A Radial Flow Microfluidic Device for Ultra‐High‐Throughput Affinity‐Based Isolation of Circulating Tumor Cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110045/1/smll201400719.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110045/2/smll201400719-sup-0001-S1.pd

A microfluidics-based method for measuring neuronal activity in Drosophila chemosensory neurons

Monitoring neuronal responses to defined sensory stimuli is a powerful and widely used approach for understanding sensory coding in the nervous system. However, providing precise, stereotypic and reproducible cues while concomitantly recording neuronal activity remains technically challenging. Here we describe the fabrication and use of a microfluidics system that allows precise temporally restricted stimulation of Drosophila chemosensory neurons with an array of different chemical cues. The system can easily be combined with genetically encoded calcium sensors, and it can measure neuronal activity at single-cell resolution in larval sense organs and in the proboscis or leg of the adult fly. We describe the design of the master mold, the production of the microfluidic chip and live imaging using the calcium sensor GCaMP, expressed in distinct types of Drosophila chemosensory neurons. Fabrication of the master mold and microfluidic chips requires basic skills in photolithography and takes ~2 weeks; the same devices can be used repeatedly over several months. Flies can be prepared for measurements in minutes and imaged for up to 1 h

Engineering brain activity patterns by neuromodulator polytherapy for treatment of disorders

Conventional drug screens and treatments often ignore the underlying complexity of brain network dysfunctions, resulting in suboptimal outcomes. Here we ask whether we can correct abnormal functional connectivity of the entire brain by identifying and combining multiple neuromodulators that perturb connectivity in complementary ways. Our approach avoids the combinatorial complexity of screening all drug combinations. We develop a high-speed platform capable of imaging more than 15000 neurons in 50ms to map the entire brain functional connectivity in large numbers of vertebrates under many conditions. Screening a panel of drugs in a zebrafish model of human Dravet syndrome, we show that even drugs with related mechanisms of action can modulate functional connectivity in significantly different ways. By clustering connectivity fingerprints, we algorithmically select small subsets of complementary drugs and rapidly identify combinations that are significantly more effective at correcting abnormal networks and reducing spontaneous seizures than monotherapies, while minimizing behavioral side effects. Even at low concentrations, our polytherapy performs superior to individual drugs even at highest tolerated concentrations

Engineering brain activity patterns by neuromodulator polytherapy for treatment of disorders

Conventional drug screens and treatments often ignore the underlying complexity of brain network dysfunctions, resulting in suboptimal outcomes. Here we ask whether we can correct abnormal functional connectivity of the entire brain by identifying and combining multiple neuromodulators that perturb connectivity in complementary ways. Our approach avoids the combinatorial complexity of screening all drug combinations. We develop a high-speed platform capable of imaging more than 15000 neurons in 50ms to map the entire brain functional connectivity in large numbers of vertebrates under many conditions. Screening a panel of drugs in a zebrafish model of human Dravet syndrome, we show that even drugs with related mechanisms of action can modulate functional connectivity in significantly different ways. By clustering connectivity fingerprints, we algorithmically select small subsets of complementary drugs and rapidly identify combinations that are significantly more effective at correcting abnormal networks and reducing spontaneous seizures than monotherapies, while minimizing behavioral side effects. Even at low concentrations, our polytherapy performs superior to individual drugs even at highest tolerated concentrations.ISSN:2041-172

Source code 2: Test datasets for OPT reconstruction and registration (part 5)

These RAR archive files contain the following: (1) acquisition data from our OPT platform of a 2 dpf fezf2 mutant embryo stained with an in situ probe for ascl1a (folder 'data\OPT', extension '.mat'). This file serves as a test dataset for our OPT reconstruction source code. (2) Unstained reference fish (URFs; folder 'data\Registration\ referenceFish') and OPT reconstructions from 8 wild-type embryos (folder 'data\Registration\TestData_th_2dpf\wt') and 8 fezf2 mutant embryos (folder 'data\Registration\TestData_th_2dpf\mt') stained with an in situ probe for tyrosine hydroxylase (th). URFs for 2 dpf and 3 dpf are provided. All th-stained embryos are 2 dpf. These images serve as a test dataset for our registration source code. Source code for both OPT reconstruction and registration is available at https://github.com/aallalou/OPT-InSitu-Toolbox (Allalou, 2017). Download all RAR archive files (parts 1 through 7) prior to extraction

Source code 2: Test datasets for OPT reconstruction and registration (part 2)

These RAR archive files contain the following: (1) acquisition data from our OPT platform of a 2 dpf fezf2 mutant embryo stained with an in situ probe for ascl1a (folder 'data\OPT', extension '.mat'). This file serves as a test dataset for our OPT reconstruction source code. (2) Unstained reference fish (URFs; folder 'data\Registration\ referenceFish') and OPT reconstructions from 8 wild-type embryos (folder 'data\Registration\TestData_th_2dpf\wt') and 8 fezf2 mutant embryos (folder 'data\Registration\TestData_th_2dpf\mt') stained with an in situ probe for tyrosine hydroxylase (th). URFs for 2 dpf and 3 dpf are provided. All th-stained embryos are 2 dpf. These images serve as a test dataset for our registration source code. Source code for both OPT reconstruction and registration is available at https://github.com/aallalou/OPT-InSitu-Toolbox. Download all RAR archive files (parts 1 through 7) prior to extraction

Source code 2: Test datasets for OPT reconstruction and registration (part 1)

These RAR archive files contain the following: (1) acquisition data from our OPT platform of a 2 dpf fezf2 mutant embryo stained with an in situ probe for ascl1a (folder 'data\OPT', extension '.mat'). This file serves as a test dataset for our OPT reconstruction source code. (2) Unstained reference fish (URFs; folder 'data\Registration\ referenceFish') and OPT reconstructions from 8 wild-type embryos (folder 'data\Registration\TestData_th_2dpf\wt') and 8 fezf2 mutant embryos (folder 'data\Registration\TestData_th_2dpf\mt') stained with an in situ probe for tyrosine hydroxylase (th). URFs for 2 dpf and 3 dpf are provided. All th-stained embryos are 2 dpf. These images serve as a test dataset for our registration source code. Source code for both OPT reconstruction and registration is available at https://github.com/aallalou/OPT-InSitu-Toolbox. Download all RAR archive files (parts 1 through 7) prior to extraction

Automated deep-phenotyping of the vertebrate brain

Abstract Here, we describe an automated platform suitable for large-scale deep-phenotyping of zebrafish mutant lines, which uses optical projection tomography to rapidly image brain-specific gene expression patterns in 3D at cellular resolution. Registration algorithms and correlation analysis are then used to compare 3D expression patterns, to automatically detect all statistically significant alterations in mutants, and to map them onto a brain atlas. Automated deepphenotyping of a mutation in the master transcriptional regulator fezf2 not only detects all known phenotypes but also uncovers important novel neural deficits that were overlooked in previous studies. In the telencephalon, we show for the first time that fezf2 mutant zebrafish have significant patterning deficits, particularly in glutamatergic populations. Our findings reveal unexpected parallels between fezf2 function in zebrafish and mice, where mutations cause deficits in glutamatergic neurons of the telencephalon-derived neocortex