  two days prior to treatment , identify homozygous male tg(dusp6:d2egfp ) zebrafish and place individually into mating tanks .
add separator and a wild type ab * female fish and leave in mating cages overnight .   the next morning
transfer embryos to 100 mm dishes with e3 ( 5 mm nacl , 0.17 mm kcl , 0.33 mm cacl2 , 0.33 mm mgso4 ) , solution and incubate at 28c for 6 hours .
remove dead and unfertilized embryos , add fresh e3 solution , and incubate overnight .  
add 200l of e3 solution with a multi - channel pipettor into each well .  
add the following compounds as listed :  123456789101112a2ldmso2lbcl ( 0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl ( 0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl ( 2.5mm)2l dmsob2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsoc2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsod2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsoe2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsof2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsog2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsoh2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2ldmso final concentrations are 1m , 5m , 10m , 20m , 25m bci in 1% dmso . cover plate in aluminum foil and incubate for 6 hours at 28c .  
add 10l of tricaine ( 0.61 mm ) to each well to anesthetize 30hpf embryos .  
choose a configuration with a 4x objective and maximum pinhole diameter , configure laser autofocus to detect plate bottom , and determine optimal z - plane to detect regions of interest .
set scan area as a single site of 2000x2000 pixels ( 4 x 4 mm ) with no binning .
configure laser power and gain such that brightest regions in positive control images do not saturate .
the instrument will automatically acquire images from all wells and archive them in a sql server database .  
upload images into definiens developer using the cellenger application module and an import filter that recognizes image format and file structure generated by the molecular devices imagexpress ultra platform .  
process images with a custom designed cognition network technology algorithm ( also termed a ruleset ) .
the algorithm , which we custom developed based on a recently published ruleset , is designed to automatically identify embryo , yolk , and head , and to quantify the brightness and area of gfp - expressing head structures .
wells in which no embryo is detected or where the head can not be unambiguously assigned ( for example , with high compound toxicity or out of focus images ) are excluded from the analysis .
configure the algorithm using several negative control images ( vehicle treated ) and positive control images ( bci treated ) such that head and yolk are correctly identified in both . set a " head structures " detection threshold as multiples of mean yolk intensity until detection of bright head structures matches visual observation .
define parameters to export . in this example , the most informative parameter was the total integrated brightness of gfp - positive regions within the head , hereafter termed " total head structures brightness " ( figure 1 ) . 

figure 1graph depicitng the dose dependent effects of bci on gfp expression in transgenic embryos .  
the definiens software uses distributed computing on several processors ( " engines " ) , enabling simultaneous analysis of multiple images .
the algorithm calculates numerical measures based on the results from the image analysis , which are saved in the database together with a copy of the processed image , with analysis applied .
after all images are analyzed , data can be visualized within cellenger or exported to third party software ( excel , graphpad prism ) for further analysis and graphical representation .
figure 2 shows archived scan images from a vehicle treated and a bci - treated well , with and without cnt analysis applied .
figure 1 documents a dose - dependent effect of bci on gfp reporter gene expression in tg(dusp6:d2egfp ) embryos .
the concentration required to elicit a half maximal response ( ec50 ) was approximately 12 m . 

( c ) image of embryo from ( a ) after running definiens ruleset to find gfp expression in the head .
( d ) image of embryo from ( b ) after running definiens ruleset to find gfp expression in the head .
note that the ruleset was able to find an expanded area of gfp expression in the treated embryo .
  two days prior to treatment , identify homozygous male tg(dusp6:d2egfp ) zebrafish and place individually into mating tanks .
add separator and a wild type ab * female fish and leave in mating cages overnight .   the next morning
transfer embryos to 100 mm dishes with e3 ( 5 mm nacl , 0.17 mm kcl , 0.33 mm cacl2 , 0.33 mm mgso4 ) , solution and incubate at 28c for 6 hours .
remove dead and unfertilized embryos , add fresh e3 solution , and incubate overnight .  
add 200l of e3 solution with a multi - channel pipettor into each well .  
add the following compounds as listed :  123456789101112a2ldmso2lbcl ( 0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl ( 0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl ( 2.5mm)2l dmsob2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsoc2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsod2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsoe2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsof2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsog2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2l dmsoh2l dmso2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2lbcl(0.1mm)2lbcl(0.5mm)2lbcl ( 1mm)2lbcl ( 2mm)2lbcl(2.5mm)2ldmso final concentrations are 1m , 5m , 10m , 20m , 25m bci in 1% dmso . cover plate in aluminum foil and incubate for 6 hours at 28c .
  add 10l of tricaine ( 0.61 mm ) to each well to anesthetize 30hpf embryos .  
choose a configuration with a 4x objective and maximum pinhole diameter , configure laser autofocus to detect plate bottom , and determine optimal z - plane to detect regions of interest .
set scan area as a single site of 2000x2000 pixels ( 4 x 4 mm ) with no binning .
configure laser power and gain such that brightest regions in positive control images do not saturate .
the instrument will automatically acquire images from all wells and archive them in a sql server database .  
upload images into definiens developer using the cellenger application module and an import filter that recognizes image format and file structure generated by the molecular devices imagexpress ultra platform .  
process images with a custom designed cognition network technology algorithm ( also termed a ruleset ) .
the algorithm , which we custom developed based on a recently published ruleset , is designed to automatically identify embryo , yolk , and head , and to quantify the brightness and area of gfp - expressing head structures .
wells in which no embryo is detected or where the head can not be unambiguously assigned ( for example , with high compound toxicity or out of focus images ) are excluded from the analysis .
configure the algorithm using several negative control images ( vehicle treated ) and positive control images ( bci treated ) such that head and yolk are correctly identified in both . set a " head structures " detection threshold as multiples of mean yolk intensity until detection of bright head structures matches visual observation .
define parameters to export . in this example , the most informative parameter was the total integrated brightness of gfp - positive regions within the head , hereafter termed " total head structures brightness " ( figure 1 ) . 

