High-performance probes for light and electron microscopy

Viswanathan, Sarada; Williams, Megan E.; Bloss, Erik B.; Stasevich, Timothy J.; Speer, Colenso M.; Nern, Aljoscha; Pfeiffer, Barret D.; Hooks, Bryan M.; Li, Wei Ping; English, Brian P.; Tian, Teresa; Henry, Gilbert L.; Macklin, John J.; Patel, Ronak; Gerfen, Charles R.; Zhuang, Xiaowei; Wang, Yalin; Rubin, Gerald M.; Looger, Loren L.

doi:10.1038/nmeth.3365

Cited by 247 publications

(261 citation statements)

References 70 publications

(68 reference statements)

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“…Each mouse received injections of three different AAV vectors (GFP, td-tomato, and smFPs; Table 1 19 ) into different locations of sensory, motor and frontal cortex (Fig. 1 and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Topographic precision in sensory and motor corticostriatal projections varies across cell type and cortical area

et al. 2018

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The striatum shows general topographic organization and regional differences in behavioral functions. How corticostriatal topography differs across cortical areas and cell types to support these distinct functions is unclear. This study contrasted corticostriatal projections from two layer 5 cell types, intratelencephalic (IT-type) and pyramidal tract (PT-type) neurons, using viral vectors expressing fluorescent reporters in Cre-driver mice. Corticostriatal projections from sensory and motor cortex are somatotopic, with a decreasing topographic specificity as injection sites move from sensory to motor and frontal areas. Topographic organization differs between IT-type and PT-type neurons, including injections in the same site, with IT-type neurons having higher topographic stereotypy than PT-type neurons. Furthermore, IT-type projections from interconnected cortical areas have stronger correlations in corticostriatal targeting than PT-type projections do. As predicted by a longstanding model, corticostriatal projections of interconnected cortical areas form parallel circuits in the basal ganglia.

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Section: Resultsmentioning

confidence: 99%

Topographic precision in sensory and motor corticostriatal projections varies across cell type and cortical area

et al. 2018

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“…These tools are based on a multicolor adaptation of the "flp-out" (22) approach that allows efficient stochastic labeling over a wide range of labeling frequencies. They also use recently developed protein reporters that improve the detection of fine neuronal processes (23). We applied these methods together with selective GAL4 drivers to characterize a family of multicolumnar local interneurons with processes in the outer half of the medulla.…”

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confidence: 99%

“…1A). The resultant proteins, named smGFPs (for "spaghetti monster GFP"), are described in detail elsewhere (23).…”

mentioning

confidence: 99%

Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system

Nern

Pfeiffer

Rubin

2015

Proc. Natl. Acad. Sci. U.S.A.

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522

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We describe the development and application of methods for high-throughput neuroanatomy in Drosophila using light microscopy. These tools enable efficient multicolor stochastic labeling of neurons at both low and high densities. Expression of multiple membrane-targeted and distinct epitope-tagged proteins is controlled both by a transcriptional driver and by stochastic, recombinase-mediated excision of transcription-terminating cassettes. This MultiColor FlpOut (MCFO) approach can be used to reveal cell shapes and relative cell positions and to track the progeny of precursor cells through development. Using two different recombinases, the number of cells labeled and the number of color combinations observed in those cells can be controlled separately. We demonstrate the utility of MCFO in a detailed study of diversity and variability of Distal medulla (Dm) neurons, multicolumnar local interneurons in the adult visual system. Similar to many brain regions, the medulla has a repetitive columnar structure that supports parallel information processing together with orthogonal layers of cell processes that enable communication between columns. We find that, within a medulla layer, processes of the cells of a given Dm neuron type form distinct patterns that reflect both the morphology of individual cells and the relative positions of their arbors. These stereotyped cell arrangements differ between cell types and can even differ for the processes of the same cell type in different medulla layers. This unexpected diversity of coverage patterns provides multiple independent ways of integrating visual information across the retinotopic columns and implies the existence of multiple developmental mechanisms that generate these distinct patterns.neuroanatomy | Drosophila | interneuron diversity | light microscopy | recombinase N ervous systems contain numerous and diverse cells displaying complex anatomical relationships. The specification and patterning of these cells must be generated by the execution of a much smaller set of instructions encoded in the genome. How many different genetic algorithms are needed? How precise are their outcomes? What types of rules do they follow? Answering such questions requires knowledge of the anatomy of neuronal processes for many different cell types, for numerous cells of the same type, and in multiple individuals. We describe here the development of a set of methods for collecting such data by light microscopy and their application in the adult visual system of Drosophila.Neuronal morphology is often sufficiently stereotyped to identify cell types. For example, many cell populations in vertebrate and invertebrate visual systems can be reliably distinguished on the basis of cell shape (1-3). In at least some cases, these anatomical cell types have been shown to correlate well with classifications based on genetic marker expression or functional properties. Important anatomical features are not limited to cell shape, but also include the spatial relationships between cells. One critical ...

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“…So far this has been limited exclusively to research applications [20,21]. Device simplification and accessible data processing will further enhance the broad application of fluorescence microscopy in enzyme-free nucleic acid detection [22,23].…”

Section: Enzyme-free Hybridization Assays: Specificity In Focusmentioning

confidence: 99%

“…Using a suitable lamp, laser, or versatile semiconductor light source (LEDs) with a low-light CCD detector, microscopy allows for the direct detection of single molecules in complex biological samples [20][21][22][23]. In fluorescence microscopy, the sensitivity is defined by multiple parameters such as quantum efficiency (QE), limiting spatial resolution, contrast, modulation transfer function (MTF), and the contrast transfer function (CTF) for the electronic detector.…”

Section: Signal Enhancement With Optical Detectorsmentioning

confidence: 99%

Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

Miotke

Barducci²,

Astakhova³

2015

Chemosensors

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Detection of low-abundance nucleic acids is a challenging task, which over the last two decades has been solved using enzymatic target amplification. Enzymatic synthesis enhances the signal so that diverse, scientifically and clinically relevant molecules can be identified and studied, including cancer DNA, viral nucleic acids, and regulatory RNAs. However, using enzymes increases the detection time and cost, not to mention the high risk of mistakes with amplification and data alignment. These limitations have stimulated a growing interest in enzyme-free methods within researchers and industry. In this review we discuss recent advances in signal-enhancing approaches aimed at nucleic acid diagnostics that do not require target amplification. Regardless of enzyme usage, signal enhancement is crucial for the reliable detection of nucleic acids at low concentrations. We pay special attention to novel nanomaterials, fluorescence microscopy, and technical advances in detectors for optical assessment. We summarize sensitivity parameters of the currently available assays and devices which makes this review relevant to the broad spectrum of researchers working in fields from biophysics, to engineering, to synthetic biology and bioorganic chemistry.

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High-performance probes for light and electron microscopy

Cited by 247 publications

References 70 publications

Topographic precision in sensory and motor corticostriatal projections varies across cell type and cortical area

Topographic precision in sensory and motor corticostriatal projections varies across cell type and cortical area

Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system

Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

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