Available Technology

A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping.

Plasmids are available from Addgene.com

• AAV-FLEX-rev-ChR2(H134R)-mCherry
• AAV-FLEX-rev-ChR2-tdtomato (recommended)
• pJ241-FLEX

These vectors are prone to recombination. This is a well known issue with these AAV vectors and is due to the inverted terminal repeats (ITRs) required for rAAV production. To minimize recombination, we propagate these plasmids in Stbl2 cells from Invitrogen. Also, to minimize recombination, cells should be cultured at 30 ºC.

Note that these cultures will grow slowly (20 h for minipreps). Better yields and culture times are obtained with 2xYT as the media. This is strongly recommended.

Because recombination may still happen occasionally, we do a panel of restriction digestions to assess whether the ITRs are in tact. Separate digestions with PvuII, Sma1, and SnaB1 should be performed. The expected patterns can be calculated from the attached sequence available on addgene.com.

Viruses for Cre-dependent optogentics based on FLEX switch available from University of Pennsylvania Vector Core

Fly stocks generated in the Rubin lab are generally deposited in the Bloomington Drosophila Stock Center. For more specialized stocks please contact us directly.

Lines have been deposited in the Bloomington Stock Center. A database showing the expression patterns of the lines and our annotation is available here.

Rubin lab Plasmid Constructs are available from Addgene, which has distributed over 430 of the plasmids listed below.

Plasmids from Pfeiffer et al. 2008 and Pfeiffer et al. 2010   

BP plasmid vector backbones are derived from pBPGUw and contain the pUC19-derived bacterial origin of replication and ampicillin resistance gene, the PhiC31 attB site, the mini-white marker for identification of transformants in Drosophila, and the DSCP basal promoter. Abbreviations:  U, DSCP basal promoter; w, mini-white marker; nls, nuclear localization signal; IVS, intervening sequence within the 5’ UTR; WPRE, a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element within the 3’ UTR; 3’ UTR Term., the transcriptional terminator; and pBP, plasmid BP backbone.

Janelia Farm Reporter Construct (JFRC) backbones are derived from pBDP. In addition, all vectors also contain a basal promoter derived from hsp70 and an SV40 transcriptional terminator.

a = Drosophila codon-optimized transgene 

 Plasmids from Nern et al PNAS 2011: Multiple new site-specific recombinases for use in manipulating animal genomes

 Plasmids from Pfeiffer et al PNAS 2012: Using translational enhancers to increase transgene expression in Drosophila 

Enables one to overlay images acquired in the same focal plane via (1) laser scanning fluorescence microscopy and (2) conventional Infrared Differential Interference Microscopy (IR-DIC).

Written by Lakshmi Ramasamy in ID&F for the Murphy Lab.

Optical flow is a key method used for quantitative motion estimation of biological structures in light microscopy. It has also been used as a key module in segmentation and tracking systems and is considered a mature technology in the field of computer vision. However, most of the research focused on 2D natural images, which are small in size and rich in edges and texture information. In contrast, 3D time-lapse recordings of biological specimens comprise up to several terabytes of image data and often exhibit complex object dynamics as well as blurring due to the point-spread-function of the microscope. Thus, new approaches to optical flow are required to improve performance for such data.

We solve optical flow in large 3D time-lapse microscopy datasets by defining a Markov random field (MRF) over super-voxels in the foreground and applying motion smoothness constraints between super-voxels instead of voxel-wise. This model is tailored to the specific characteristics of light microscopy datasets: super-voxels help registration in textureless areas, the MRF over super-voxels efficiently propagates motion information between neighboring cells and the background subtraction and super-voxels reduce the dimensionality of the problem by an order of magnitude. We validate our approach on large 3D time-lapse datasets of Drosophila and zebrafish development by analyzing cell motion patterns. We show that our approach is, on average, 10x faster than commonly used optical flow implementations in the Insight Tool-Kit (ITK) and reduces the average flow end point error by 50% in regions with complex dynamic processes, such as cell divisions.

The publication of the optical flow algorithm is available in the literature section above (Amat, Myers and Keller 2013, Bioinformatics).

Structured process for the manual count of particles (e.g. cell bodies) in 2D and 3D images of any kind with graphical mark-up in the image.
For flexibility reasons this tool was implemented as macro-set for fiji/ImageJ (version 1.47h).

For installation
- download and decompress the file behind the download link below,
- copy the result into the 'macros' folder of your fiji/ImageJ,
- restart fiji/imageJ,
- install tool into fiji/imageJ from the menu: Plugins>Macros>Install...

Successful installation will generate two new buttons ('RGB' and '?') in fiji/imageJ.
The '?' button will display more help on the function of the tool.

Author: Arnim Jenett
Feb 2013

This archive contains our custom software tools for registration and fusion of simultaneous multi-view (SiMView) image data. Two different versions of the code are included (sub-folders “1p-SiMView” and “2p-SiMView”), for processing one-photon SiMView data sets (asynchronous bi-directional illumination) and two-photon SiMView (synchronous bi-directional illumination) data sets, respectively.

All algorithms were developed and tested in the Matlab computer language (version R2011b, The Mathworks). In addition to the Matlab core installation, the Image Processing Toolbox is required to execute the programs. Multi-threaded execution through the job management scripts furthermore requires the Parallel Computing Toolbox. Software compatibility was verified for PCs with a Windows 7 64-bit operating system.

