Glial cells exist throughout the nervous system, and play essential roles in various aspects of neural development and function. Distinct types of glia may govern diverse glial functions. To determine the roles of glia requires systematic characterization of glia diversity and development. In the adult Drosophila central brain, we identify five different types of glia based on its location, morphology, marker expression, and development. Perineurial and subperineurial glia reside in two separate single-cell layers on the brain surface, cortex glia form a glial mesh in the brain cortex where neuronal cell bodies reside, while ensheathing and astrocyte-like glia enwrap and infiltrate into neuropils, respectively. Clonal analysis reveals that distinct glial types derive from different precursors, and that most adult perineurial, ensheathing, and astrocyte-like glia are produced after embryogenesis. Notably, perineurial glial cells are made locally on the brain surface without the involvement of gcm (glial cell missing). In contrast, the widespread ensheathing and astrocyte-like glia derive from specific brain regions in a gcm-dependent manner. This study documents glia diversity in the adult fly brain and demonstrates involvement of different developmental programs in the derivation of distinct types of glia. It lays an essential foundation for studying glia development and function in the Drosophila brain.

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 observation of biological processes in their natural in vivo context is a key requirement for quantitative experimental studies in the life sciences. In many instances, it will be crucial to achieve high temporal and spatial resolution over long periods of time without compromising the physiological development of the specimen. Here, we discuss the principles underlying light sheet-based fluorescence microscopes. The most recent implementation DSLM is a tool optimized to deliver quantitative data for entire embryos at high spatio-temporal resolution. We compare DSLM to the two established light microscopy techniques: confocal and two-photon fluorescence microscopy. DSLM provides up to 50 times higher imaging speeds and a 10-100 times higher signal-to-noise ratio, while exposing the specimens to at least three orders of magnitude less light energy than confocal and two-photon fluorescence microscopes. We conclude with a perspective for future development.

A derivative of rhodamine 110 has been designed and assessed as a probe for cytochrome P450 activity. This probe is the first to utilize a ’trimethyl lock’ that is triggered by cleavage of an ether bond. In vitro, fluorescence was manifested by the CYP1A1 isozyme with k(cat)/K(M)=8.8x10(3)M(-1)s(-1) and K(M)=0.09microM. In cellulo, the probe revealed the induction of cytochrome P450 activity by the carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin, and its repression by the chemoprotectant resveratrol.

A long-standing goal of biology is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 hours of development. Multiview in vivo imaging at 1.5 billion voxels per minute provides "digital embryos," that is, comprehensive databases of cell positions, divisions, and migratory tracks. Our analysis of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo’s cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.

At excitatory synapses, decreases in cleft [Ca] arising from activity-dependent transmembrane Ca flux reduce the probability of subsequent transmitter release. Intense neural activity, induced by physiological and pathological stimuli, disturb the external microenvironment reducing extracellular [Ca] ([Ca](o)) and thus may impair neurotransmission. Increases in [Ca](o) activate the extracellular calcium sensing receptor (CaSR) which in turn inhibits nonselective cation channels at the majority of cortical nerve terminals. This pathway may modulate synaptic transmission by attenuating the impact of decreases in [Ca](o) on synaptic transmission. Using patch-clamp recording from isolated cortical terminals, cortical neuronal pairs and isolated neuronal soma we examined the modulation of synaptic transmission by CaSR. EPSCs were increased on average by 88% in reduced affinity CaSR-mutant (CaSR(-/-)) neurons compared with wild-type. Variance-mean analysis indicates that the enhanced synaptic transmission was due largely to an increase in average probability of release (0.27 vs 0.46 for wild-type vs CaSR(-/-) pairs) with little change in quantal size (23 +/- 4 pA vs 22 +/- 4 pA) or number of release sites (11 vs 13). In addition, the CaSR agonist spermidine reduced synaptic transmission and increased paired-pulse depression at physiological [Ca](o). Spermidine did not affect quantal size, consistent with a presynaptic mechanism of action, nor did it affect voltage-activated Ca channel currents. In summary, reduced CaSR function enhanced synaptic transmission and CaSR stimulation had the opposite effect. Thus CaSR provides a mechanism that may compensate for the fall in release probability that accompanies decreases in [Ca](o).

