Home

Zooprocess for ZOOSCAN MANUAL - Observatoire Océanologique

1. Fichier Edition Affichage Favoris Outils Pr c dente J Rechercher ydossiers C4 AG GZ XxX A Exe Adresse G Zooscan Zooscan_scan _workirg_demo_1 gt OK J Dossiers T C otjunt1st330 J X ji B rg_demo_1_zoocont jpg 15779Ko Fichier JPG 23 03 2006 11 00 C otjunt 1st435 2 rg_demo_1_z001 jpg 17 239Ko Fichier JPG 23 03 2006 11 08 C O1mar22st230 rg_ demo_ 1 rg_demo_1_vis1 jpq 15193Ko Fichier JPG 23 03 2006 11 01 C O1mar22st330 a rg_demo_1_sep gif 27 Ko ACDSee6 GIF Image 23 03 2006 11 01 C O1mar22st435 S lectionnez un l ment pour obtenir 38 rg_demo_1_out1 gif 122 Ko ACDSee6 GIF Image 23 03 2006 11 02 J otmayo2st33a une description rq_demo_1_msk1 gif 384Ko ACDSee6 GIF Image 23 03 2006 11 02 i Cal O1mayO2st435 Voir aussi rg_demo_1_meta txt 1Ko Texte seulement 23 03 2006 10 53 a O1may16st230 Mes documents rg_demo_1_meas txt 15Ko Texte seulement 23 03 2006 11 05 i a O1may16st330 Favoris r seau rg_demo_1_log txt 3Ko Texte seulement 23 03 2006 11 05 Ga O1may16st435 Poste de travail rg_demo_1_ident txt 4Ko Texte seulement 23 03 2006 11 08 i TE O1may28st230 aaro demo_1_dat1 pid 18Ko Fichier PID 23 03 2006 11 05 2 O1may28st330 i rq_demo_1_9 jpq 7 Ko Fichier JPG 23 03 2006 11 05 fa O1may28st435 l rg_demo_1_86 jpg 6Ko Fichier JPG 23 03 2006 11 05 C otnovi2st230 rg_demo_1_85 jpg 7 Ko Fichier JPG 23 03 2006 11 05 J O1nov12st330 rg_demo_1_84 jpq 7Ko Fichi
2. cccccccccccesesssseeeecccceeeeeesseeeeeceeeeeaes 13 5 3 Prepare sample to be SCanned ccccccccsssessseccceccceaeeeeseeccceeeeeaaeeseececeeessaaaaeseeeeeess 14 5 4 SCAN ANG PROC SSS SAMI CS cese E EE 14 Ia OE oh E N E E N S A S E 15 5 6 Separate WOU Mna ODOC Seeron Er E 17 auf POC CSS AAI AOC Si pec eeeseh ciate E E E ne sesysbacaees 17 Acure Ana Fes TOM ZO OSC AU cease cots cena E E E EE A 17 6 1 DADE ONG WOT osc sas css cee pe czencacapeaienccatssaescabouite seeabanohnee vecarceniateetoasaaxacacasstaneieeecrestantaces 17 02 Resolution and orey MCV CL SCANS esusen aE E EREEREER 17 6 3 Color Balance and grey level Scale ic cecccscccccceeceeeeeeeeeeeeeeeeaaeeeseeeeeeeeeeeaeaenees 18 6 3 1 Using default MANUAL balance professional Vuescan edition 18 6 4 About the log and the metadata files eeecsseccccceeceeeeeseeecceeeeeaaeeseeeceeeeesaeas 18 6 5 Scan menus CESCH PH Oi sespe E a 19 6 5 1 ocan Conis VICTI 55 hertcnoraseritasth rane ncseviahlesannetnce is auateinaa nceuiah den inernostiasuianes 19 6 5 2 Scan proced re Warning eriaren aent i E a 19 6 5 3 Scan EOC CIS airar ea caedesv ata adie son heed weeded eatieacia sacar 20 6 5 4 Plankton SCannine Procequte ss jeaciuteetseerteieiienlrtot saute alneteieuenlonnecel 20 6 5 5 Savin Mae TO WIS Kas wetrenaa hatin staan hehe na a aeetnnge eiceamenadeade mums 23 6 5 6 Pimeda Oaa a len aies yew ee aececacbacicewsieeenaes yew uecececbanieeseegieasees 23 6 5 7
3. Projectis trial dat LIJT folderis HMZooscantrial_datZoostcan_configi SAM W A f setto BEFORE you may experience memory problem with 2400 dpi images when lJ memory set below 1640 Mo lf setto BEFORE the 8 bit final image conversion quality is increased lf gamma 1 contrast enhanced lf gamma 1 gt image smoothed lf gamma 1 gt Mo correction Select gamma option BEFORE applying LUT Gamma 0 5 5 0 E Automatic Zooscan Grey Level Adjustment lf setto NWO the min and max values will be utilized Automatic median grey adjustment yes Select ratio 1 16 Select OD range 1 3 gt Absolute o0scan Grey Level Adjustment not used if setto YES above Minimum 16 bit scale fo Maximum 16 bit scale 85536 Obits images spliting limit Moy 300 overlaping 0 07 typical 0 070 OK Cancel 12 9 CHANGE CHECK Zooscan version amp parameters The Zooscan model is selected when you first install Zooprocess on the computer If you want to use another Zooscan model you must change the Zooscan version using this tool The next created project will use this new configuration as the calibration tools will do The previous files in the main Zooscan calibration folder are backed up using the previous model name ext
4. SKIP allready processed images SELECT PARTICLE TREATMENT PARAMETRES Threshold 243 0 Min ESD mm 4 Max ESD mm 100 SAVE thumbnail images of organisms SAVE also FALSE tagged object thumbnail images V KEEP Tag informations from previous process if exists V Force doubloon check for frame 2 images Use single image tool to process manual identification Cancel Do not enable the SKIP already processed image option 12 2 Manual measurements on selected vignettes This tool modified v 4 00 allows filling a length txt file containing up to two manual length measurements for the organisms of an image This tool is not as safe as the next one that works on all vignettes successively If a previous length txt file exists you can either keep the previous measurements or reset them to zero The manual measurements can be useful to process halo metric functions to convert some of the automatic measurements from ImageJ to meaningful ones widely used by scientists 70 uf Manual Measurements v 3 14 x Project essais_duplicates_sep If exists the measurements file _length tt must also bein the same folder Press ROLL mouse button on the vignette to activate the measure tool Number of lengths to measure on each organism izl Erase previous measurements from length tet file lf gamma 1 contrast enhanced lf gamma 1 gt image smoothed lf gamma 1 Mo gamma correction S
5. The graph is displayed when you have selected the axes Click on the EXIT green area or close the graph to end process lolx 520250 pixels RGB 5048 Img g_demo_1 Units scientific Resolution 2400 3 Major mm Do you want to plot another graphs for the same PID file Mo Cancel 60 11 2 4 View vignette from an ident file This is a helpful tool to visualize vignettes of organisms identified manually using the Zooprocess provided function above in the Process manual It is similar to the view vignette from text file tool allowing you to sort organisms by name or any other parameter from the ident file The ident txt vis1 jpg and dat1 pid files must be in the same folder and must remain in the selected project subfolder which is displayed at the top of the window below VIGNETTES from IDENT data file l x YIS IDENT and PID files must bein the same folder i Enhance contrast Y i SAVE thumbnail images oforganisms i KEEP vignette opened 7 OFEN IMAGE to process in EVWZo0scan_pointh_ra Open visd jpg file ONLY OK Cancel 11 3STATS and GRAPHS You can create there basic standard graphs and histograms from the PID file data section configure your own graphs or process basic stats The resulting graphs and histograms can be saved in the result folder of the selected project 61 STATS and GRAPHS x fa parametre doesnt exist in the file ts data willbe set equivalentto N PLOT
