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140 Publications
Showing 61-70 of 140 resultsPulsed lasers are key elements in nonlinear bioimaging techniques such as two-photon fluorescence excitation (TPE) microscopy. Typically, however, only a percent or less of the laser power available can be delivered to the sample before photoinduced damage becomes excessive. Here we describe a passive pulse splitter that converts each laser pulse into a fixed number of sub-pulses of equal energy. We applied the splitter to TPE imaging of fixed mouse brain slices labeled with GFP and show that, in different power regimes, the splitter can be used either to increase the signal rate more than 100-fold or to reduce the rate of photobleaching by over fourfold. In living specimens, the gains were even greater: a ninefold reduction in photobleaching during in vivo imaging of Caenorhabditis elegans larvae, and a six- to 20-fold decrease in the rate of photodamage during calcium imaging of rat hippocampal brain slices.
Commentary: Na Ji came to me early in her postdoc with an idea to reduce photodamage in nonlinear microscopy by splitting the pulses from an ultrafast laser into multiple subpulses of reduced energy. In six weeks, we constructed a prototype pulse splitter and obtained initial results confirming the validity of her vision. Further experiments with Jeff Magee demonstrated that the splitter could be used to increase imaging speed or reduce photodamage in two photon microscopy by one to two orders of magnitude. This project is a great example of how quickly one can react and exploit new ideas in the Janelia environment.
A new type of wide-field fluorescence microscopy is described, which produces 100-nm-scale spatial resolution in all three dimensions, by using structured illumination in a microscope that has two opposing objective lenses. Illumination light is split by a grating and a beam splitter into six mutually coherent beams, three of which enter the specimen through each objective lens. The resulting illumination intensity pattern contains high spatial frequency components both axially and laterally. In addition, the emission is collected by both objective lenses coherently, and combined interferometrically on a single camera, resulting in a detection transfer function with axially extended support. These two effects combine to produce near-isotropic resolution. Experimental images of test samples and biological specimens confirm the theoretical predictions.
The obesogenic effect of a high-fat (HF) diet is counterbalanced by stimulation of energy expenditure and lipid oxidation in response to a meal. The aim of this study was to reveal whether muscle nonshivering thermogenesis could be stimulated by a HF diet, especially in obesity-resistant A/J compared with obesity-prone C57BL/6J (B/6J) mice. Experiments were performed on male mice born and maintained at 30 degrees C. Four-week-old mice were randomly weaned onto a low-fat (LF) or HF diet for 2 wk. In the A/J LF mice, cold exposure (4 degrees C) resulted in hypothermia, whereas the A/J HF, B/6J LF, and B/6J HF mice were cold tolerant. Cold sensitivity of the A/J LF mice was associated with a relatively low whole body energy expenditure under resting conditions, which was normalized by the HF diet. In both strains, the HF diet induced uncoupling protein-1-mediated thermogenesis, with a stronger induction in A/J mice. Only in A/J mice: 1) the HF diet augmented activation of whole body lipid oxidation by cold; and 2) at 30 degrees C, oxygen consumption, total content, and phosphorylation of AMP-activated protein kinase (AMPK), and AICAR-stimulated palmitate oxidation in soleus muscle was increased by the HF diet in parallel with significantly increased leptinemia. Gene expression data in soleus muscle of the A/J HF mice indicated a shift from carbohydrate to fatty acid oxidation. Our results suggest a role for muscle nonshivering thermogenesis and lipid oxidation in the obesity-resistant phenotype of A/J mice and indicate that a HF diet could induce thermogenesis in oxidative muscle, possibly via the leptin-AMPK axis.
Many perceptual processes and neural computations, such as speech recognition, motor control and learning, depend on the ability to measure and mark the passage of time. However, the processes that make such temporal judgements possible are unknown. A number of different hypothetical mechanisms have been advanced, all of which depend on the known, temporally predictable evolution of a neural or psychological state, possibly through oscillations or the gradual decay of a memory trace. Alternatively, judgements of elapsed time might be based on observations of temporally structured, but stochastic processes. Such processes need not be specific to the sense of time; typical neural and sensory processes contain at least some statistical structure across a range of time scales. Here, we investigate the statistical properties of an estimator of elapsed time which is based on a simple family of stochastic process.
We describe a class of models that predict how the instantaneous firing rate of a neuron depends on a dynamic stimulus. The models utilize a learnt pointwise nonlinear transform of the stimulus, followed by a linear filter that acts on the sequence of transformed inputs. In one case, the nonlinear transform is the same at all filter lag-times. Thus, this "input nonlinearity" converts the initial numerical representation of stimulus value to a new representation that provides optimal input to the subsequent linear model. We describe algorithms that estimate both the input nonlinearity and the linear weights simultaneously; and present techniques to regularise and quantify uncertainty in the estimates. In a second approach, the model is generalized to allow a different nonlinear transform of the stimulus value at each lag-time. Although more general, this model is algorithmically more straightforward to fit. However, it has many more degrees of freedom than the first approach, thus requiring more data for accurate estimation. We test the feasibility of these methods on synthetic data, and on responses from a neuron in rodent barrel cortex. The models are shown to predict responses to novel data accurately, and to recover several important neuronal response properties.
