Available Technology

Freely available reagents, software and instruments developed at Janelia Farm.

Reagents

Cre-dependent viral vector for optogenetics

We have developed a widely used Cre-dependent viral vector utilizing a genetic element called a FLEX switch.

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

Plasmids are available from Addgene.com

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

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

Line name Insertion Site Bloomington Stock Number Reference Chr
pJFRC1-10XUAS-mCD8::GFP attP2 32184 Pfeiffer et al. 2010 3
pJFRC2-10XUAS-IVS-mCD8::GFP attP2 32185 Pfeiffer et al. 2010 3
" attP40 32186 " 2
" su(Hw)attP1 32187 " 3
" su(Hw)attP5 32188 " 2
" su(Hw)attP8 32189 " 1
pJFRC3-1XUAS-IVS-mCD8::GFP attP2 32190 " 3
pJFRC4-3XUAS-IVS-mCD8::GFP attP2 32191 " 3
pJFRC5-5XUAS-IVS-mCD8::GFP attP2 32192 " 3
pJFRC6-15XUAS-IVS-mCD8::GFP attP2 32193 " 3
pJFRC7-20XUAS-IVS-mCD8::GFP attP2 32194 " 3
pJFRC8-40XUAS-IVS-mCD8::GFP attP2 32195 " 3
pJFRC12-10XUAS-IVS-myr::GFP attP2 32197 Pfeiffer et al. 2010 3
" attP40 32198 " 2
" su(Hw)attP5 32199 " 2
" su(Hw)attP1 32200 " 3
" su(Hw)attP8 32196 " 1
pJFRC13-10XUAS-IVS-GFP attP2 32201 Pfeiffer et al. 2010 3
pJFRC14-10XUAS-IVS-GFP-WPRE attP2 32202 Pfeiffer et al.2010 3
pJFRC15-13XLexAop2-mCD8::GFP attP2 32203 " 3
" attP40 32205 " 2
" su(Hw)attP8 32204 " 1
pJFRC16-16XLexAop2-mCD8::GFP attP2 32206 " 3
pJFRC17-26XLexAop2-mCD8::GFP attP2 32207 " 3
pJFRC18-8XLexAop2-mCD8::GFP attP2 32208 Pfeiffer et al. 2010 3
pJFRC19-13XLexAop2-IVS-myr::GFP attP2 32209 " 3
" attP40 32210 " 2
" su(Hw)attP1 32212 " 3
" su(Hw)attP8 32211 " 1
pJFRC20-8XLexAop2-IVS-GAL80-WPRE attP2 32213 Pfeiffer et al. 2010 and unpublished 3
" attP40 32214 " 2
" su(Hw)attP1 32215 " 3
" su(Hw)attP5 32216 " 2
" su(Hw)attP8 32217 " 1
pJFRC21-10XUAS-IVS-mCD8::RFP attP2 32218 " 3
" attP40 32219 " 2
" su(Hw)attP8 32220 " 1
pJFRC22-10XUAS-IVS-myr::tdTomato attP2 32221 " 3
" attP40 32222 " 2
" su(Hw)attP8 32223 " 1
pJFRC23-10XUAS-IVS-myr::Dronpa attP2 32224 Vaziri et al 2008; Pfeiffer et al. 2010 3
" attP40 32225 " 2
pJFRC24-10XUAS-IVS-myr::tdEos attP2 32226 Pfeiffer et al. 2010 and unpublished 3
" attP40 32227 " 2
P{20XUAS-GCaMP3} attP2 32236 Tian et al. 2009; Pfeiffer et al. 2010 3
PBac{20XUAS-GCaMP3} VK00005 32237 " 3
P{20XUAS-GCaMP3}su(Hw) su(Hw)attP8 32234 " 1
P{20XUAS-GCaMP3} attP18 32235 " 1
pJFRC24-10XUAS-IVS-myr::tdEos; pJFRC24-10XUAS-IVS-myr::tdEos attP40;attP2 32228 Pfeiffer et al. 2010 and unpublished 2;3
pJFRC15-13XLexAop2-mCD8::GFP, pJFRC21-10XUAS-IVS-mCD8::RFP su(Hw)attP8, attP18 32229 " 1,1
pJFRC34-5XUAS-DSCP-E86tetLC attP2   " 3
pJFRC39-10XUAS-FRT>STOP>FRT-E86tetLC attP2   " 3
pJFRC26-13XLexAop2-IVS-dTrpA1-WPRE VK00005   " 3
pJFRC31-13XLexAop2-GCamp3-WPRE, pJFRC31-13XLexAop2-GCamp3-WPRE attP2; VK00005   " 3,3
pJFRC40-13XLexAop2-FRT>STOP>FRT-myrGFP attP2   " 3
pJFRC153-20XUAS-IVS-B2::PEST  attP2   Nern et al. 2011 3
pJFRC154-3XUAS-IVS-B2::PEST attP40   Nern et al. 2011 2
pJFRC155-1XUAS-DSCP-B2::PEST/TM3 attP2   Nern et al. 2011 3
pJFRC156-21XUAS-B2RT>-dSTOP-B2RT>-myr::RFP  attP2   Nern et al. 2011 3