6. 1 2 oS Marc PICHERAL CNRS UPMC LOV Station Zoologique BP 28 La Darse 06234 Villefranche mer Tel 4 93 76 38 08 Fax 4 93 76 38 34 Email marc picheral obs vlfr fr Zooprocess for ZOOSCAN MANUAL Laboratoire d Oc anologie de Villefranche sur mer updated for version 7 00 2010 06 29 POO UE CUS MAINA I a a E E E E E E E gaayeas te1Pisuahecers 4 About Zooscan scanners and software ssesesensesesesesesesesenseseseseesesesesensesesessesesesense 4 2 1 What are the differences between a scanner and a Zooscan cccccceceessseeeeeeeeees 4 2 2 Why using dedicated software for imaging plankton with scanner or Zooscan 4 Install Zooscan drivers and Vuescan eeeeessssssseoeeeessssssseerersssssseseteresssssssereessssssseeerees 5 3 1 MEAS A A E e E eseoeudeenes 5 3 2 Install VUESCAN optional sicsit cccssev canershencscoveaniesiiesodesessatnecsbnodscuveasiecietedesesainens 5 Launch Zooprocess for ZOOSCAN 0s sensssczeacerstesetsieceatendviovas snc scaanvenctescnseccmdeeddaneauntesxauntesetess 6 4 1 P een ne oe ee ee ee oe eee 6 4 2 CEC AE TIES PO Of C0 A E E E A E E A A 8 4 3 TAS OR TOOD eea See dcen seat T ecestop decissweeaensouedeeneoueacaus E 9 Basic sample scanning and processing procedures ccccccccessseseeseccceceeeeeeesseeeeeeeeeaas 12 5 1 Create NEW project or Import existing Project eeeeccccccccceeeeeeeeeeeeeeeeeeeeeeeeees 13 3 2 SCAN CONVERT Background Image
7. If real RAW image option is selected version above 3 01 the image conversion is done automatically before the PROCESS see related chapter The scanning procedure is exactly the same in both menus The process will be described in the PROCESS Converted Image process one section below The SCAN Sample with Zooscan for archive no process menu allows scanning any sample cell at any resolution up to 2400 dpi The images will be processed later 6 2 Resolution and grey level scale If you have installed the professional edition of Vuescan the Zooprocess 3 01 and above versions allow to save real raw 16 bit images Zooprocess allows 16 bit to 8 bit conversion of these raw images using user defined parameters This later option is now the standard way to proceed for new series of samples It allows a complete absolute inter calibration of different Zooscans if the default parameters are Kept see calibration below Zooprocess is functioning correctly with all the resolutions including and below 2400 dpi All frame size can be treated at resolutions up to 2400 dpi as single images with extension _1 17 6 3 Color Balance and grey level scale 6 3 1 Using default MANUAL balance professional Vuescan edition The default settings contained in the files created during the Zooprocess installation and the create project tool set the system to scan in real RAW mode as described below You must thu
8. When the image process ends Zooprocess starts the particle process on the resulting corrected image If the vis file process is skipped Zooprocess opens the vis file from the sample subfolder Particle process flowchart 1 Image segmentation using the threshold upper value 2 Detection of objects sized above the minimum ESD limit and below the maximum limit analysis 1 3 Measurement of all the ImageJ parameters on all of the selected organisms plus fractal dimension and skeleton area Analysis 2 New parameters to be added there later on 4 Basic third analysis to create the resulting _meas txt file analysis 3 DO NOT TRY ANY ACTION ON IMAGEJ or USE ANY OTHER SOFTWARE while processing 9 5 Separation process 9 5 1 Important note for 2400 dpi images This tool is ONLY available in the SINGLE Image process It is actually v 6 15 not recommended to utilize it See chapter dedicated to other separation tools later in this manual The separation tool of the versions released before v2 07 doesn t support the doubloons detected in the second frame of a divided image It is thus not usable with 2400 dpi images and remains a good solution ONLY for images up to 1200 dpi Do not use the separation tool from the Single image process tool See the new Separation from vignettes tool added in version 2 09 38 9 5 2 Process description If selected the separation process begins by the first analysi
9. ce eeeccsecccccceeeeeeeseeeceeceeeaaeeeneeeceeees 62 11 3 3 Process only BASIC STATS of a sample ce ccccssssssseeeeeceeceaneeeseeeeeees 63 A gt Viewin With Outlines aectsiiudiatnneciesaemacareccuusand dane caiaieeenounecuuandtoreca 64 12 PMC UG WEA OO aa cinerea ise eerie rset edits E aatas a epee inate dete aaa un aataein eee ced 65 IZ SEPARATION cactocestncie cence nce eae ee eae 65 12 1 1 Separation mandatory flowchalt sormissa rA ERENER A Te 65 22 Separatton irom B W MSk 1M AGE bai ssetesiah heb ianere ser iataacbinte wsaiabaetiinerneswasaaakls 65 12 1 3 Separation usine Vignettes nessis site a a E E 66 12 1 4 Removing a Separation MAaSK ccecccscccccccccesseseseccccceeeaeeseseeeceeeesssauaeseeeseees 69 12L5 Process newly Separated Iman eS isch sce cantare E R eas 69 12 2 Manual measurements on selected vignettes cccccccccccsssssseeececeeeeeeeeeseeeeeeeeeeaaas 70 12 3 Manual measurements on all vignettes sec wnesccorrieeimhiccenweeasntor eect I 2A Clocalopened mase in ei r E E N 75 123 Proe e MAN ASSIA MI aa e a 75 V L rete NEW Pror ene a cae barn aaa 75 1232 Remove amex sune projecteren E 79 12 6 Editmetadata from raw TONG sssacecoscerestouesiddeareedestove sanded ondeentomsatdearrebsedoniwaddverebes 79 VF EDR COnmeuraiOn eee N 80 Ce EDTEOTIGC caaan N 83 12 9 CHANGE CHECK Zooscan version amp PparaMeteTS ccccccccccececeesseeseeeeeeeeeaaas 85 13 Procedure for automatic 1dentificatio
