Supplementary Protocol
Contents
1. FluoreScore3D exe lt 2D position info file from view 1 gt lt 2D position info file from view 2 gt lt proj ection Matrices file proj mat lt txL gt lt output 3D positions csv gt Example Piucrescore 0 exe viewl pos csv View2 pos csVv projmat txt 0 Sdposscsv Make sure projmat txt is in the same folder with videos or give the FluoreScore3D exe the full path to projMat txt For example D GFP ExperimentI projMat txt 3 4 Batch processing 3 4 1 Short experiments If the experiments are less than one hour we will have one video file for each view After finding parameters using FluoreScoreGUI processing videos FluoreScoreCMD FluoreScoreSQ and FluoreScore3D can be called in a BAT file to facilitate processing To do so create a new file with Notepad and copy paste the commands in the table below and save the file with bat extension Make sure you change the processing parameters and filenames according to your experiment Moreover when there is only one fly we not only get GFP data but also 3Dposition of the fly over the time In this case we call FluoreScore3D to reconstruct 3D position of the fly for number of flies echo on FluoreScoreCMD exe AviFilecam0O avi 15 AviFilecaml avi 15 n aL AviFilecam0O mask jpg 160 80 570 560 AviFilecaml mask jpg 230 100 550 530 FluoreScoreSQ exe viewO csv viewl csv finalOutput csv 1 20 FPilucrescores ex view pos csv viewl pos csv projmat txt 3dpos csv for2. These videos show how the processing is done 15
3. e g ctrlFileName myctrifile Name of Experiment which willl be shown as a title for the figures e g experimentName 100727 GFP VS non GFP If there are a case and control input TRUE otherwise input FALSE N mber or input GrP files iL oie muliiple Number of flies Length of input videos in frame SOOO tore MOS 00 eG Window oize Tor smoothing frame base e g smoothingWindowSize 1801 for about 1 minute window 4 minutes 7201 Note the number should be odd Name For the Dain ihat OU wani to plot If mule ipo ke bin information is needed one can simply input any bin name separated by nameofbin nameofbin g3Num Or g4NUM e g g3Num g4Num gAvg g4SumtrlAvgt b4Sum OMe Genie tse any COMommMaeloOn mOr eany Ota t b ee dh 27 37 47 Sum Num Avg Name of the Din that you want to add As you are Joling tO add 2 or more sign will Dbe there e g CombinedSum g3Sumt g4Sum If you donot want to add bim then make Combinedr lag FALSE otherwise TRUE it VOU vant TO proceso muliiple files simultaneously you can enter start file number and end file number For example 12 StartDummy 1 and DummyFileNumber 15 Programwill process all the files from 1 to 15 To choosing Bin G4 G1 start with the highest bin that has pixels for any sample If that bin does not contain pixels for each sample add the next highest bin and so on Here is an e
4. 2 To set threshold use slider If the output filename has not been indicated it will ask for an output file name Enter that and continue Threshold can be set between 0 and 127 The larger the threshold is the less noise there will be To find an appropriate threshold you should see the output video Make sure that under show box input video and processed area are checked Adjust threshold such that flies are detected blue color in the majority of frames and background scattered pixels are eliminated Record the value used Use this value across any additional samples so data is analyzed consistently 3 To set the processing mask open the input video and under the analyze menu choose mask Draw free hand closed loop around areas to be excluded and then close Use this option to exclude regions of glare or other background from the analysis After you have closed all the masking windows a jpg file with name videofilename_mask jpg will appear in the folder that contains the original video files To select region of interest ROI choose Custom ROI under the Analyze menu A window with a frame of video will pop up Right click on image to select the upper left point and don t release right click until you reach the lower right point of the rectangular ROI You can do this as many times as you want until you get your desired ROI Once you are done close the frame window A text file called videofilename _roiPoints txt will be created in the sam
5. Number of flies N N lt gt 1 echo on FluoreScoreCMD exe AviFilecam0O avi 15 AviFilecaml avi 15 n 1 Avilitecam0 mask Jpg 100 80 570 360 AviFilecaml mask jpg 230 100 330 330 FluoreScoreSQ exe view0O csv viewl csv finalOutput osy view0 pos csv 10 viewl pos csv 1 20 3 4 2 Long experiments To facilitate processing experiments that are long e g 24 hours experiments user can use the bat file that is provided in FSAnalyzer called FluoreScoreBatch bat The usage of that is as below You should define your processing parameters e g mask image and ROTD in the file Command line options FluoreScoreBatch bat lt first hour gt lt last hour gt Example FluoreScoreBatch bat 1 24 11 4 Data Analysis FSAnalyzer 4 1 Short Experiments We do some basic statistical analysis and plotting using R Open fsAnalyzer_shortExperiments_main r The parameters that need to be set from setwd to nameofbin are listed below PARAMETER FUNCTION setwd caseFileName ctrlilFileName experimentName doCompare fileNumber flyNumber videoLength smoothingWindowSize binName CombinedSum CombinedFlag DummyFileNumber StartDummy See Vork ng rector ye PO Re older menise Crom 7 aoh contains all the CSV riles within double guor lon e g setwd H GFP 100924 Name of Case Cov file No need Co pur VCS e g caseFileName mycasefile ata Name or semi CSV file No need tO oUt 5