Landmark correspondences can be used for various tasks in image processing such as image alignment, reconstruction of panoramic photographs, object recognition and simultaneous localization and mapping for mobile robots. The computer vision community knows several techniques for extracting and pairwise associating such landmarks using distinctive invariant local image features. Two very successful methods are the Scale Invariant Feature Transform (SIFT)1 and Multi-Scale Oriented Patches (MOPS).2
We implemented these methods in the Java programming language3 for seamless use in ImageJ.4 We use it for fully automatic registration of gigantic serial section Transmission Electron Microscopy (TEM) mosaics. Using automatically detected landmark correspondences, the registration of large image mosaics simplifies to globally minimizing the displacement of corresponding points.
We present here an introduction to automatic landmark correspondence detection and demonstrate our implementation for ImageJ. We demonstrate the application of the plug-in on diverse image data.

In order to study anatomy of organisms with high-resolution there is an increasing demand to image large specimen in three dimensions (3D). Confocal microscopy is able to produce high-resolution 3D images, but these are limited by its relatively small field of view compared to the size of large biological specimens. To overcome this drawback, motorized stages moving the sample are used to create a tiled scan of the whole specimen. The physical coordinates provided by the microscope stage are not precise enough to allow reconstruction (”Stitching”) of the whole image from individual image stacks.
We developed an algorithm, as well as an ImageJ plug-in, based on the Fourier Shift Theorem that computes all possible translations (x, y, z) between two 3D images at once, yielding the best overlap in terms of the cross correlation measure. Apart from the obvious gain in computation time it has the advantage that it cannot be trapped in local minima as it simply computes all possible solutions. Computing the overlap between two adjacent image stacks is fast (12 seconds for two 512x512x89 images on a Intel ® Core2Duo with 2.2GHz) making it suitable for real time use, i.e. computing the output image during acquisition of the individual image stacks.
To compensate the possible shading- and brightness differences we apply a smooth linear intensity transition between the overlapping stacks. Additionally we extended the to generic 3D registration using gradient based rotation detection on top of the phase correlation method. We demonstrate the performance of our 3D stitching plug-in on several tiled confocal images and show an example of its application for 3D registration.

Neuron models, in particular conductance-based compartmental models, often have numerous parameters that cannot be directly determined experimentally and must be constrained by an optimization procedure. A common practice in evaluating the utility of such procedures is using a previously developed model to generate surrogate data (e.g., traces of spikes following step current pulses) and then challenging the algorithm to recover the original parameters (e.g., the value of maximal ion channel conductances) that were used to generate the data. In this fashion, the success or failure of the model fitting procedure to find the original parameters can be easily determined. Here we show that some model fitting procedures that provide an excellent fit in the case of such model-to-model comparisons provide ill-balanced results when applied to experimental data. The main reason is that surrogate and experimental data test different aspects of the algorithm’s function. When considering model-generated surrogate data, the algorithm is required to locate a perfect solution that is known to exist. In contrast, when considering experimental target data, there is no guarantee that a perfect solution is part of the search space. In this case, the optimization procedure must rank all imperfect approximations and ultimately select the best approximation. This aspect is not tested at all when considering surrogate data since at least one perfect solution is known to exist (the original parameters) making all approximations unnecessary. Furthermore, we demonstrate that distance functions based on extracting a set of features from the target data (such as time-to-first-spike, spike width, spike frequency, etc.)–rather than using the original data (e.g., the whole spike trace) as the target for fitting-are capable of finding imperfect solutions that are good approximations of the experimental data.

Widefield fluorescence microscopy is seeing dramatic improvements in resolution, reaching today 100 nm in all three dimensions. This gain in resolution is achieved by dispensing with uniform Köhler illumination. Instead, non-uniform excitation light patterns with sinusoidal intensity variations in one, two, or three dimensions are applied combined with powerful image reconstruction techniques. Taking advantage of non-linear fluorophore response to the excitation field, the resolution can be further improved down to several 10 nm. In this review article, we describe the image formation in the microscope and computational reconstruction of the high-resolution dataset when exciting the specimen with a harmonic light pattern conveniently generated by interfering laser beams forming standing waves. We will also discuss extensions to total internal reflection microscopy, non-linear microscopy, and three-dimensional imaging.