10. f E 2 o x 520250 pixels RGB 504k Img rgq_demo_1 Units scientific Res 2400 dpi 200 process List save Copy 11 3 3 Process only BASIC STATS of a sample If you select this tool you will get some basic information about your sample If you want a more complete result file use the Plot 13 graphs amp 12 histograms tool You will have in addition the STD of each parameter and a 256 class sorted histogram Both resulting data can be saved in the project result folder using the same name stats txt name 63 Log File Edit PARAMETRE EFFECTIF MEAM MIM MAR Area 86 10425 7093632121213 Esd 860 9734 0 3002 4 1577 Major 86 1 74100 3643 11 3091 Minor 86 0 6201 0 1677 2 6065 Elong 86 2 7911 1 0601 16 7887 Feretd6 2 40276 0 3917 16 8979 Circ 860 1743 0 040 59 Ming datto 231 WedianSb21 7 4419 155 242 Mean 86 211 9071 1560 61 241 51 Areaexc gb 10425 7093632121213 Fractal g6 1 25771 131 59 Minor 86 0 6201 0 1677 2 6065 11 4 View Image with outlines Useful tool to visualize the image and the outlines issued from the particle process of the image It shows exactly the shape of the organisms computed after image adjustment grey level and background subtract and threshold Both the vis1 jpg image and the outl gif images must be in the same folder f gamma 1 gt contrast enhanced f gamma 1 gt image smoothed f gamma 1 gt Mo gamma correction SET gamma 0 6 5 i500 C
11. gt Median filter implemented in the conversion normalisation process gt Edit LUT tool modified accordingly 99 1 VY YY YYYYY A VVVVV WV Bug fixed in the Create project tool concerning the selection of the new parameters RollingBall 50 subtract method validated for single frame 2400dpi images Bug fixed in the Find OD position tool Bug fixed for images containing less than 26 objects Gamma value added in vignettes legend in both Extract vignettes tools New tool added to create training from identified vignettes 8 Version 6 10 Upgrade to ImageJ 1 410 Large frame samples can now be processed at 2400dpi as single image Edit Lut tool modified accordingly Check mouse tool modified and moved in the Calibration menu Metadata form simplified Calibration menu includes now the check mouse tool Diagnostic menu added to include o OD process o Background analysis 15 9 Version 6 11 gt gt VVVVV Bug fixed in the filtering of large frame images at 2400dp1 Extract vignettes for PKId and Extract vignettes according to prediction enhanced for o Complete clear outside option o Possibility to remove the legend o Flipping option added o Bmp and Tif option added for the vignettes Large frame samples can now be processed at 2400dpi as single image Background analysis improved Process OD improved Load identification from folders improved to add statistics on the prediction efficiency Create p
12. scanning In case of imported images this section can be reduced or increased depending of the configuration file loaded see calibration section Time at the moment of the scan v4 0 Refer to Vuescan manual for the fields description 8 or 16 bit image image scan resolution This offset is added in frame _2 log file after the image division It is utilized to compute the OD position and to check for doubloons All the red lines below are needed to compute the OD position and to check for doubloons not used in raw image v3 01 48 TIFFFile 1 1 if a neutral or none color balance selected TIFFSizeReduction 1 TIFFFileName essai20070131 2400 1 tif TIFFFileType 1 TIFFCompression 0 TIFFProfile 0 JPEGFile 0 DetaultPolder E 200stan_e s6a1520070131 4200sean scan RawFile 1 1 if a RAW image saving option or manual color balance selected RawFileName E Zooscan_essais20070131 Zooscan_scan _raw essai20070131 2 400_raw_1 tif RawFileType 2 Raw image saved in 16 bits RawCompression 0 Copyright Zooscan_CNRS_2006 LogFile 0 Options 2 Prefs GraphType 2 ExternalViewer 0 ExternalKditor 0 WindowxOffset 10 WindowyYyOffset 10 WindowxXSize 1008 WindowYSize 990 GuidedMode 0 StartupTip 0 LUT Section added in v3 01 Color_Balance manual can be set to manual neutral or none Min 340 Max 21340 Gamma 1 Order before Adjust yes Modeset 194
13. Batch image process enhanced The images and the vignettes are no longer visible in batch mode dividing the processing time by 4 gt Default process configuration file name includes now the word both instead of narrow to avoid confusion gt Extract vignette according to prediction enhanced volume selection random folder Duplicates are no longer extracted gt Load vignettes bug corrected for the dat1 txt files gt Roll button set to 8 by default when Zooprocess is installed or project created gt New shape and texture parameters available for new projects gt Large frame images can now be scanned at 2400 dpi for archive They are converted in 1200 dpi during the conversion process We hope to be able to process them at 2400 dpi in the future gt Edit metadata tool modified to updates also the log and pid files of the sample if exist gt Narrow frame images acquired at 2400 dpi can now be processed as a unique frame reducing by two the number of resulting files suppressing the duplicates and reducing the processing time gt Edit LUT file tool modified to allow user to set the size limit for the splitting of the images in two frames 15 6 Version 5 07 gt Bug fixed in Single Image Process 15 7 Version 5 08 gt Default parameters modified for Hydroptic V2 o D tection limit 0 85 o Median 1 filter added gt Process again all background tool created to allow post processing using new normalisation setting
14. EDIT LUT file chapter of this manual and in Convert Raw images to 8 bit calibrated images 6 4 About the log and the metadata files When Zooprocess scan a RAW image using Vuescan it saves the RAW image a metadata file containing all the data concerning the sample collection and a log file containing the scanning parameters in the _raw folder of the project These files should NEVER be deleted as Zooprocess needs the Image section of the LOG file to compute the position of the OD cell and the doubloons if the images have been divided The metadata file from the _raw folder can be edited again more easily using the dedicated tool In case of process problem with ImageJ your LOG file may be corrupted version above 2 00 are safer If you are advised that the LOG file may be corrupted please check that the file in the project subfolder starts by PID and contain the Image section down to Author or 18 more If not check that the temp_log txt file in the Zooscan_temp folder is related to the last treated image If it is and its content 1s OK copy it in the Image subfolder and rename it 6 5 Scan menus description 6 5 1 Scan Config Menu SCAN CONFIG MENU 5 x SELECT SCAN CONFIG FILE in HiV ooscan_advooscan_contigh Select the correct frame size narrowllarge Your Scanning configuration filename starts with vuescan_zooscan and contains narrow or large SELECT Scan Config File vuescan_zoosca