pJFRC156-21XUAS-B2RT>-dSTOP-B2RT>-myr::RFP  attP40   Nern et al. 2011 2
pJFRC156-21XUAS-B2RT>-dSTOP-B2RT>-myr::RFP  VK00005   Nern et al. 2011 3
pJFRC157-20XUAS-IVS-B3::PEST attP2   Nern et al. 2011 3
pJFRC158-3XUAS-IVS-B3::PEST/CyO attP40   Nern et al. 2011 2
pJFRC159-1XUAS-DSCP-B3::PEST  attP2   Nern et al. 2011 3
pJFRC160-21XUAS-B3RT>-dSTOP-B3RT>-myr::RFP attP2   Nern et al. 2011 3
pJFRC160-21XUAS-B3RT>-dSTOP-B3RT>-myr::RFP attP40   Nern et al. 2011 2
pJFRC160-21XUAS-B3RT>-dSTOP-B3RT>-myr::RFP VK00005   Nern et al. 2011 3
pJFRC161-20XUAS-IVS-KD::PEST attP2   Nern et al. 2011 3
pJFRC162-3XUAS-IVS-KD::PEST attP40   Nern et al. 2011 2
pJFRC163-1XUAS-DSCP-KD::PEST attP2   Nern et al. 2011 3
pJFRC164-21XUAS-KDRT>-dSTOP-KDRT>-myr::RFP attP2   Nern et al. 2011 3
pJFRC164-21XUAS-KDRT>-dSTOP-KDRT>-myr::RFP attP40   Nern et al. 2011 2
pJFRC164-21XUAS-KDRT>-dSTOP-KDRT>-myr::RFP VK00005   Nern et al. 2011 3
pJFRC165-20XUAS-IVS-R::PEST attP2   Nern et al. 2011 3
pJFRC166-3XUAS-IVS-R::PEST attP40   Nern et al. 2011 2
pJFRC167-1XUAS-DSCP-R::PEST attP2   Nern et al. 2011 3
pJFRC168-21XUAS-RSRT>-dSTOP-RSRT>-myr::RFP attP2   Nern et al. 2011 3
pJFRC168-21XUAS-RSRT>-dSTOP-RSRT>-myr::RFP/CyO attP40   Nern et al. 2011 2
pJFRC168-21XUAS-RSRT>-dSTOP-RSRT>-myr::RFP VK00005   Nern et al. 2011 3
pJFRC170-3XUAS-IVS-Cre::PEST attP40   Nern et al. 2011 2
pJFRC171-1XUAS-DSCP-Cre::PEST/TM3 attP2   Nern et al. 2011 3
pJFRC172-10XUAS-loxP>-dSTOP-loxP>-myr::GFP attP2   Nern et al. 2011 3
pJFRC172-10XUAS-loxP>-dSTOP-loxP>-myr::GFP attP40   Nern et al. 2011 2
pJFRC173-20XUAS-IVS-Dre::PEST attP2   Nern et al. 2011 3
pJFRC176-10XUAS-rox>-dSTOP-rox>-myr::GFP attP2   Nern et al. 2011 3
pJFRC176-10XUAS-rox>-dSTOP-rox>-myr::GFP attP40   Nern et al. 2011 2
pJFRC150-20XUAS-IVS-Flp1::PEST attP2   Nern et al. 2011 3
pJFRC151-3XUAS-IVS-Flp2::PEST attP40   Nern et al. 2011 2
pJFRC152-20XUAS-IVS-Flp1 attP2   Nern et al. 2011 3
pJFRC177-10XUAS-FRT>-dSTOP-FRT>-myr::GFP attP2   Nern et al. 2011 3
pJFRC177-10XUAS-FRT>-dSTOP-FRT>-myr::GFP attP40   Nern et al. 2011 2
pJFRC27-13XLexAop2-IVS-GCamp3-p10 attP2   Pfeiffer et al. 2012 3
pJFRC27-13XLexAop2-IVS-GCamp3-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC27-13XLexAop2-IVS-GCamp3-p10 su(Hw)attP5   Pfeiffer et al. 2012 2
pJFRC27-13XLexAop2-IVS-GCamp3-p10 attP40   Pfeiffer et al. 2012 2
pJFRC28-10XUAS-IVS-GFP-p10 attP2   Pfeiffer et al. 2012 3
pJFRC29-10XUAS-IVS-myr::GFP-p10 attP2   Pfeiffer et al. 2012 3
pJFRC57-13XLexAop2-IVS-GFP-p10 attP2   Pfeiffer et al. 2012 3
pJFRC57-13XLexAop2-IVS-GFP-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC57-13XLexAop2-IVS-GFP-p10 su(Hw)attP5   Pfeiffer et al. 2012 2
pJFRC59-13XLexAop2-IVS-myr::GFP-p10 attP2   Pfeiffer et al. 2012 3
pJFRC59-13XLexAop2-IVS-myr::GFP-p10 VK00005   Pfeiffer et al. 2012 3
 pJFRC59-13XLexAop2-IVS-myr::GFP-p10 su(Hw)attP5   Pfeiffer et al. 2012 2
pJFRC65-13XLexAop2-IVS-GFP-aequorin-p10 attP2   Pfeiffer et al. 2012 3
pJFRC65-13XLexAop2-IVS-GFP-aequorin-p10
VK00005   Pfeiffer et al. 2012 3
pJFRC65-13XLexAop2-IVS-GFP-aequorin-p10 su(Hw)attP5   Pfeiffer et al. 2012 2
pJFRC80-10XUAS-IVS-Syn21-GFP attP2   Pfeiffer et al. 2012 3