At the beginning of the final larval (fifth) instar of Manduca sexta, imaginal precursors including wing discs and eye primordia initiate metamorphic changes, such as pupal commitment, patterning and cell proliferation. Juvenile hormone (JH) prevents these changes in earlier instars and in starved final instar larvae, but nutrient intake overcomes this effect of JH in the latter. In this study, we show that a molecular marker of pupal commitment, broad, is up-regulated in the wing discs by feeding on sucrose or by bovine insulin or Manduca bombyxin in starved final instar larvae. This effect of insulin could not be prevented by JH. In vitro insulin had no effect on broad expression but relieved the suppression of broad expression by JH. This effect of insulin was directly on the disc as shown by its reduction in the presence of insulin receptor dsRNA. In starved penultimate fourth instar larvae, broad expression in the wing disc was not up-regulated by insulin. The discs became responsive to this action of insulin during the molt to the fifth instar together with the ability to become pupally committed in response to 20-hydroxyecdysone. Thus, the Manduca bombyxin acts as a metamorphosis-initiating factor in the imaginal precursors.
The structure of aquaporin-0 (AQP0) has recently been determined by electron crystallography of two-dimensional (2D) crystals and by X-ray crystallography of three-dimensional (3D) crystals. The electron crystallographic structure revealed nine lipids per AQP0 monomer, which form an almost complete bilayer. The lipids adopt a wide variety of conformations and tightly fill the space between adjacent AQP0 tetramers. The conformations of the lipid acyl chains appear to be determined not only by the protein surface but also by the acyl chains of adjacent lipid molecules. In the X-ray structure, the hydrophobic region of the protein is surrounded by a detergent micelle, with two ordered detergent molecules per AQP0 monomer. Despite the different environments, the electron crystallographic and X-ray structures of AQP0 are virtually identical, but they differ in the temperature factors of the atoms that either contact the lipids in the 2D crystals or are exposed to detergents in the 3D crystals. The temperature factors are higher in the X-ray structure, suggesting that the detergent-exposed AQP0 residues are less ordered than the corresponding ones contacting lipids in the 2D crystals. An examination of ordered detergent molecules in crystal structures of other aquaporins and of lipid molecules in 2D and 3D crystals of bacteriorhodopsin suggests that the increased conformational variability of detergent-exposed residues compared to lipid-contacting residues is a general feature.
Phase-sensitive sum-frequency vibrational spectroscopy was used to study water/vapor interfaces of HCl, HI, and NaOH solutions. The measured imaginary part of the surface spectral responses provided direct characterization of OH stretch vibrations and information about net polar orientations of water species contributing to different regions of the spectrum. We found clear evidence that hydronium ions prefer to emerge at interfaces. Their OH stretches contribute to the "ice-like" band in the spectrum. Their charges create a positive surface field that tends to reorient water molecules more loosely bonded to the topmost water layer with oxygen toward the interface, and thus enhances significantly the "liquid-like" band in the spectrum. Iodine ions in solution also like to appear at the interface and alter the positive surface field by forming a narrow double-charge layer with hydronium ions. In NaOH solution, the observed weak change of the "liquid-like" band and disappearance of the "ice-like" band in the spectrum indicates that OH(-) ions must also have excess at the interface. How they are incorporated in the interfacial water structure is, however, not clear.
A long-standing conjecture in neuroscience is that aspects of cognition depend on the brain’s ability to self-generate sequential neuronal activity. We found that reliably and continually changing cell assemblies in the rat hippocampus appeared not only during spatial navigation but also in the absence of changing environmental or body-derived inputs. During the delay period of a memory task, each moment in time was characterized by the activity of a particular assembly of neurons. Identical initial conditions triggered a similar assembly sequence, whereas different conditions gave rise to different sequences, thereby predicting behavioral choices, including errors. Such sequences were not formed in control (nonmemory) tasks. We hypothesize that neuronal representations, evolved for encoding distance in spatial navigation, also support episodic recall and the planning of action sequences.
Probing chromatin structure with nucleases is a well-established method for determining the accessibility of DNA to gene regulatory proteins and measuring competency for transcription. A hallmark of many silent genes is the presence of translationally positioned nucleosomes over their promoter regions, which can be inferred by the sensitivity of the underlying DNA to nucleases, particularly micrococcal nuclease. The quality of this data is highly dependent upon the nuclear preparation, especially if the digestion products are analyzed by high-resolution detection methods such as reiterative primer extension. Here we describe a method to isolate highly purified nuclei from the budding yeast Saccharomyces cerevisiae and the use of micrococcal nuclease to map the positions of nucleosomes at the RNR3 gene. Nuclei isolated by this procedure are competent for many of the commonly used chromatin mapping and detection procedures.