15. Ident tem Tag BX BY Width He fields must exist in the datafile Label lt gt Image name tem lt gt Organism N As many additional identification columns can be included File header must include the column names Image resolution iti i Enhance contrast i SAVE thumbnail images of organisms 7 i KEEP vignette opened 7 Sart identlist on IMAGE MAME Open TEXT file lmagename must be inside the text file only or space separators are allowed OK Cancel The next window displayed is similar to the one for the data section of the pid file and the opened image name and full path is displayed 11 2 3 View vignette from 2D graph You will build a 2D graph using data issued from the data section of any PID file and select the vignette to be displayed by clicking onto the graph or drawing a rectangle around the data points of the vignettes to be displayed This routine is not using any project architecture Do not close any of the opened images It will be done automatically during the process for the vignettes and at the end for the vis1 jpg one You can add outlines to your vignettes and select the axes limit for the graph Please follow the instructions 58 VIEW IGNETTES from a ZD graph 3 11 m m m 0511_80100_large1 SELECT GRAPH AXES If you have selected the manual scaling you will have to set the axes limit SELECT GRAPH LIMITS 59
16. REMOVED GREY circle Objects to be KEPT Click on the objects with the let mouse button to change status Click outside of the objects with the roll mouse button to END DO NOT PRESS CANCEL OK Cancel Zooprocess simultaneously draw circles and label number on the upper left corner of the objects in the image The image is then magnified to 100 On this example the object 5 chaetognath is kept 1 grey circle the object 11 that we separated from the chaetognath is tagged O black circle and so on 42 temp_vis1 tif 75 _ 5 j 3239410584 pixels o bit 33MB If you press the ROLL button on the image the TAG CONTROL is displayed You can end tag edition or go on editing tags by pressing CANCEL TAG CONTROL x OK ta END Tag edition CANCEL to EDIT the TAG OK Cancel If you press OK the log window is completed with the data from the Result window If you have not selected the Manual Identification menu the following window will be displayed 43 po x PROCESS Other tile OK cancel Note be careful when tagging small objects contained in the bounding rectangle of a larger one you may also tag the large one A solution is to first tag the small one and then check and un tag the large one 9 7 Vignette extraction Note we today recommend v 6 15 to use the dedicated tools associated with the use of Plankton Identifier and to extract vignettes using the prediction identification
17. REPLACING preservative by water OPTIONAL SIEVING and Flume work SPLITING IMAGING with Zooscan number of organisms is too high it can be sieved in two different size class aliquots and divided again N1 must be higher than N2 The different aliquots are then scanned using the Zooscan after a careful separation 5 4 Scan and process samples You have now two main basic options 1 The first one is to scan sample and process it immediately by using the SCAN CONVERT and PROCESS Sample with Zooscan It can be very fast and simple if you use the 1200 dpi scanning resolution but you may feel that you lose time during process mainly if you scan samples successively 2 If you scan samples at high resolution 2400 dpi or you don t want to wait during process use the SCAN Sample with Zooscan for archive no process tool to scan samples and process them later during nighttime using the CONVERT amp PROCESS Images in batch mode tool keeping the default parameters to avoid reprocessing already processed samples You will scan an image using either vuescan_zooscan_2400dpi_framel_narrow ini vuescan_zooscan_1200dpi_frame2_large ini vuescan_zooscan_2400dpi_frame2_large ini according to the size of frame you are using and the way you created your project Image will be converted using the LUT TXT data It will be processed using the default process_install_both_config txt file written by the project creation tool Y
18. You thus don t need to keep the converted TIF images in the Zooscan_scan folder if you have their RAW source images and Log and Meta archived into the _raw folder Zooprocess can process any TIF image stored in the Zooscan_scan folder of your project A meta and a log files are requested anyhow If you want to process a converted ZIP archived image you will first have to extract it into the Zooscan_scan folder and copy both the meta and the log files into either the same Zooscan_scan folder or into the image subfolder of the _work folder The resulting vis1 image is saved as a TIF zipped archive _vis1 zip Zooprocess is capable to read both vis1 jpg and vis1 zip files and reads first the zip one if both exist Zooprocess allows image processing using pre configured parameters stored in a config file The process writes files using the same folder arrangement than ZooImage plug in The difference is that the ZIM file is replaced by a PID and the naming convention is not checked and the separators are in PID instead of TAB in ZIM data section The batch mode is similar to the one by one mode except that you don t have to check the metadata of the samples and a suivi log file 1s created in the Zooscan_temp folder The process flowchart is 1 Check of the metadata for the selected file in zooscan_ scan 2 Selection of the operations to be carried out 3 Image process macro Zoosca
