RESUMEN
Multiphoton microscopy is a powerful technique based on complex quantum mechanical effects. Thanks to the development of turnkey mode-locked laser systems, multiphoton microscopy is now available for everyone to use without extreme complexity. In this short introduction, we describe qualitatively the important concepts underlying the most commonly used type of multiphoton microscopy (two-photon excitation). We elucidate how those properties lead to the powerful results that have been achieved using this technique. As with any technique, two-photon excitation microscopy has limitations that we describe, and we provide examples of particular classes of experiments where two-photon excitation microscopy is advantageous over other approaches. Finally, we briefly describe other useful multiphoton microscopy approaches, such as three-photon excitation and second harmonic generation imaging.
Asunto(s)
Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Investigación Biomédica/métodos , Corteza Cerebral/citología , Humanos , RatonesRESUMEN
AIMS/HYPOTHESIS: Mutations that render ATP-sensitive potassium (K(ATP)) channels insensitive to ATP inhibition cause neonatal diabetes mellitus. In mice, these mutations cause insulin secretion to be lost initially and, as the disease progresses, beta cell mass and insulin content also disappear. We investigated whether defects in calcium signalling alone are sufficient to explain short-term and long-term islet dysfunction. METHODS: We examined the metabolic, electrical and insulin secretion response in islets from mice that become diabetic after induction of ATP-insensitive Kir6.2 expression. To separate direct effects of K(ATP) overactivity on beta cell function from indirect effects of prolonged hyperglycaemia, normal glycaemia was maintained by protective exogenous islet transplantation. RESULTS: In endogenous islets from protected animals, glucose-dependent elevations of intracellular free-calcium activity ([Ca(2+)](i)) were severely blunted. Insulin content of these islets was normal, and sulfonylureas and KCl stimulated increased [Ca(2+)](i). In the absence of transplant protection, [Ca(2+)](i) responses were similar, but glucose metabolism and redox state were dramatically altered; sulfonylurea- and KCl-stimulated insulin secretion was also lost, because of systemic effects induced by long-term hyperglycaemia and/or hypoinsulinaemia. In both cases, [Ca(2+)](i) dynamics were synchronous across the islet. After reduction of gap-junction coupling, glucose-dependent [Ca(2+)](i) and insulin secretion was partially restored, indicating that excitability of weakly expressing cells is suppressed by cells expressing mutants, via gap-junctions. CONCLUSIONS/INTERPRETATION: The primary defect in K(ATP)-induced neonatal diabetes mellitus is failure of glucose metabolism to elevate [Ca(2+)](i), which suppresses insulin secretion and mildly alters islet glucose metabolism. Loss of insulin content and mitochondrial dysfunction are secondary to the long-term hyperglycaemia and/or hypoinsulinaemia that result from the absence of glucose-dependent insulin secretion.
Asunto(s)
Señalización del Calcio/fisiología , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Animales , Animales Recién Nacidos , Calcio/metabolismo , Señalización del Calcio/genética , Diabetes Mellitus/etiología , Secreción de Insulina , Canales KATP/genética , Ratones , Ratones Transgénicos , Canales de Potasio de Rectificación Interna/genéticaRESUMEN
In this work, we report a method to observe soft X-ray radiographs at nanoscale of various kind of samples, biological and metallic, stored in a thin layer of lithium fluoride, employing scanning near-field optical microscopy with an optical resolution that reaches 50 nm. Lithium fluoride material works as a novel image detector for X-ray nano-radiographs, due to the fact that extreme ultraviolet radiation and soft X-rays efficiently produce stable point defects emitting optically stimulated visible luminescence in a thin surface layer. The bi-dimensional distribution of the so-created defects depends on the local nanostructure of the investigated sample.