Synaptic molecular imaging in spared and deprived columns of mouse barrel cortex with array tomography

Weiler, Nicholas; Collman, Forrest; Vogelstein, Joshua T.; Burns, Randal; Smith, Stephen J.

doi:10.1038/sdata.2014.46

Cited by 12 publications

(23 citation statements)

References 121 publications

(103 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When two effective antibodies are available for use in a specific application, a common question is “which one is better?” To answer this question, we created five AT datasets, each with two previously validated antibodies used at concentrations previously determined to yield optimal immunolabeling. These antibody pairs were evaluated alongside an antibody for a different synaptic target protein, thoroughly validated for AT in prior studies (Micheva et al, 2010 ; Weiler et al, 2014 ). An example slice of each dataset is shown in Figure 6 .…”

Section: Resultsmentioning

confidence: 99%

“…A synapse can be unambiguously identified via electron microscopy, but this approach is too time-consuming and expensive to be practical for large scale (~ 100 antibodies against a target protein) antibody screening tests. A more efficient strategy is to double label the same sample with another antibody already known to localize at synapses, and measure colocalization (Micheva et al, 2010 ; Weiler et al, 2014 ). While effective, this method presents a number of challenges.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Computational Synaptic Antibody Characterization Tool for Array Tomography

et al. 2018

Self Cite

View full text Add to dashboard Cite

Application-specific validation of antibodies is a critical prerequisite for their successful use. Here we introduce an automated framework for characterization and screening of antibodies against synaptic molecules for high-resolution immunofluorescence array tomography (AT). The proposed Synaptic Antibody Characterization Tool (SACT) is designed to provide an automatic, robust, flexible, and efficient tool for antibody characterization at scale. SACT automatically detects puncta of immunofluorescence labeling from candidate antibodies and determines whether a punctum belongs to a synapse. The molecular composition and size of the target synapses expected to contain the antigen is determined by the user, based on biological knowledge. Operationally, the presence of a synapse is defined by the colocalization or adjacency of the candidate antibody punctum to one or more reference antibody puncta. The outputs of SACT are automatically computed measurements such as target synapse density and target specificity ratio that reflect the sensitivity and specificity of immunolabeling with a given candidate antibody. These measurements provide an objective way to characterize and compare the performance of different antibodies against the same target, and can be used to objectively select the antibodies best suited for AT and potentially for other immunolabeling applications.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Computational Synaptic Antibody Characterization Tool for Array Tomography

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…, Micheva & Smith 2007 Adoption of mechanized microscope components alleviated some of this burden, but setting up position lists remained tedious. Plugins to commercial microscope control software have allowed some automation of position lists, but tend to only work on very well cleaned ribbons with minimal irregularities [ Weiler et al 2014 ]. Other custom software solutions have been described, ZEISS Correlative Array Tomography software for example, but position list creation remains a mostly manual process, that takes up to 20-30 minutes per ribbon.…”

Section: Discussionmentioning

confidence: 99%

MosaicPlanner: Hardware Agnostic Array Tomography Acquisition Software

Serafin

Gliko

Smith

et al. 2018

Preprint

View full text Add to dashboard Cite

Array tomography (AT) is a technique for acquiring high resolution highly multiplexed imagery from series of ultra-thin sections arranged as an array on a rigid substrate. Specialized microscope control has been required to utilize AT as an imaging technique, which is often time consuming, and yields small volume data sets. Here we present MosaicPlanner, an open source software platform for light level AT, that streamlines the acquisition process and utilizes the general microscope control API provided by Micro-Manager, allowing AT data to be acquired on a wide variety of microscope hardware. This report provides a description of the MosaicPlanner software design, and platform improvements that were implemented to increase the acquisition speed of high volume, multiplexed AT datasets.

show abstract

“…Unlike a typical application of array tomography which seeks to catalogue large numbers of neural elements within a volume of tissue (Kleinfeld et al, 2011; Weiler et al, 2014), this application takes the opposite approach of identifying the full extent of a single element within that tissue, in this case the most reduced element of a neural circuit, the connection made between just one neuron and one other. It also differs somewhat from a similar EM-based approach of Lichtman (Kasthuri et al, 2015; Kaynig et al, 2015) in that instead of using a ‘saturated reconstruction’ approach, we selectively label the elements we wish to study by injecting them with fluorescent markers.…”

Section: Discussionmentioning

confidence: 99%

Array tomography of physiologically-characterized CNS synapses

Valenzuela

Király

Liu

et al. 2016

Journal of Neuroscience Methods

View full text Add to dashboard Cite

Background The ability to correlate plastic changes in synaptic physiology with changes in synaptic anatomy has been very limited in the central nervous system because of shortcomings in existing methods for recording the activity of specific CNS synapses and then identifying and studying the same individual synapses on an anatomical level. New method We introduce here a novel approach that combines two existing methods: paired neuron electrophysiological recording and array tomography, allowing for the detailed molecular and anatomical study of synapses with known physiological properties. Results The complete mapping of a neuronal pair allows determining the exact number of synapses in the pair and their location. We have found that the majority of close appositions between the presynaptic axon and the postsynaptic dendrite in the pair contain synaptic specializations. The average release probability of the synapses between the two neurons in the pair is low, below 0.2, consistent with previous studies of these connections. Other questions, such as receptor distribution within synapses, can be addressed more efficiently by identifying only a subset of synapses using targeted partial reconstructions. In addition, time sensitive events can be captured with fast chemical fixation. Comparison with existing methods Compared to existing methods, the present approach is the only one that can provide detailed molecular and anatomical information of electrophysiologically-characterized individual synapses. Conclusions This method will allow for addressing specific questions about the properties of identified CNS synapses, even when they are buried within a cloud of millions of other brain circuit elements.

show abstract

Synaptic molecular imaging in spared and deprived columns of mouse barrel cortex with array tomography

Cited by 12 publications

References 121 publications

A Computational Synaptic Antibody Characterization Tool for Array Tomography

A Computational Synaptic Antibody Characterization Tool for Array Tomography

MosaicPlanner: Hardware Agnostic Array Tomography Acquisition Software

Array tomography of physiologically-characterized CNS synapses

Contact Info

Product

Resources

About