19. ZIP raw image option is available only if you have selected the MANUAL color balance WAIT more than 30 seconds between the PREVIEW end and the SCAN Do NOT press OK before pressing SCAN in Vuescan When you have pressed the OK button in that menu Zooprocess will wait until the image file is created in the _raw folder It displays the waiting message in its status bar and the LOG window displays the scanning parameters and the time needed to complete the process from the moment you pressed OK Zooprocess waits now for the image file from Vuescan 22 Log me ioii File Edit JPEGFile 0 Copyright zoo0scan_ChRS_2006 LogFile 0 Options 2 DetaultFolderEvZooscanizooscan scan Prefs Windows afs at 1 0 Window Size 990 GuidedwMode 0 StartupTip 0 Window afs et 1 0 WindowsSize 1008 Options 2 RefreshFasto RefreshEachsScan 0 Externalviewer O ExternalEditor U PreviewMem 1 290 scanwlem 1 290 Graphlype 3 Raw Image Size 129 Mo 1 WAITING for sample_1 tit SAVING FROM ZOOSCANM TO DISK 0 51 0 SAVING FROM ZOO0SCAN TO DISK 1 51 2 SAVING FROM ZOO8CAN TO DISK 2 51 4 SAVING FROM ZOOSCAN TO DISK 3 51 6 SAVING FROM ZOOSCAN TO DISK 4 51 8 SAVING FROM ZOOSCAN TO DISK 5 51 10 SAVING FROM ZOOSCANM TO DISK 6 51 12 4 F 6 5 5 Saving Image to Disk When the image file appears it is not yet completely filled by Vuescan You have to wait for SAVING FROM ZOOSCAN to DISK The Status Bar shows the time rem
20. able to reprocess images using the right background image You can remove the frames used to average the final blank A log file is recorded with the image and indicates the color balance and LUT settings It also records all the scanning parameters plus the median grey level of each of the background images This last information may be useful to check the Zooscan characteristics over the time using the diagnostic tools 21 8 Convert Raw images to 8 bit calibrated images 8 1 Introduction The true RAW images are saved in the 66 _raw sub folder of the selected project In the following chapter of this manual the RAW image is the one from the _raw folder while the raw one comes from the Zooscan_scan folder The raw word is added to the image filename in the _raw folder These file are then zipped to save drive space during the process Metadata and Log files are also saved there with the images Two tools can perform the conversion one is included in the CONVERT amp PROCESS Images in batch mode process and the second is CONVERT RAW image to 8 bit calibrated image The resulting images are the same but the first tool can perform all the successive tasks without user intervention 8 2 Zooprocess conversion Zooprocess will check 1f a lut txt file exists in the Zooscan_config folder of your project and verify the setting from the file This lut txt file is automatically created wi
21. all the graphs and stats files created during the processes e Zooscan_projectname PID_process e Zooscan_projectname PID_process Learning_set where the learning set is to be created using folder sorted vignettes Plankton Identifier e Zooscan_projectname PID_process Pid_results where PID will save the results of the process e Zooscan_projectname PID_process Pid_to_process where PID files to be analysed using PID will be copied e Zooscan_projectname PID_process Datl_extracted where txt files used to extract vignettes according to prediction will be copied e Zooscan_projectname PID_process Datl_validated where txt files of validated vignettes will be copied e Zooscan_projectname PID_process Pid_predicted where user can move the pid files that have already been predicted e Zooscan_projectname PID_process Sorted_vignettes where the vignettes will be sorted according to predictions by the EXTRACT vignettes in folders according to prediction tool Predictions will be validated from these folders using the LOAD identifications from sorted vignettes e Zooscan_projectname PID_process Unsorted_vignettes_pid where you will place the vignettes and their PID files to be sorted in the learning set folders The Extract vignettes for Plankton Identifier tool will extract vignette hereby The last project used is automatically selected by Zooprocess except if you decide to change it A lut txt file will also be written in the project con
22. and abort process saving nothing RS t RI Press YES to process next one Press NO to end and SAVE Press CANCEL to ABORT and save nothing Mo Cancel Notes If you want to draw more than a single line on a vignette draw the first one press Yes to process next one click on PREV to go back to the previous image and draw an additional line Up to 3 lines can be added in a single vignette If you process frame 2 the vignettes of the doubloons duplicates are not displayed as the software considers that you processed them in frame 1 You can move forward or backward in the vignettes to check the job already done 68 If you press No to end and Save and if you have drawn at least one separation line this menu is displayed DO NOT press Cancel as your work would be lost RS ti i Press YES to process image now Press NO to end now and process image later Mo Cancel If you press YES the image will be processed again using the settings of the config file that you have chosen above If you press No the separation mask will be saved You will then have to process Image as usual Do not forget to select the separation tool in single mode In that case accept the mask as it is when asked to EDIT the mask DO NOT EDIT it The separation mask will be included automatically in the Batch process is you keep the default settings 12 1 4 Removing a separation mask There is no special tool to remove existing separa
23. and process it in the same project Some tools close Zooprocess themselves after process to reset memory If you want to create or select another project press Cancel to close Zooprocess and launch it again 11 7OOPROCESS x OK Cancel 5 Basic sample scanning and processing procedures Refer to the dedicated chapters of the manual for more detailed information e Corrected VIS img e Segmented img e Outlines img N lized 1 ormalized img e PID log meta meas Metadata Neca Log file tadat Log i PID file gt dat1 txt file HEADER Scanning amp Process Log Metadata Normalized Img Log file BLANK img 1 line of variables object Minimum Digital ARCHIVE This procedure uses the default parameters that are saved during the installation of Zooprocess and the creation of the working projects These parameters should be kept for all projects insuring inter comparability of the data issued from the different Zooscans Always check that you have selected or created the proper project This will insure that you will always get the same scanning parameters and that you won t have to re enter the default metadata for your sample 12 5 1 Create NEW project or Import existing project This operation is to be done only if you want to create a new project You can also use it to import an existing project from USB drive for example 5 2 SCAN CONVERT Background Image The default option