6. Supplementary Material Using GFP video to track 3D movement and conditional gene expression in free moving flies Reza Ardekani Yichuan Michelle Huang Prathamesh Sancheti Ramunas Stanciauskas Simon Tavar John Tower Experimental protocol Hardware requirements 1 Desktop computer with FW800 PCI E cards to connect the cameras 2 Two video cameras Grasshopper type GRAS 20S4C Point Grey Research Scottsdale AZ 3 2x 8mm Megapixel fixed focal lens Edmund Optics Inc Barrington NJ 4 Two blue LED lights Blue 3 Watt LED MR16 bulb Super Bright LEDs Inc St Louis MO 5 MDF GFP filter set Thorlabs Inc Newton NJ 6 Two MF469 35 filters pass range 450nm 490nm 7 Two MF525 39 filters pass range 500nm 550nm 8 Standard glass Drosophila culture vial as observation chamber 9 Aluminum Breadboard 24 x 24 x 1 2 MB2424 Thorlabs Inc Newton NJ 10 2 x Mounting Post Length 14 P14 Thorlabs Inc Newton NJ 11 2x 1 5 Mounting Post Bracket C1505 Thorlabs Inc Newton NJ 12 2 x 3 One Arm Package Two Knuckles 3 Shaft NT53 885 Edmond Optics Inc Barrington NJ 13 2 x Mounting Post Base 02 40 X 0 50 High PB2 Thorlabs Inc Newton NJ 14 2 x Dovetail Optical Rail 3 RLAO300 Thorlabs Inc Newton NJ 15 2 x Extended Rail Carrier 1 x 2 8 32 Tapped Holes 1 4 Counterbore RC1 Thorlabs Inc Newton NJ 16 Cardboard box Software requirements 1 Flycap SDK provid
7. X2 F440 11 8 1624x1224 COD YUV 422 Max Speed 5800 SNC S800 lzoch 5800 and 30 Htz 30 fps Cancel Configure Selected Click OK Choose default Codec amera 1 started without format mode fps 36 68 J Do you want to use default codec F F D 7873 CYAN 7 Yua e by entering Y 4 Recording starts once it is over a Done message will appear You must copy files to another folder or change file names if you want to record videos in the same folder Otherwise if open VideoGrabber from the same folder old videos will be over written 2 Camera Calibration 1 Create a checkerboard as below which contains NxN blocks with known dimension e g 5mmx5mm and mark each corner we chose N 10 2 Take five pictures using each camera In each picture let the checkerboard be inclined with different direction angel as shown below Checkerboard position 1 position 2 position 3 position 4 position 5 3 Import all images into Calibration tool and calibrate each camera as described in following table 4 Copy HelpCalib exe into the folder that contains result of calibration Double click on HelpCalib exe to run the program A file named projMat txt will be created in the same folder Close the program by pressing any key projMat txt is exactly what we need in FluoreScore3D exe STEP 1 Click on the logo as left twice in Ft te order to create two differe
8. e folder that contains the video file Repeat the same process for the second video 3 Processing videos In this section we describe the command line options for FluoreScoreCMD FluoreScoreSQ and FluoreScore3D 3 1 FluorScoreCMD Command line options FluoreScoreCMD exe lt first view video file avi gt lt threshold for first view between 0 and 127 gt lt second view video file avi gt lt threshold for second view between 0 and 127 gt lt Display y for yes n for no gt lt Number of frames to be processed 1 to process the whole video gt lt mask Image for viewl Jpg gt lt ROIleftUpperxX gt lt ROILeftUpperY gt lt ROIrightDownxX gt lt ROIrightDownY gt lt mask Image for view2 jpg gt lt ROIleftUpperx gt lt ROILeftUpperY gt lt ROIrightDownx gt lt ROIrightDownY gt Example FluoreScoreCMD exe AviFilecam0O avi 15 AviFilecaml avi 15 n 1 Avilbilecam0 mask Jpg 160 80 370 360 AviFilecaml mask jpg 230 100 330 330 3 2 FluoreScoreSQ Command line options FluoreScoreSQ exe lt first view info file csv gt lt second view info file csv gt lt combined output csv lt flyNumber should be 1 gt lt threshold for the size of flies in pixel gt Example FluoreScoreSQ exe viewO csv viewl csv finalOutput csv 1 20 For threshold for size of the fly we use 15 20 for eyeless GFP eyes 20 for esg GFP gut and 50 for hsp70GFP whole body 3 3 FluoreScore3D Command line options
