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
Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 micros were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 +/- 1 microm. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods.
Asunto(s)
Microscopía Electrónica/instrumentación , Microscopía Electrónica/métodos , Animales , Tampones (Química) , Células COS , Chlorocebus aethiops , Factor de Crecimiento Epidérmico/metabolismo , Diseño de Equipo , Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Oro/química , Procesamiento de Imagen Asistido por Computador , Nanopartículas del Metal/química , Nanotecnología , Unión Proteica , Silicio/químicaRESUMEN
Over the past decade, two-photon microscopy has successfully made the transition from the laser laboratory into a true biological research setting. This has been due in part to the recent development of turnkey ultrafast laser systems required for two-photon microscopy, allowing ease of use in nonspecialist laboratories. The advantages of two-photon microscopy over conventional optical sectioning techniques are for greater imaging depths and reduced overall phototoxicity, as such enabling noninvasive intra-vital imaging of cellular and subcellular processes. Greater understanding of these advantages has allowed this technique to be more effectively utilized in a biological research setting. This review will cover the recent widespread uses of two-photon microscopy and highlight the wide range of physiological studies enabled in fields such as neurosciences, developmental biology, immunology, cancer biology, and endocrinology.
Asunto(s)
Células/ultraestructura , Microscopía de Fluorescencia por Excitación Multifotónica , Animales , Fenómenos Fisiológicos Celulares , Humanos , Neoplasias/patologíaRESUMEN
Human HaCaT cells, exposed for 24 h to a 1 mT (rms) 50 Hz sinusoidal magnetic field in a temperature-regulated solenoid, suffer detectable changes in their biochemical properties and shapes. By using infrared wavelength-selective scanning near-field optical microscopy, we observed changes in the distribution of the inner chemical functional groups and in the cell morphology with a resolution of 80-100 nm.
Asunto(s)
Campos Electromagnéticos , Queratinocitos , Piel/citología , Línea Celular , Humanos , Rayos Infrarrojos , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Queratinocitos/ultraestructura , Microscopía de Sonda de BarridoRESUMEN
Due to its surface sensitivity and high spatial resolution, scanning near-field optical microscopy (SNOM) has a significant potential to study the lateral organization of membrane domains and clusters. Compared to other techniques, infrared near-field microscopy in the spectroscopic mode has the advantage to be sensitive to specific chemical bonds. In fact, spectroscopic SNOM in the infrared spectral range (IR-SNOM) reveals the chemical content of the sample with a lateral resolution around 100 nm (Cricenti et al., 1998a, 1998b, 2003). Model lipid membranes were studied by IR-SNOM at several wavelengths. Topographical micrographs reveal the presence of islands at the surface and the optical images indicate the formation of locally ordered multiple bilayers - both critically important features for biotechnology and medical applications.
Asunto(s)
Lípidos de la Membrana/química , Microscopía Confocal/métodos , Espectrofotometría Infrarroja/métodos , Membrana Dobles de Lípidos/química , Microscopía Confocal/instrumentación , Modelos Biológicos , Fosfatidilcolinas/química , Espectrofotometría Infrarroja/instrumentaciónRESUMEN
AIMS/HYPOTHESIS: ATP-sensitive K(+) (K(ATP)) channels couple glucose metabolism to insulin secretion in pancreatic beta cells. In humans, loss-of-function mutations of beta cell K(ATP) subunits (SUR1, encoded by the gene ABCC8, or Kir6.2, encoded by the gene KCNJ11) cause congenital hyperinsulinaemia. Mice with dominant-negative reduction of beta cell K(ATP) (Kir6.2[AAA]) exhibit hyperinsulinism, whereas mice with zero K(ATP) (Kir6.2(-/-)) show transient hyperinsulinaemia as neonates, but are glucose-intolerant as adults. Thus, we propose that partial loss of beta cell K(ATP) in vivo causes insulin hypersecretion, but complete absence may cause insulin secretory failure. MATERIALS AND METHODS: Heterozygous Kir6.2(+/-) and SUR1(+/-) animals were generated by backcrossing from knockout animals. Glucose tolerance in intact animals was determined following i.p. loading. Glucose-stimulated insulin secretion (GSIS), islet K(ATP) conductance and glucose dependence of intracellular Ca(2+) were assessed in isolated islets. RESULTS: In both of the mechanistically distinct models of reduced K(ATP) (Kir6.2(+/-) and SUR1(+/-)), K(ATP) density is reduced by approximately 60%. While both Kir6.2(-/-) and SUR1(-/-) mice are glucose-intolerant and have reduced glucose-stimulated insulin secretion, heterozygous Kir6.2(+/-) and SUR1(+/-) mice show enhanced glucose tolerance and increased GSIS, paralleled by a left-shift in glucose dependence of intracellular Ca(2+) oscillations. CONCLUSIONS/INTERPRETATION: The results confirm that incomplete loss of beta cell K(ATP) in vivo underlies a hyperinsulinaemic phenotype, whereas complete loss of K(ATP) underlies eventual secretory failure.