24. awd acelsbietnencaieteannaben saa A sam ueleaaes 100 D CUSTOM OM a r E cnsecherimtedone 100 KAOS N STS TO a aaa cae cease E S 100 15 11 VErSIOn O IS aeaee a E AO 100 19 12 Verion OF icere a 101 15 13 WW CUSTOM Gol nar E E 101 DIe VenO O O er E E S 101 15 15 VCE SUOMI OO csr a setscet tase te eaten es esas uh seat eatin bas E 101 1 About this manual The new Zooprocess 7 00 version is now fully compatible with the Zooscan the Underwater Vision Profiler and the Flowcam A new Zooprocess manual is now available on top of this Zooscan renamed one from which I have removed the unnecessary chapters This manual has been written as a reference document for the Zooprocess software User can find all the necessary information to fully understand all the available tools User can anyhow only install the software and process his samples using the default parameters as described in the BASIC sample scanning and processing procedures chapter e If you want to install Zooprocess read chapters 1 2 3 and 4 e If you have to create a project read chapter 4 1 first e If your system is already installed and your project created read chapters 4 2 and 4 3 first User should also rely on the Short operator guide available on the website 2 About Zooscan scanners and software 2 1 What are the differences between a scanner and a Zooscan Scanner is used in Zooscan but a Zooscan is much more than a scanner e tis waterproof and constr
25. dati txt or txt file Mes documents 20090519 1616 to validate fae aie Bureau Mes documents oe Poste de iT Norm de fichier tangles PID _process Sorted_vignettes 20090512 2119 to_validate Select Favoris Fichiers du type Tous les fichiers Annuler If you select to UPDATE all dat txt files in Pid_results folder you will be allowed to save a unique datafile containing the data from all the datl txt file The datl txt files will be updated to include a new prediction column named according to the date and time of the process If the _datl txt filename differs from the image name another file named imagename_datl txt will be created Do not forget to remove the previous _dat1 txt files for the same image READ IDENTIFICATION from folder sorted vignettes version 6 11 Label Item amp pred_ fields must exist in the datafile Label lt gt Image name ltem lt gt Organism N File header must include the column names Open a TEXT or CSV file or select dat txt option Imagename must be inside the text file Only tabs or space separators are allowed tabs separated files can size up to 1500 rows ones can size more than 60 000 rows UPDATE all dati tt files in Pid_results folder V Save all data as a unique datafile named Id from sorted vignettes 20090524 2211 tt if UPDATE all dat1 ttfiles in Pid results folder is selected V Convert all Id to lowerca
26. folder OD measurement adjust grey level to the reference value if selected Find the transparent frame borders Subtract background background image or rolling ball Adjust the image quality version 4 03 natural or log gamma2 Clear outside of the transparent frame limits and add black borders Important notes on the subtract background methods All the images from a same project must be processed using the same background option and image quality adjustment Do not mix Rolling ball with any of the other methods except if no background can be used for a problematic image All the images that will be processed for identification using the same training set must be scanned and processed using the same parameters The options that can be set in the configuration file are 1 Rolling ball 2 Image SELECT background user selects manually the background that will be subtracted to a single image or all the batch processed ones 3 Image CLOSEST the background scanned at the closest date to the image scan will be automatically selected by Zooprocess 4 Image RECOVER a If the image is processed for the first time the CLOSEST function is applied b If the image is reprocessed the background image used for the first process is re used If it doesn t exist the SELECT method is applied in single image process and the CLOSEST is selected in batch mode Important notes on the Image quality adjustment methods All the ima
27. for processing images containing large opaque organisms This tool allows you to scan a background image blank to be subtracted from the sample image The method should be well tuned and controlled but we got the same results same measurements 1 on images processed both methods successively rolling and blank The background image MUST be scanned and processed AS the plankton images will be e Same frame e Same color balance e Same LUT file option The macro creates a Zooscan_back folder in you project folder if it doesn t exist yet It uses the vuescan ini file from the Zooscan_config folder of the project to scan images at 1200 dpi that are averaged into one final background image The final image will have a 300 dpi resolution nevertheless is the vuescan ini scan resolution 25 SCAN BACKGROUND CONFIG MENU Project essais duplicates _sep ZAQQSCAM installed modelis Biotam VUESCAM installed version is 6 3 25 The SCAN file is the same that you will use to later scan images Select the right size offrame narrows large according to the file name Your configuration filename contains vwescan and either narrow or large words SELECT SCAN CONFIG FILE in Etifooscan_essais duplicates sepiooscan_contigh vuescan_zooscan_1200dpi_frame 2_large ini OK Cancel The whole background scanning and process takes one minutes per image and should be performed at least once a week better everyday if th