figure 1graph depicitng the dose dependent effects of bci on gfp expression in transgenic embryos .  
the definiens software uses distributed computing on several processors ( " engines " ) , enabling simultaneous analysis of multiple images . the algorithm calculates numerical measures based on the results from the image analysis , which are saved in the database together with a copy of the processed image , with analysis applied .
after all images are analyzed , data can be visualized within cellenger or exported to third party software ( excel , graphpad prism ) for further analysis and graphical representation .
figure 2 shows archived scan images from a vehicle treated and a bci - treated well , with and without cnt analysis applied .
figure 1 documents a dose - dependent effect of bci on gfp reporter gene expression in tg(dusp6:d2egfp ) embryos .
the concentration required to elicit a half maximal response ( ec50 ) was approximately 12 m . 

( c ) image of embryo from ( a ) after running definiens ruleset to find gfp expression in the head .
( d ) image of embryo from ( b ) after running definiens ruleset to find gfp expression in the head .
note that the ruleset was able to find an expanded area of gfp expression in the treated embryo .
a major factor limiting the throughput of zebrafish chemical screens is the lack of methodology to systematically process 96-well plates for imaging and analysis . because images of multicellular organisms are complex , low contrast , and heterogeneous in nature
, existing image analysis algorithms fail to detect and quantify specific structures within the organism .
most published zebrafish chemical screens therefore involve visual examination of individual wells by dedicated personnel throughout the procedure .
this usually prevents the generation of numerical readouts and a complete archiving of screen images and data .
furthermore , manual evaluation eliminates several key advantages of image - based screening , namely the ability to conduct retrospective analyses of screen performance , to examine phenotypic changes that were not the primary focus of the screening campaign ( e.g. , toxicity or developmental defects ) and to cross - reference screening data with past or future screens using the same compound library . in this report
we highlight methodology for automated imaging and analysis of zebrafish embryos in multiwell plates without user intervention .
we automated image capture on an imagexpress ultra laser scanning confocal reader ( molecular devices , sunnyvale , ca ) to image tg(dusp6:d2egfp ) embryos in 96 well plates and developed an image analysis algorithm based on definiens ' cognition network technology that quantified gfp expression in the heads of transgenic embryos .
the method delivered graded responses and quantified the in vivo activity of a small molecule activator of fgf signaling .
, pittsburgh pa ) documenting that it is possible to implement automated image capture of zebrafish embryos on a variety of commercially available plate readers .
the development of methodology to automatically analyze multicellular organism images eliminated the need for visual scoring by a human observer and enabled the generation and archiving of numerical data and images for retrospective analysis and comparisons with future screens .
 we demonstrate the application of image - based high - content screening ( hcs ) methodology to identify small molecules that can modulate the fgf / ras / mapk pathway in zebrafish embryos . 
 the zebrafish embryo is an ideal system for in vivo high - content chemical screens . 
 the 1-day old embryo is approximately 1 mm in diameter and can be easily arrayed into 96-well plates , a standard format for high throughput screening . during the first day of development , embryos are transparent with most of the major organs present , thus enabling visualization of tissue formation during embryogenesis . 
 the complete automation of zebrafish chemical screens is still a challenge , however , particularly in the development of automated image acquisition and analysis . 
 we previously generated a transgenic reporter line that expresses green fluorescent protein ( gfp ) under the control of fgf activity and demonstrated their utility in chemical screens 1 . to establish methodology for high throughput whole organism screens 
 , we developed a system for automated imaging and analysis of zebrafish embryos at 24 - 48 hours post fertilization ( hpf ) in 96-well plates 2 . in this video 
 we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule ( bci ) that hyperactivates fgf signaling 3 . 
 the plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a molecular devices imagexpress ultra laser scanning confocal hcs reader . 
 images are processed by definiens developer software using a cognition network technology algorithm that we developed to detect and quantify expression of gfp in the heads of transgenic embryos . in this example 
 we highlight the ability of the algorithm to measure dose - dependent effects of bci on gfp reporter gene expression in treated embryos . 
Protocol
1) Set up zebrafish matings and BCI treatment.
2) Automated imaging of 96-well plates and analysis.
Representative Results:
Discussion