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Hiperinsulinismo/genética , Pérdida de Heterocigocidad , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/deficiencia , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Canales de Potasio de Rectificación Interna/deficiencia , Canales de Potasio de Rectificación Interna/genética , Animales , Glucemia/metabolismo , Insulina/genética , Insulina/metabolismo , Secreción de Insulina , Cinética , Ratones , Ratones Noqueados , Canales de Potasio/genética , Receptores de Droga , Receptores de SulfonilureasRESUMEN
To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.
Asunto(s)
Glucosa/metabolismo , Músculo Esquelético/metabolismo , 3-O-Metilglucosa/metabolismo , Adulto , Anciano , Transporte Biológico Activo , Diferenciación Celular , Células Cultivadas , Femenino , Hexoquinasa/metabolismo , Humanos , Hipoglucemiantes/farmacología , Insulina/farmacología , Cinética , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Músculo Esquelético/citología , NADP/metabolismo , Fosforilación , Ácido Pirúvico/farmacología , Estimulación QuímicaRESUMEN
Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopy and quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60 min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface.
Asunto(s)
Proteínas Portadoras , Núcleo Celular/enzimología , Glucoquinasa/metabolismo , Glucógeno Hepático/biosíntesis , Hígado/enzimología , Transporte Activo de Núcleo Celular , Animales , Células Cultivadas , Péptidos y Proteínas de Señalización Intracelular , Cinética , Perfusión , Proteínas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be approximately 30 microM. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be approximately 7 microM. Likewise, the NAD(P)H increase of the mitochondrial domain is approximately 60 microM and is delayed with respect to the change in cytoplasmic NAD(P)H by approximately 20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.
Asunto(s)
Glucosa/farmacología , Islotes Pancreáticos/fisiología , Mitocondrias/metabolismo , NADP/metabolismo , Animales , Células Cultivadas , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Matriz Extracelular , Femenino , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Cinética , Ratones , Ratones Endogámicos , Mitocondrias/efectos de los fármacos , Espectrometría de FluorescenciaRESUMEN
The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study thin samples may be limited by increased photobleaching.
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Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Fenómenos Biofísicos , Biofisica , Dextranos , Fluoresceínas , Colorantes Fluorescentes , Indoles , Fotoquímica , FotonesAsunto(s)
Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , NADP/análisis , NAD/análisis , Animales , Células Cultivadas , Glucosa/metabolismo , Hexoquinasa/metabolismo , Procesamiento de Imagen Asistido por Computador , Islotes Pancreáticos/citología , Rayos Láser , Ratones , Músculos/citología , Páncreas/citología , FotonesRESUMEN
We describe the use of modified versions of the Aequora victoria green fluorescent protein (GFP) to simultaneously follow the expression and distribution of two different proteins in the nematode, Caenorhabditis elegans. A cyan-colored GFP derivative, designated CFP, contains amino acid (aa) substitutions Y66W, N146I, M153T and V163A relative to the original GFP sequence and is similar to the previously reported "W7" form. A yellow-shifted GFP derivative, designated YFP, contains aa substitutions S65G, V68A, S72A and T203Y and is similar to the previously described "I0C" variant. Coding regions for CFP and YFP were constructed in the context of a high-activity C. elegans expression system. Previously characterized promoters and localization signals have been used to express CFP and YFP in C. elegans. Filter sets designed to distinguish YFP and CFP fluorescence spectra allowed visualization of the two distinct forms of GFP in neurons and in muscle cells. A series of expression vectors carrying CFP and YFP have been constructed and are being made available to the scientific community.