28. for removing the background heterogeneity is to scan a background blank and process conversion it as a normal image This blank will then be smoothed and subtracted from the plankton image Follow the steps and do not change any setting If the Zooscan_back folder doesn t exist yet it will be automatically created We recommend scanning 2 images that will be automatically averaged in a background DO NOT forget to wait more than 30 seconds after each scan ends and before the next one The background operation should be performed frequently once a week once a day at the beginning of the working day It has NOT to be done before each sample scan Zooprocess will process the samples using the closest background We recommend to scan daily background to insure proper supervision of the Zooscan system and insure that the Zooscan is well cleaned before scanning any sample This is an example of a normal Background image It is clean no object and the borders of the transparent frame are visible on the four sides of the image There are no bubbles below the OD reference on the right side You cannot see the meniscus on the sides because you have filled it above the first step of the frame 13 5 3 Prepare sample to be scanned Sample is first rinsed under the flume to replace preservative by fresh or salted water If the splitter N1 aliquots V N2 aliquots 4 Objects separation 100um sieve Scanning Filling Metadata for
29. from config file units for the Results from MinSize converted to area in pixel from maxSize converted to area in pixel You ve selected to work with separation mask No existing mask an empty one is created now 51 Analysis_Method from_global_image Analysis method Zooscan_particle_analysis txt macro This macro i15 to be modified to avoid memory problem and to measure more parameters than the actual version modified in version 1 02 CRITICAL Analysed_Objects 86 Results from the initial particle analysis Analysis_Method from_global_image Successive analysis because we have splitted or organism Analysed_Objects 86 We have only cut a small part which area is below the Max Area It is thus not added as a new object TAG CREATE No existing tag in the Results file We create now the Tag column and all organisms are set to 1 true as default value TAG EDIT Tag edition Results SAVED meas txt file recorded now in the sample subfolder Date_end 20060323 1105 End of Process date and time Data Data section The _meas txt file is loaded and printed in the PID Since version 3 11 the tagged 0 and tagged 2 doubloons objects are included in the data section and the meas txt file Item Label Area Mean StdDev Mode Min Max X Y XM YM Perim BX BY Width Heig ht Major Minor Angle Circ Feret IntDen Median Skew Kurt sArea XStart YStar t lag Lbeag_cemo_1794772235 797 24 247 24471557 25172950 6
30. made using a dedicated learning set The vignette extraction is performed if selected just after the Particle process Depending on the process chosen it will be done either e After the Particle Analysis e After the Separation e After the Tag Zooprocess loads the _visl jpg or the _vis1l zip file and uses the Results table to create thumbnail images and store them in the sample sub folder All the organisms listed and tagged 1 Gf tag exists in the PID file are extracted in this function If you want to force Zooprocess to extract the false tagged objects too 0 or 2 you must select this function in the CONFIG menu for Process above The size of the vignettes is issued from the BX BY width and height data of the Results The dimensions are increased of 20 to be sure that the whole organism is displayed even if the treatment did not outlined well the appendices Zooprocess adds a bar scale of the length defined in the config file 1 mm This scale is displayed at the bottom of the vignette in an added area If the object is smaller than the scale bar the vignette is enlarged to include this scale The vignette contrast may be enhanced according to the config file gamma parameter This option will not change the objects measurements in the pid data file The resolution issued from the scanning information is used there to scale the image and the corners of the box containing the organism are displayed 44 rg_demo_1_15
31. more secured 2 Version 4 03 Improvement of the IHM in Extract vignettes to folders tool New order of the Scan and Process tools in the main Zooprocess window Image process can now be set either to neutral or log gamm2 to better adjust the image quality It also permits to get same results with both rolling ball and image background subtract methods Modification of the Create Project tool to include this new function Modification of the Calibrate threshold tool for the same reason Modification of the single and the batch process tools o to allow skipping already converted processed images o the closest background image selection replaces the last option Conversion macro secured for large images Adding of the checking tool based on segmented images Zooprocess installation simplified and Z icon start added in the main ImageJ toolbar 3 Version 5 00 Zooprocess 5 00 is now ready for the future Hydroptic v1 and Hydroptic v2 Zooscans systems Bug fixed in the case of particle re processing in batch and single mode Prepared to be updated for the new Vuescan 8 4 57 version v5 00 still work with Vuescan 8 3 23 Calibrations tools improved to help users checking their systems Bug fixed in the case of particle re processing in batch and single mode Rolling ball setting is now adjusted automatically according to the image resolution Bug fixed in the SCAN amp PROCESS tool the log was not correct in the raw f