pJFRC81-10XUAS-IVS-Syn21-GFP-p10 attP2   Pfeiffer et al. 2012 3
pJFRC82-20XUAS-IVS-Syn21-GFP-p10 attP2   Pfeiffer et al. 2012 3
pJFRC83-10XUAS-IVS-L21-GFP attP2   Pfeiffer et al. 2012 3
pJFRC84-10XUAS-IVS-AcNPV-GFP attP2   Pfeiffer et al. 2012 3
pJFRC85-10XUAS-IVS-EoNPV-GFP attP2   Pfeiffer et al. 2012 3
pJFRC86-10XUAS-IVS-TMV-GFP attP2   Pfeiffer et al. 2012 3
pJFRC90-20XUAS-IVS-Syn21-mPA-p10 attP2   Pfeiffer et al. 2012 3
pJFRC90-20XUAS-IVS-Syn21-mPA-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC90-20XUAS-IVS-Syn21-mPA-p10 su(Hw)attP5   Pfeiffer et al. 2012 2
pJFRC91-20XUAS-IVS-Syn21-mSPA-GFP-p10 attP2   Pfeiffer et al. 2012 3
pJFRC91-20XUAS-IVS-Syn21-mSPA-GFP-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC91-20XUAS-IVS-Syn21-mSPA-GFP-p10 su(Hw)attP5   Pfeiffer et al. 2012 2
pJFRC92-20XUAS-IVS-Syn21-mC3PA-GFP-p10 attP2   Pfeiffer et al. 2012 3
pJFRC92-20XUAS-IVS-Syn21-mC3PA-GFP-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC93-13XLexAop2-IVS-Syn21-mPA-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC93-13XLexAop2-IVS-Syn21-mPA-p10 su(Hw)attP1   Pfeiffer et al. 2012 3
pJFRC93-13XLexAop2-IVS-Syn21-mPA-p10 su(Hw)attP8   Pfeiffer et al. 2012 X
pJFRC94-13XLexAop2-IVS-Syn21-mSPA-GFP-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC94-13XLexAop2-IVS-Syn21-mSPA-GFP-p10 su(Hw)attP1   Pfeiffer et al. 2012 3
pJFRC94-13XLexAop2-IVS-Syn21-mSPA-GFP-p10 su(Hw)attP8   Pfeiffer et al. 2012 X
pJFRC95-13XLexAop2-IVS-Syn21-mC3PA-GFP-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC95-13XLexAop2-IVS-Syn21-mC3PA-GFP-p10 su(Hw)attP1   Pfeiffer et al. 2012 3
pJFRC96-20XUAS-IVS-GFP-aequorin-p10 attP2   Pfeiffer et al. 2012 3
pJFRC96-20XUAS-IVS-GFP-aequorin-p10
VK00005   Pfeiffer et al. 2012 3
pJFRC96-20XUAS-IVS-GFP-aequorin-p10 attP40   Pfeiffer et al. 2012 2
pJFRC97-20XUAS-IVS-GCamp3-p10 attP2   Pfeiffer et al. 2012 3
pJFRC97-20XUAS-IVS-GCamp3-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC97-20XUAS-IVS-GCamp3-p10 su(Hw)attP1   Pfeiffer et al. 2012 3
pJFRC98-20XUAS-IVS-Shibire-ts1-p10 attP2   Pfeiffer et al. 2012 3
pJFRC98-20XUAS-IVS-Shibire-ts1-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC99-20XUAS-IVS-Syn21-Shibire-ts1-p10 attP2   Pfeiffer et al. 2012 3
pJFRC99-20XUAS-IVS-Syn21-Shibire-ts1-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC99-20XUAS-IVS-Syn21-Shibire-ts1-p10 su(Hw)attP1   Pfeiffer et al. 2012 3
pJFRC100-20XUAS-TTS-Shibire-ts1-p10 attP2   Pfeiffer et al. 2012 3
pJFRC100-20XUAS-TTS-Shibire-ts1-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC100-20XUAS-TTS-Shibire-ts1-p10 su(Hw)attP1   Pfeiffer et al. 2012 3
pJFRC100-20XUAS-TTS-Shibire-ts1-p10 su(Hw)attP5   Pfeiffer et al. 2012 2
pJFRC101-20XUAS-IVS-Syn21-Shibire-ts1-GFP-p10 attP2   Pfeiffer et al. 2012 3
pJFRC101-20XUAS-IVS-Syn21-Shibire-ts1-GFP-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC104-13XLexAop2-IVS-Syn21-Shibire-ts1-p10 attP2   Pfeiffer et al. 2012 3
pJFRC104-13XLexAop2-IVS-Syn21-Shibire-ts1-p10 VK00005   Pfeiffer et al. 2012 3
pJFRC104-13XLexAop2-IVS-Syn21-Shibire-ts1-p10 su(Hw)attP1   Pfeiffer et al. 2012 3