9. ed by Point grey research along with cameras 2 Camera Calibration Tool freely available for download at http www cs ucl ac uk staff Dan Stoyanov calib 3 VideoGrabber freely available at http code google com p video grabber 4 FluoreScoreSuite which has following modules a FluoreScoreGUI version 1 00 b FluoreScoreCMD version 1 00 C FluoreScoreSQ version 1 00 d FluoreScore3D version 1 00 e CalibHelp version 1 00 f fsAnalyzer version 1 00 USER MANUAL 1 Video recording 1 Create a new folder e g under H GFP e g name it DATE 4 9 11 2 Copy VideoGrabber exe to the folder created in step 1 3 Double click VideoGrabber exe to open it A Command line window will open Enter inputs as mentioned below Input 9000 for Smin ideoGrabber 7200 for 4min etc 30 Enter number of frames to capture for each camera HAH fps ps We want to record videos using openCV hence hit a One can choose a to open display windows But here we choose b Get prompt of of cameras detected 2 Enter 2 hit return Number of cameras to use select Camera Then the FlyCap Rane ene Grasshopper GAAS 2054C Z430633 1lbede led configuration for both Grasshopper GRAS 2054C S060745 Theda eed cameras at this time Number of cameras detected 2 Make sure that they Selected Camera Information have 800x600 resolution Bus Node 3 0 Sensor Sony IC
10. nt cameras Insert new camera STEP 2 Import images into each of camera fo Please import them one by one and in the Add Images same order for both cameras STEP 3 Double click on first image and set parameters for the camera Calibration Object Properties iii Ey STEP 4 Select grid of image Click on image from cornerl gt corner 2 gt corner 3 gt corner Bi Select rid 4 Make sure that the order is same for all images Calibration Tool will extract the points of intersection for grids Make sure these points are not off from intersections STEP 5 Do STEP 3 for all images STEP 6 Select camera 1 Click on Calibrate TR i i go tz JB button It will generate intrinsic and extrinsic alibrate parameters of the camera Do the same operation for camera 2 STEP 7 Click on SAVE icon to save pen project Once the project is saved multiple files will be created which include intrinsic extrinsic parameters for each camera Camera Calibration steps 3 Finding processing parameters Goal Indicating threshold masking area and ROI for one experiment A FluoreScore V 2 0 File Analyze Help V Denoising V Save data per silh Input File D Reza gfp experiments analysis new batch of videos Eyeless Single Male a1 0000 avi Output File FluoreScoreGUI 1 Open FluorescoreGUI and under the file menu click on open and indicate the input video
11. to process 13 5 Vial setup 1 Adjust luminance focus of both cameras Try to make the setting as close as possible 2 Adjust distance between platform and lights For 24 hours 48 hours videos make sure to put back both lights and make the distance between light and vial to be 15cm 3 When during experiment try to put vial in exactly the same place There is a pattern of circle on platform O Camera 6 Supplementary package This package contains three folders 1 PlooSONE_ExperimentData This folder contains data files csv for three experiments 255B with drug Elav GS with drug and 255B without drug toPlot r generates figure 6 in the paper 2 Running Sample This folder contains Input files View a AVI file eyeless_al800 avi View b AVI file eyeless_b1800 avi Projection matrices for 3D reconstruction projMat Intermediate output files eyeless_al800 csv eyeless_b1800 csv pos2d_a csv pos2d_b csv 14 Final output files GFP signal for the experiment combinedGFPOutput csv 3D position of fly pos3D csv Rename run txt to run bat and run it it will generate final and intermediate output files from input files Use plot3D r to see the 3D path Figure 4 in the paper 3 Sample footage This folder contains two footages one for single fly and the other one for multiple flies FluoreScoreSuite_Multiple_Flies avi and FluoreScoreSuite_SingleFly avi
12. xample of input parameters setwd H GFP 4 11 1I1 07 2_FysM paper j Cascrileneme lt Cj 2 Fo etr FileName lt Cleo Ma experimentName lt 4 11 11 C7 2 name of experiment doCompare lt TRUE fileNumber lt 1 flyNumber lt 2 videoLength lt 7200 length of video in frame smoothingWindowSize lt 900 window size for Smoothing binName lt o3Sum o4Sum name of the bin that you are interested i e g4num CombinedSum lt g3Sumrgq4Sum CombinedFlag lt TRUE 4 2 Long Experiments In long experiments i e 24 or 48 hours we are mostly interested in measuring the change of GFP signal in time To do so open fsAnalyzer_longExperiments_main r PARAMETER FUNCTION experiment name This will be used to name the figures Filename Prefix csv filenames should be an a specific format PathToCSVFiles Path to the folder that contains scsv files generated by EPluerescoresurrte Usually the same folder that contains videos firstHour Pimcst hour to process LastHour Last Neus TO OLeCcecss It will generate a tif in the same folder of csv files that contains plot of average G1 G3 and G4 for the course of time Here is an example of input parameters experiment name 255B filename Prefix Ply gt p Cev here the filenames are Fly Lscsv Fly 2 csv pathToCSVFiles G newFluoreScoreTest 5 12 11 firstHour 1 First hour to process lastHour 24 last hour
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