful for characterizing small dynamic systems such as biological cells. In addition to its practical utility, however, FCS provides a window on mesoscopic systems in which fluctuations from steady states not only provide the basis for the measurement but also can have important consequences for the behavior and evolution of the system. For example, a new and potentially interesting field for FCS studies could be the study of nonequilibrium steady states, especially in living cells.

Analysis of biological samples by capillary electrophoresis with laser induced fluorescence detection.

In this paper an overview is provided on practical difficulties as well as applications of capillary electrophoresis coupled to laser induced fluorescence detection methods in the field of analysis of biological samples. Various methodological approaches elaborated for determination of small molecules, peptides and proteins are outlined. Besides giving an overview on detection based on native fluorescence, immune and enzyme assays, the main focus is the problematics of sample derivatization and achievable detection sensitivities in the analysis of real biological samples. The characteristics and applicability of the most commonly used labeling reagents are discussed in details.

Investigating biological processes at the single molecule level using luminescent quantum dots.

In this report we summarize the progress made in the past several years on the use of luminescent QDs to probe biological processes at the single molecule level. We start by providing a quick overview of the basic properties of semiconductor nanocrystals, including synthetic routes, surface-functionalization strategies, along with the main attributes of QDs that are of direct relevance to single molecule studies based on fluorescence detection. We then detail some valuable insights into specific biological processes gained using single QDs. These include progress made in probing biomolecular interactions, tracking of protein receptors both in vitro and in live cells, and single particle resonance energy transfer. We will also discuss the advantages offered and limitations encountered by single QD fluorescence as an investigative tool in biology.

Fluorescence as a tool for advanced diagnostics.

Fluorescence is a versatile technique with applications ranging from ultra-low-cost tests that are available over the counter via point of care instruments for use in the doctor's office to state-of-the-art clinical analysers. This article describes fluorescence, draws on a number of examples to illustrate its versatility and looks at what the future has in store for this powerful technique.

Interrogating single molecules.

The development of nanotechnology relies heavily on the ability to observe and probe at molecular scales. Thus, breakthroughs in instrumentation and techniques play a significant role in the advancement in nanoscience. In this article, we survey recent developments in observing, trapping, and probing single molecules in solution. We give particular attention to patents that cover enabling instrumentation in each of these related areas including, respectively, fluorescence imaging, optical tweezers, and anti-Brownian traps. We conclude by highlighting a broad trend in the literature and patent base from observation toward active interrogation of single molecules in solution.