GAL4 Lines

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

Plasmids

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   

Name Addgene
Plasmid
ID
5' UTR IVS Transgene 3' UTR WPRE 3' UTR Term. Ref

pBDP

17566

 

none

 

 

Pfeiffer et al. 2008

pBPGUw

17575

 

GAL4a

 

hsp70

Pfeiffer et al. 2008

pBPGw

17574

 

GAL4a

 

hsp70

Pfeiffer et al. 2008

pBPGAL4.1Uw

26226

 

GAL4a

 

hsp70

Pfeiffer et al. 2010

pBPGAL4.2Uw-2

26227

 

GAL4a

 

SV40

Pfeiffer et al. 2010

pBPGAL4.2::VP16Uw

26228

 

GAL4::VP16a

 

hsp70

Pfeiffer et al. 2010

pBPGAL4.2::p65Uw

26229

 

GAL4::p65a

 

hsp70

Pfeiffer et al. 2010

pBPnlsLexA::GADflUw

26232

 

nlsLexA::GADfla

 

hsp70

Pfeiffer et al. 2010

pBPLexA::p65Uw

26231

 

LexA::p65a

 

hsp70

Pfeiffer et al. 2010

pBPnlsLexA::p65Uw

26230

 

nlsLexA::p65a

 

hsp70

Pfeiffer et al. 2010

pBPZpGAL4DBDUw

26233

 

Zip-GAL4DBDa

 

hsp70

Pfeiffer et al. 2010

pBPp65ADZpUw

26234

 

p65AD-Zipa

 

hsp70

Pfeiffer et al. 2010

pBPGAL80Uw-4

26235

+

GAL80a

+

hsp70

Pfeiffer et al. 2010

pBPGAL80Uw-6

26236

+

GAL80a

+

SV40

Pfeiffer et al. 2010

pJFRC-MUH

26213

 

 

 

 

Pfeiffer et al. 2010

pJFRC12-10XUAS-IVS-myr::GFP

26222

+

myr::GFP

 

SV40

Pfeiffer et al. 2010

pJFRC14-10XUAS-IVS-GFP-WPRE

26223

+

GFP

+

SV40

Pfeiffer et al. 2010

pJFRC18-8XLexAop2-mCD8::GFP

26225

 

mCD8::GFP

 

SV40

Pfeiffer et al. 2010

pJFRC19-13XLexAop2-IVS-myr::GFP

26224

+

myr::GFP

 

SV40

Pfeiffer et al. 2010

pJFRC2-10XUAS-IVS-mCD8::GFP

26214

+

mCD8::GFP

 

SV40

Pfeiffer et al. 2010

pJFRC2-INS

26215

 

mCD8::GFP

 

SV40

Pfeiffer et al. 2010

pJFRC3-1XUAS-IVS-mCD8::GFP

26216

 

mCD8::GFP

 

SV40

Pfeiffer et al. 2010

pJFRC4-3XUAS-IVS-mCD8::GFP

26217

 

mCD8::GFP

 

SV40

Pfeiffer et al. 2010

pJFRC5-5XUAS-IVS-mCD8::GFP

26218

 

mCD8::GFP

 

SV40

Pfeiffer et al. 2010

pJFRC6-15XUAS-IVS-mCD8::GFP

26219

 

mCD8::GFP

 

SV40

Pfeiffer et al. 2010

pJFRC7-20XUAS-IVS-mCD8::GFP

26220

 

mCD8::GFP

 

SV40

Pfeiffer et al. 2010

pJFRC8-40XUAS-IVS-mCD8::GFP

26221

 

mCD8::GFP

 

SV40

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

Name Addgene
Plasmid
ID

pJFRC150-20XUAS-IVS-Flp1::PEST

32132

pJFRC151-3XUAS-IVS-Flp2::PEST

32133

pJFRC153-20XUAS-IVS-B2::PEST

32134

pJFRC156-21XUAS-B2RT>-dSTOP-B2RT>-myr::RFP

32135

pJFRC157-20XUAS-IVS-B3::PEST

32136

pJFRC159-1XUAS-DSCP-B3::PEST

32137

pJFRC158-3XUAS-IVS-B3::PEST

32138

pJFRC160-21XUAS-B3RT>-dSTOP-B3RT>-myr::RFP

32139

pJFRC161-20XUAS-IVS-KD::PEST

32140

pJFRC164-21XUAS-KDRT>-dSTOP-KDRT>-myr::RFP

32141

pJFRC165-20XUAS-IVS-R::PEST

32142

pJFRC168-21XUAS-RSRT>-dSTOP-RSRT>-myr::RFP

32143

pJFRC170-3XUAS-IVS-Cre::PEST

32144

pJFRC172-10XUAS-loxP>-dSTOP-loxP>-myr::GFP

32145

pJFRC173-20XUAS-IVS-Dre::PEST

32146

pJFRC176-10XUAS-rox>-dSTOP-rox>-myr::GFP

32147

pBPhsFlp1

32148

pJFRC177-10XUAS-FRT>-dSTOP-FRT>-myr::GFP

32149

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

Name Addgene Plasmid ID

pJFRC28-10XUAS-IVS-GFP-p10

36431

pJFRC81-10XUAS-IVS-Syn21-GFP-p10

36432

Software

A spherical harmonics framework for whole-embryo mechanics modeling

We provide the following computer code and programs:

1. Matlab classes and utility functions for calculating spherical harmonics basis functions and their derivatives, and also for generating, mapping, manipulating and viewing surface meshes and SPHARM objects.

2. C/C++ classes and utility functions for calculating spherical harmonics basis functions and their derivatives, and for generating and representing SPHARM objects and the SPHARM-MECH shell object.

3. SHAPE (Spherical HArmonics Parameterization Explorer): PC application with VTK/QT GUI for manipulating and viewing surfaces and testing the accuracy of C/C++ classes.