The publication of the SiMView technology framework is available in the literature section above (Tomer, Khairy, Amat and Keller 2012, Nature Methods).

Lead author:  Sean Eddy

Brain function relies on communication between large populations of neurons across multiple brain areas, a full understanding of which would require knowledge of the time-varying activity of all neurons in the central nervous system. Here we use light-sheet microscopy to record activity, reported through the genetically encoded calcium indicator GCaMP5G, from the entire volume of the brain of the larval zebrafish in vivo at 0.8 Hz, capturing more than 80% of all neurons at single-cell resolution. Demonstrating how this technique can be used to reveal functionally defined circuits across the brain, we identify two populations of neurons with correlated activity patterns. One circuit consists of hindbrain neurons functionally coupled to spinal cord neuropil. The other consists of an anatomically symmetric population in the anterior hindbrain, with activity in the left and right halves oscillating in antiphase, on a timescale of 20 s, and coupled to equally slow oscillations in the inferior olive.

The publication of the whole-brain functional imaging project is available in the literature section above (Ahrens, Orger, Robson, Li and Keller 2013, Nature Methods).

Lead author:  Eric Nawrocki

The Multi-Worm Tracker is available on Sourceforge.

Frank Midgley (Janelia Scientific Computing) worked with Vivek Jayaraman, Mitya Chklovskii and others at Janelia on this freely available software tool for neural circuit visualization. It allows users to dynamically represent connectivity and information flow at different levels of a nervous system, and can also serve as a front-end for storage of other types of data (e.g., physiological or anatomical). More information available at: Neuroptikon.org

FlyFizz is an evolving webspace dedicated to enabling the exchange of information relating to one growing subfield of Drosophila brain physiology: understanding how neural circuits generate behavior by applying electrophysiological and optical imaging techniques, particularly in behaving flies. We hope this space will become a repository for supplemental information regarding published techniques, as well as a community forum for discussion, software distribution and job postings.

Learn more at FlyFizz.org

For additional GENIE Project reagent information, please visit the GENIE Project Community Forum.

Plasmids available from Addgene.org:

CMV-GCaMP6s (improved SNR, slower kinetics)

CMV-GCaMP6m (improved SNR, intermediate kinetics)

CMV-GCaMP6f (improved SNR, faster kinetics)

CMV-GCaMP5G

CMV-GCaMP3

Viruses available from University of Pennsylvania Viral Vector Core:

AAV2/1.hSynap.GCaMP6s.WPRE.SV40

AAV2/1.hSynap.GCaMP6m.WPRE.SV40

AAV2/1.hSynap.GCaMP6f.WPRE.SV40

AAV2/1.CAG.GCaMP6s.WPRE.SV40

AAV2/1.CAG.GCaMP6m.WPRE.SV40

AAV2/1.CAG.GCaMP6f.WPRE.SV40

AAV2/1.hSynap.flex.GCaMP6s.WPRE.SV40

AAV2/1.hSynap.flex.GCaMP6m.WPRE.SV40

AAV2/1.hSynap.flex.GCaMP6f.WPRE.SV40

AAV2/1.CAG.flex.GCaMP6s.WPRE.SV40

AAV2/1.CAG.flex.GCaMP6m.WPRE.SV40

AAV2/1.CAG.flex.GCaMP6f.WPRE.SV40

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AAV2/1.hSynap.GCaMP5G.WPRE.SV40

AAV2/1.hSynap.Flex.GCaMP5G.WPRE.SV40

AAV2/1.CAG.Flex.GCaMP5G.WPRE.SV40

AAV2/5.hSynap.GCaMP5G.WPRE.SV40

AAV2/9.hSynap.GCaMP5G.WPRE.SV40

AAV2/9.hSynap.Flex.GCaMP5G.WPRE.SV40

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AAV2/1.hSynap.GCaMP3.3.SV40

AAV2/5.hSynap.GCaMP3.3.SV40

AAV2/9.hSynap.GCaMP3.3.SV40

AAV2/1.hSynap.Flex.GCaMP3.3.SV40

AAV2/5.hSynap.Flex.GCaMP3.3.SV40

AAV2/9.hSynap.Flex.GCaMP3.3.SV40

Mice available from The Jackson Laboratory:

Rosa26-CAG-lox-stop-lox-GCaMP3-WPRE

Thy1-GCaMP3

Flies available from Bloomington Stock Center:

P{20XUAS-IVS-GCaMP6s}attP40

P{20XUAS-IVS-GCaMP6m}attP40

P{20XUAS-IVS-GCaMP6f}attP40

PBac{20XUAS-IVS-GCaMP6s}VK00005

PBac{20XUAS-IVS-GCaMP6m}VK00005

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P{20XUAS-IVS-GCaMP5G}attP40

PBac{20XUAS-IVS-GCaMP5G}VK00005

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P{UAS-GCaMP3.T}attP40

P{20XUAS-GCaMP3}su(Hw)attP8

P{20XUAS-GCaMP3}attP18

P{20XUAS-GCaMP3}attP2

PBac{20XUAS-GCaMP3}VK00005

C. elegans distributed upon request:

mec-4::nls-RSET-GCaMP6s:SL2:nls-TagRFP::unc-54utr