Asunto(s)
Caenorhabditis elegans/genética , Expresión Génica , Proteínas Luminiscentes/genética , Sustitución de Aminoácidos , Animales , Animales Modificados Genéticamente , Biotecnología , Estudios de Evaluación como Asunto , Proteínas Fluorescentes Verdes , Músculos/metabolismo , Plásmidos/genética , Espectrometría de FluorescenciaRESUMEN
Two-photon excitation microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Since two-photon excitation occurs only at the focal point of the microscope, it inherently provides three-dimensional resolution. This localization of excitation also minimizes photobleaching and photodamage, which are the ultimate limiting factors in imaging living cells. Furthermore, no pinhole is required to attain three-dimensional discrimination, so the efficiency of fluorescence collection is increased. These advantages allow experiments on thick living samples that would not be possible with other imaging techniques. The cost and complexity of the lasers required for two-photon excitation microscopy have limited its use, but appropriate turn-key lasers have now been introduced, and their cost should decrease. Finally, the recent introduction of commercial two-photon excitation laser-scanning microscope systems allows a much larger group of researchers access to this state-of-the-art methodology.
Asunto(s)
Microscopía Fluorescente/métodos , Animales , Rayos Láser , FotonesRESUMEN
We describe a method for assaying protein interactions that offers some attractive advantages over previous assays. This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green fluorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green fluorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission. By using proteins encoded by circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers. BRET should be particularly useful for testing protein interactions within native cells, especially with integral membrane proteins or proteins targeted to specific organelles.
Asunto(s)
Proteínas Bacterianas/metabolismo , Relojes Biológicos/fisiología , Ritmo Circadiano/fisiología , Espectrometría de Fluorescencia/métodos , Proteínas Bacterianas/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano , Clonación Molecular , Cianobacterias , Dimerización , Transferencia de Energía , Escherichia coli/genética , Luciferasas/genética , Mediciones Luminiscentes , Proteínas Luminiscentes/genética , Unión Proteica , Proteínas Recombinantes de FusiónRESUMEN
The relationship between glucokinase (GK) and glucose-stimulated metabolism, and the potential for metabolic coupling between beta cells, was examined in isolated mouse islets by using a recombinant adenovirus that expresses Cre recombinase (AdenoCre) to inactivate a conditional GK gene allele (gklox). Analysis of AdenoCre-treated islets indicated that the gklox allele in approximately 30% of islet cells was converted to a nonexpressing variant (gkdel). This resulted in a heterogeneous population of beta cells where GK was absent in some cells. Quantitative two-photon excitation imaging of NAD(P)H autofluorescence was then used to measure glucose-stimulated metabolic responses of individual islet beta cells from gklox/lox mice. In AdenoCre-infected islets, approximately one-third of the beta cells showed markedly lower NAD(P)H responses. These cells also exhibited glucose dose responses consistent with the loss of GK. Glucose dose responses of the low-responding cells were not sigmoidal and reached a maximum at approximately 5 mM glucose. In contrast, the normal response cells showed a sigmoidal response with an KcatS0.5 of approximately 8 mM. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in beta cells within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.
Asunto(s)
Adenoviridae/genética , Glucoquinasa/genética , Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/enzimología , Animales , Glucoquinasa/metabolismo , Técnicas In Vitro , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADP/metabolismoRESUMEN
We describe the quantitative monitoring of TATA-binding protein (TBP) localization and expression in living Saccharomyces cerevisiae cells. We replaced the endogenous TBP with a green fluorescent protein (GFP) x TBP fusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM). When GFP x TBP expression was altered by using various promoters, the levels measured by LSCM correlated well with the levels determined by immunoblot of whole cell extract protein. These results show that GFP x TBP imaging not only offers a method of measurement equivalent to a more conventional technique but also provides real-time quantitation in living cells and subcellular localization information. Time-lapse confocal imaging of GFP x TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0.7) between mother and daughter cells. Based on this and data from a mutant which underexpresses GFP x TBP, we suggest that intracellular levels of TBP are near rate-limiting for growth and viability.