4. SPHARM-MECH (generalization of the SPHARM approach for mechanics): PC application with VTK/QT GUI for performing mechanics simulations. Note: Please see README.TXT in the root folder of the SPHARM-MECH archive for information on SPHARM-MECH memory requirements and typical start-up performance.

Ctrax

Ctrax is an open-source, freely available, machine vision program for estimating the positions and orientations of many walking flies, maintaining their individual identities over long periods of time.

Ctrax was designed to allow high-throughput, quantitative analysis of behavior in freely moving flies. Our primary goal in this project is to provide quantitative behavior analysis tools to the neuroethology community; thus, we've endeavored to make the system adaptable to other labs' setups. We have assessed the quality of the tracking results for our setup, and found that it can maintain fly identities indefinitely with minimal supervision, and on average for 1.5 fly-hours automatically.

To further compensate for identity and other tracking errors, we provide the FixErrors Matlab GUI that identifies suspicious sequences of frames and allows a user to correct any tracking errors. We also distribute the BehavioralMicroarray Matlab Toolbox for defining and detecting a broad palette of individual and social behaviors. This software inputs the trajectories output by Ctrax and computes descriptive statistics of the behavior of each individual fly. We provide software for three proof-of-concept experiments to show the potential of the Ctrax software and our behavior detectors.

Ctrax is available for download at http://ctrax.sourceforge.net/.

DISF (Dual Image Source Fusion)

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.

Fast and robust optical flow for time-lapse microscopy using super-voxels

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).

Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data

The comprehensive reconstruction of cell lineages in complex multicellular organisms is a central goal of developmental biology. We present an open-source computational framework for the segmentation and tracking of cell nuclei with high accuracy and speed. We demonstrate its (i) generality by reconstructing cell lineages in four-dimensional, terabytesized image data sets of fruit fly, zebrafish and mouse embryos acquired with three types of fluorescence microscopes, (ii) scalability by analyzing advanced stages of development
with up to 20,000 cells per time point at 26,000 cells per minute on a single computer workstation and (iii) ease of use by adjusting only two parameters across all data sets and providing visualization and editing tools for efficient data curation. Our approach achieves on average 97.0% linkage accuracy across all species and imaging modalities. Using our system, we performed the first cell lineage reconstruction of early Drosophila melanogaster nervous system development, revealing neuroblast dynamics throughout an entire embryo.

Fiji Cell Counter

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

High-throughput multiview image registration for SiMView microscopy

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).

HMMER

Profile hidden Markov models for biological sequence analysis.

Lead author:  Sean Eddy

 

Image processing and analysis of whole-brain functional recordings

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).

Infernal

RNA structure alignment and database search using covariance models.

Lead author:  Eric Nawrocki

 

JAABA: The Janelia Automatic Animal Behavior Annotator

JAABA is a machine learning-based system that enables researchers to automatically compute interpretable, quantitative statistics describing video of behaving animals. Through our system, users encode their intuition about the structure of behavior by labeling the behavior of the animal, e.g. walking, grooming, or following, in a small set of video frames. JAABA uses machine learning techniques to convert these manual labels into behavior detectors that can then be used to automatically classify the behaviors of animals in large data sets with high throughput. Our system combines an intuitive graphical user interface, a fast and powerful machine learning algorithm, and visualizations of the classifier into an interactive, usable system for creating automatic behavior detectors. JAABA is complementary to video-based tracking methods, and we envision that it will facilitate extraction of detailed, scientifically meaningful measurements of the behavioral effects in large experiments.

JAABA is an open-source, freely available program developed by members of the Branson lab. It is available for download at: http://jaaba.sourceforge.net/

Multi-Worm Tracker

Behavioral tracking software.

The Multi-Worm Tracker is available on Sourceforge.

Neuroptikon

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

spikeGL

A software project

stimGL

A software project

Tools/Instruments

FlyFizz

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

GCaMP calcium indicators

Plasmids available from Addgene.org:

Name Description Addgene Plasmid ID Reference

CMV-GCaMP5G

 

31788

Akerboom et al., 2012

CMV-GCaMP3

 

22692

Tian et al., 2009

pCMV-GCaMP6s

slow, sensitive

40753

Chen et al., 2013

pCMV-GCaMP6m

medium

40754

Chen et al., 2013

pCMV-GCaMP6f

fast

40755

Chen et al., 2013

 

Viruses available from University of Pennsylvania Viral Vector Core:

Name Penn Catalog Number Penn Map and Sequence Number Reference

AAV1.hSynap.GCaMP5G(GCaMP3-T302L.R303P.D380Y).WPRE.SV40

AV-1-PV2478

Penn Vector P2478

Akerboom et al., 2013

AAV5.hSynap.GCaMP5G(GCaMP3-T302L.R303P.D380Y).WPRE.SV40

AV-5-PV2478

Penn Vector P2478

Akerboom et al., 2013

AAV9.hSynap.GCaMP5G(GCaMP3-T302L.R303P.D380Y).WPRE.SV40

AV-9-PV2478

Penn Vector P2478

Akerboom et al., 2013

AAV9.CAG.GCaMP5G(GCaMP3-T302L.R303P.D380Y).WPRE.SV40

AV-9-PV2643

Penn Vector P2643

Akerboom et al., 2013

AAV1.CAG.Flex.GCaMP5G(GCaMP3-T302L.R303P.D380Y).WPRE.SV40

AV-1-PV2541

Penn Vector P2541

Akerboom et al., 2013

AAV9.CAG.Flex.GCaMP5G(GCaMP3-T302L.R303P.D380Y).WPRE.SV40

AV-9-PV2541

Penn Vector P2541

Akerboom et al., 2013

AAV1.hSynap.Flex.GCaMP5G(GCaMP3-T302L.R303P.D380Y).WPRE.SV40

AV-1-PV2540

Penn Vector 2540

Akerboom et al., 2013

AAV5.hSynap.Flex.GCaMP5G(GCaMP3-T302L.R303P.D380Y).WPRE.SV40

AV-5-PV2540

Penn Vector P2540

Akerboom et al., 2013

AAV9.hSynap.Flex.GCaMP5G(GCaMP3-T302L.R303P.D380Y).WPRE.SV40

AV-9-PV2540

Penn Vector P2540

Akerboom et al., 2013

AAV1.hSynap.GCaMP3.WPRE.SV40

AV-1-PV1627

Penn Vector P1627

Tian et al., 2013

AAV5.hSynap.GCaMP3.WPRE.SV40

AV-5-PV1627

Penn Vector P1627

Tian et al., 2013

AAV9.hSynap.GCaMP3.WPRE.SV40

AV-9-PV1627

Penn Vector P1627

Tian et al., 2013

AAV1.hSynap.Flex.GCaMP3.WPRE.SV40

AV-1-PV1921

Penn Vector P1921

Tian et al., 2013

AAV5.hSynap.Flex.GCaMP3.WPRE.SV40

AV-5-PV1921

Penn Vector P1921

Tian et al., 2013

AAV9.hSynap.Flex.GCaMP3.WPRE.SV40

AV-9-PV1921

Penn Vector P1921

Tian et al., 2013

AAV1.Syn.GCaMP6f.WPRE.SV40

AV-1-PV2822

Penn Vector P2822

Chen et al., 2013

AAV1.Syn.GCaMP6s.WPRE.SV40

AV-1-PV2824

Penn Vector P2824

Chen et al., 2013

AAV1.Syn.GCaMP6m.WPRE.SV40

AV-1-PV2823

Penn Vector P2823

Chen et al., 2013

AAV5.Syn.GCaMP6m.WPRE.SV40

AV-5-PV2823

Penn Vector P2823

Chen et al., 2013

AAV5.Syn.GCaMP6f.WPRE.SV40

AV-5-PV2822

Penn Vector P2822

Chen et al., 2013

AAV5.Syn.GCaMP6s.WPRE.SV40

AV-5-PV2824

Penn Vector P2824

Chen et al., 2013

AAV9.Syn.GCaMP6f.WPRE.SV40

AV-9-PV2822

Penn Vector P2822

Chen et al., 2013

AAV9.Syn.GCaMP6m.WPRE.SV40

AV-9-PV2823

Penn Vector P2823

Chen et al., 2013

AAV9.Syn.GCaMP6s.WPRE.SV40

AV-9-PV2824

Penn Vector P2824

Chen et al., 2013

AAV1.CAG.GCaMP6s.WPRE.SV40

AV-1-PV2833

Penn Vector P2833

Chen et al., 2013

AAV5.CAG.GCaMP6s.WPRE.SV40

AV-5-PV2833

Penn Vector P2833

Chen et al., 2013

AAV9.CAG.GCaMP6s.WPRE.SV40

AV-9-PV2833

Penn Vector P2833

Chen et al., 2013

AAV1.CAG.Flex.GCaMP6f.WPRE.SV40

AV-1-PV2816

Penn Vector 2816

Chen et al., 2013

AAV1.CAG.GCaMP6f.WPRE.SV40 

AV-1-PV3081

Penn Vector P3081

Chen et al., 2013

AAV9.CAG.GCaMP6m.WPRE.SV40

AV-5-PV3080

Penn Vector P3080

Chen et al., 2013

AAV9.CAG.GCaMP6f.WPRE.SV40

AV-9-PV3081

Penn Vector P3081

Chen et al., 2013

AAV1.hSyn.Flex.iGluSnFR.WPRE.SV40

AV-1-PV2724

Penn Vector P2724

Marvin et al., 2013

AAV5.hSyn.Flex.iGluSnFR.WPRE.SV40

AV-5-PV2724

Penn Vector P2724

Marvin et al., 2013

AAV9.hSyn.Flex.iGluSnFR.WPRE.SV40

AV-9-PV2724

Penn Vector P2724

Marvin et al., 2013

AAV1.CAG.Flex.iGluSnFR.WPRE.SV40

AV-1-PV2725

Penn Vector P2725

Marvin et al., 2013

AAV5.CAG.Flex.iGluSnFR.WPRE.SV40

AV-5-PV2725

Penn Vector P2725

Marvin et al., 2013

AAV9.CAG.Flex.iGluSnFR.WPRE.SV40

AV-9-PV2725

Penn Vector P2725

Marvin et al., 2013

 

Mice available from The Jackson Laboratory:

Name Jax Stock Number Reference

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

014538

Zariwala et al., 2010

 

024275

 

 

 

024339

 

 

 

024276

 

 

Collaborators available at The Jackson Laboratory:

Name Jax Stock Number Reference

B6;129S6-Gt(ROSA)26Sortm96(CAG-GCaMP6s)Hze/J

024106

 

 

B6;129S-Igs7tm93.1(tetO-GCaMP6f)Hze/J

024103

 

 

Gt(ROSA)26Sortm5(ACTB-tTA)Luo Igs7tm93.1(tetO-GCaMP6f)Hze/HzeJ

024107

 

 

B6;DBA-Tg(tetO-GCaMP6s)2Niell/J

024742

 

 

B6;129S-Gt(ROSA)26Sortm95.1(CAG-GCaMP6f)Hze/J

024105

 

 

B6;129S6-Polr2atm1(CAG-GCaMP5g,tdTomato)Tvrd/J

024477

 

 

 

Flies available from Bloomington Stock Center:

Line name Insertion Site Bloomington Stock Number Reference Chr
P{20XUAS-IVS-GCaMP5G} attP40 42037 Akerboom et al., 2012  2
P{20XUAS-IVS-GCaMP5G} VK00005 42038 Akerboom et al., 2012  3

P{UAS-GCaMP3.T}

attP40

32116

Tian et al., 2009

 2

P{20XUAS-GCaMP3} su(Hw)attP8 32234 Tian et al., 2009  1
P{20XUAS-GCaMP3} attP18 32235 Tian et al., 2009  1
P{20XUAS-GCaMP3} attP2 32236 Tian et al., 2009  3
PBac{20XUAS-GCaMP3} VK00005 32237 Tian et al., 2009  3
P{20XUAS-IVS-GCaMP6s}
attP40 42746  Chen et al., 2013  2

P{20XUAS-IVS-GCaMP6f}

attP40

42747

Chen et al., 2013

 2

P{20XUAS-IVS-GCaMP6m}

attP40

42748

Chen et al., 2013

 2

PBac{20XUAS-IVS-GCaMP6s}

VK00005

42749  

Chen et al., 2013

 3

PBac{20XUAS-IVS-GCaMP6m}

VK00005

42750

Chen et al., 2013

 3

w[1118]; P{y[+t7.7] w[+mC]=13XLexAop2-IVS-GCaMP6s-SV40}

su(Hw)attP1

44273

Chen et al., 2013

 3

w[1118]; P{y[+t7.7] w[+mC]=13XLexAop2-IVS-GCaMP6s-p10}

su(Hw)attP1

44274

Chen et al., 2013

 3

w[1118]; P{y[+t7.7] w[+mC]=13XLexAop2-IVS-GCaMP6m-p10}

su(Hw)attP1

44275

Chen et al., 2013

 3

P{y[+mDint2] w[+mC]=13XLexAop2-IVS-GCaMP6m-p10}

VK00005/TM3, Sb[1]

44276

Chen et al., 2013

 3

w[1118]; P{y[+t7.7] w[+mC]=13XLexAop2-IVS-GCaMP6f-p10}

su(Hw)attP5

44277

Chen et al., 2013

 2

w[1118]; P{y[+t7.7] w[+mC]=13XLexAop2-IVS-GCaMP6m-SV40}

su(Hw)attP1

44588

Chen et al., 2013

 3

w[1118]; P{y[+t7.7] w[+mC]=13XLexAop2-IVS-GCaMP6s-SV40}

su(Hw)attP5

44589

Chen et al., 2013

 2

w[1118]; P{y[+t7.7] w[+mC]=13XLexAop2-IVS-GCaMP6s-p10}

su(Hw)attP5

44590

Chen et al., 2013

 2

GCaMP6 calcium indicators

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

 

Plasmids available from Addgene.org:

Name Description Addgene Plasmid ID Reference

CMV-GCaMP6s

improved SNR, slower kinetics

40753

Chen et al., 2013

CMV-GCaMP6m

improved SNR, intermediate kinetics

40754

Chen et al., 2013

CMV-GCaMP6f

improved SNR, faster kinetics

40755

Chen et al., 2013

 

Viruses available from University of Pennsylvania Viral Vector Core:

Name Penn Catalog Number Penn Map and Sequence Number Reference

AAV1.Syn.GCaMP6f.WPRE.SV40

AV-1-PV2822

Penn Vector P2822

Chen et al., 2013

AAV1.Syn.GCaMP6s.WPRE.SV40

AV-1-PV2824

Penn Vector P2824

Chen et al., 2013

AAV1.Syn.GCaMP6m.WPRE.SV40

AV-1-PV2823

Penn Vector P2823

Chen et al., 2013

AAV5.Syn.GCaMP6m.WPRE.SV40

AV-5-PV2823

Penn Vector P2823

Chen et al., 2013

AAV5.Syn.GCaMP6f.WPRE.SV40

AV-5-PV2822

Penn Vector P2822

Chen et al., 2013

AAV5.Syn.GCaMP6s.WPRE.SV40

AV-5-PV2824

Penn Vector P2824

Chen et al., 2013

AAV9.Syn.GCaMP6f.WPRE.SV40

AV-9-PV2822

Penn Vector P2822

Chen et al., 2013

AAV9.Syn.GCaMP6m.WPRE.SV40

AV-9-PV2823

Penn Vector P2823

Chen et al., 2013

AAV9.Syn.GCaMP6s.WPRE.SV40

AV-9-PV2824

Penn Vector P2824

Chen et al., 2013

AAV1.CAG.GCaMP6s.WPRE.SV40

AV-1-PV2833

Penn Vector P2833

Chen et al., 2013

AAV5.CAG.GCaMP6s.WPRE.SV40

AV-5-PV2833

Penn Vector P2833

Chen et al., 2013

AAV9.CAG.GCaMP6s.WPRE.SV40

AV-9-PV2833

Penn Vector P2833

Chen et al., 2013

AAV1.CAG.Flex.GCaMP6f.WPRE.SV40

AV-1-PV2816

Penn Vector 2816

Chen et al., 2013

AAV1.CAG.Flex.GCaMP6m.WPRE.SV40

AV-1-PV2817

Penn Vector 2817

Chen et al., 2013

AAV1.CAG.Flex.GCaMP6s.WPRE.SV40

AV-1-PV2818

Penn Vector 2818

Chen et al., 2013

AAV1.Syn.Flex.GCaMP6f.WPRE.SV40

AV-1-PV2819

Penn Vector 2819

Chen et al., 2013

AAV1.Syn.Flex.GCaMP6m.WPRE.SV40

AV-1-PV2820

Penn Vector 2820

Chen et al., 2013

AAV1.Syn.Flex.GCaMP6s.WPRE.SV40

AV-1-PV2821

Penn Vector 2821

Chen et al., 2013

AAV5.CAG.Flex.GCaMP6f.WPRE.SV40

AV-5-PV2816

Penn Vector 2816

Chen et al., 2013

AAV5.CAG.Flex.GCaMP6m.WPRE.SV40

AV-5-PV2817

Penn Vector 2817

Chen et al., 2013

AAV5.CAG.Flex.GCaMP6s.WPRE.SV40

AV-5-PV2818

Penn Vector 2818

Chen et al., 2013

AAV5.Syn.Flex.GCaMP6f.WPRE.SV40

AV-5-PV2819

Penn Vector 2819

Chen et al., 2013

AAV5.Syn.Flex.GCaMP6m.WPRE.SV40

AV-5-PV2820

Penn Vector 2820

Chen et al., 2013

AAV5.Syn.Flex.GCaMP6s.WPRE.SV40

AV-5-PV2821

Penn Vector 2821

Chen et al., 2013

AAV9.CAG.Flex.GCaMP6f.WPRE.SV40

AV-9-PV2816

Penn Vector 2816

Chen et al., 2013

AAV9.CAG.Flex.GCaMP6m.WPRE.SV40

AV-9-PV2817

Penn Vector 2817

Chen et al., 2013

AAV9.CAG.Flex.GCaMP6s.WPRE.SV40

AV-9-PV2818

Penn Vector 2818

Chen et al., 2013

AAV9.Syn.Flex.GCaMP6f.WPRE.SV40

AV-9-PV2819

Penn Vector 2819

Chen et al., 2013

AAV9.Syn.Flex.GCaMP6m.WPRE.SV40

AV-9-PV2820

Penn Vector 2820

Chen et al., 2013

AAV9.Syn.Flex.GCaMP6s.WPRE.SV40

AV-9-PV2821

Penn Vector 2821

Chen et al., 2013

 

 

Mice available from The Jackson Laboratory:

Name Description Jax Stock Number Reference

Thy1-GCaMP6s-WPRE (founder line GP4.3)

improved SNR, slower kinetics, variable neuronal expression pattern

024275

-

Thy1-GCaMP6f-WPRE (founder line GP5.5)

improved SNR, faster kinetics, variable neuronal expression pattern

024276

-

Thy1-GCaMP6f-WPRE (founder line GP5.11)

improved SNR, faster kinetics, variable neuronal expression pattern

024339

-

 

 

Flies available from Bloomington Stock Center:

Line name Insertion Site Bloomington Stock Number Reference Chr
P{20XUAS-IVS-GCaMP6s} attP40 42476 Chen et al., 2013 2
P{20XUAS-IVS-GCaMP6m} attP40 42748 Chen et al., 2013 2
P{20XUAS-IVS-GCaMP6f} attP40 42747 Chen et al., 2013 2
PBac{20XUAS-IVS-GCaMP6s} VK00005 42749 Chen et al., 2013 3
PBac{20XUAS-IVS-GCaMP6m} VK00005 42750 Chen et al., 2013 3
P{13XLexAop2-IVS-GCaMP6s-SV40} su(Hw)attP1 44273 Chen et al., 2013 3
P{13XLexAop2-IVS-GCaMP6m-SV40} su(Hw)attP1 44588 Chen et al., 2013 3
P{13XLexAop2-IVS-GCaMP6s-SV40} su(Hw)attP5 44589 Chen et al., 2013 2
P{13XLexAop2-IVS-GCaMP6s-p10} su(Hw)attP1 44274 Chen et al., 2013 3
P{13XLexAop2-IVS-GCaMP6m-p10} su(Hw)attP1 44275 Chen et al., 2013 3
P{13XLexAop2-IVS-GCaMP6s-p10} su(Hw)attP5 44590 Chen et al., 2013 2
P{13XLexAop2-IVS-GCaMP6f-p10} su(Hw)attP5 44277 Chen et al., 2013 2
P{13XLexAop2-IVS-GCaMP6m-p10} VK00005 44276 Chen et al., 2013 3

 

C. elegans distributed upon request (contact Program Scientist):

Name

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

Modeling and Experimental Analysis of Hippocampal Neurons and Microcircuits

SEPTEMBER 1, 2012 – AUGUST 31, 2013