32. of vignettes in each sub folder It allows a better estimation of the results from the Plankton Identifier confusion matrix You are first asked to select the source folder This folder must contain sub folders with sorted vignettes and all the PID files for these vignettes You can select either an existing learning set or a folder containing validated vignettes where you will have added the PID files EE _ x PROCESS INFORMATION Create a sub learnigset from sorted vignettes A user defined number of vignettes from each id folder is randomly copied into the new Learningset folders The PID files are also copied Press YES to continue E select SOURCE folder Sorted vignettes folders POS 17_ 7 119_te_valelabe 200806 19_1615_te_valelahe p 20005 19_ 16 16_te_valldabe jo SOS li al to vadat Pa 104 a ldd vabdated Pichia chi ppi Tours kes fichiers z You are the asked to define the destination directory to place the resulting learning set and to select the number of vignettes to be randomly selected If the number of vignettes of a folder is not sufficient all vignettes will be selected 93 14 Zooscan diagnostic FOOSCAN DIAGNOSTIC tools Check Normalization with OD discus 14 1 Analyse background images Copy all RAW background into a dedicated folder and process the tool to get the following image for the RAW images upper graphs and the normalized images bottom graphs 60000
33. post processing you don t need to install the Zooscan drivers that are the scanner drivers e Connect the Zooscan to the computer e When the Zooscan scanner is detected insert the provided Epson CD into your PC and let the computer find the more suitable drivers e You can now scan with the system 3 2 Install VUESCAN optional Note Hydroptic provides NOW a professional licence number with their Zooscans e Zooprocess 5 01 requires Vuescan version 8 3 23 or 8 4 57 Execute vuesca83 exe 8 3 23 or vuescan84 exe 8 4 57 from the Zooscan_Install zip archive or from the download section of the website The licence number of the previous version remains usable if you update to 8 4 57 e Install Vuescan on the root of one of the computer hard drives Follow standard installation e If it is a first installation buy Vuescan Professional license from www hamrick com reg html Enter license number in the Help menu of vuescan e Copy the vuescan bmp file from the Zooscan_Install zip archive into the Vuescan folder replace existing file 4 Launch Zooprocess for ZOOSCAN 4 1 First start CLOSE ALL OTHER APPLICATIONS BEFORE LAUNCHING ZOOPROCESS LET ZOOPROCESS WORK DO NOT TRY ANYTHING DURING PROCESS When started for the first time Zooprocess automatically creates the proper folders and configuration files on the selected drive We recommend installing the Zooprocess root folder on the C drive and the proje
34. recover the measurements from the previous measurement file and some measurements exist for a vignette This vignette is skipped and you are advised by this message a 8 Cx Vignette 10 32 skipped Measurements recovered If you are processing a frame 2 of a sample the duplicates are skipped and this message displayed EE tit Duplicate or false organism Vignette 25 34skipped Press YES to process next measurement Press NWO to end and SAVE measurements Press CANCEL to ABORT and save nothing Mo Cancel 74 12 4 Close all opened images This tool can be useful to close opened images even if Zooprocess automatically close opened images before any memory requiring process 12 5 Projects management 12 5 1 Create NEW project 12 5 1 1 Previous version without project organization If you ve already worked with previous versions of Zooprocess which did not allow you to sort your files by projects you will have to do some manual adjustments in your folders to import your previous data into the new system In that previous case launch Zooprocess and create a new project in the main menu select Create NEW project tool and enter the project name when asked for All the necessary folders will be automatically created on the selected drive Do not create any scanning file during the procedure Exit Zooprocess Copy your files from the main Zooprocess _raw and _work folders into the created pr
35. size of the pixel size of the pixel see ImageJ only for Stack unit for all the measurements background subtract value CRITICAL minimum ECD to keep particle during analysis maximum ECD to keep particle during analysis length of the line to be drawn on the vignettes position of the OD reference for measuring the grey value if greycor gt 2 The distance in inch from the 82 right side of the maximum image scan able with the Zooscan not the frame This value is set in the calibration procedures of Zooprocess CRITICAL doyabspos_inch 4 02 position of the OD reference for measuring the grey value if greycor gt 2 The distance in inch from the top side of the maximum image that can be scanned with the Zooscan without the frame This value is set in the calibration procedures of Zooprocess CRITICAL bleft 16 value for the LEFT mouse button click broll 10 value for the ROLL mouse button click bright 4 value for the RIGHT mouse button click contrast_pourcent 1 5 gamma for vignette enhancement version 3 11 doubloonxy_inch 0 05 tolerance for the doubloon position in X and Y doubloonarea_pourcent 0 4 tolerance for the doubloon position area greylimit 10 if the measured grey level differs from the reference greyref set value show a popup control menu to user Help avoid correcting too much the grey level of an image This value is to be set according to our shared experience of Zooscans frame both information on the f
36. subfolder e LOG file inform about the existence of correct LOG file in the subfolder e Par metadata file is related to the CNRS Zooscan previous image process only e Separation mask exists if you have already treated the image and separated connected objects 34 Tag data exists as a column in the existing _meas txt file if you have already tagged objects or if your image is the second frame of a splitted large image Ident data exists only if you have already entered the manual identification process which creates a _ident txt file in the sample subfolder The SELECT IMAGE TREATMENT PARAMETRES section allows user to define the operations to be performed Defaults options are automatically displayed according to the files and data already processed An information is displayed here about the background subtract method selected in the configuration file Correct grey level if the greycor parameter in the config file is different than 0 you can choose to adjust the image to the greyref value of the config file Note that if the difference between the measured grey level of the OD reference in the image and the greyref value is higher than the greylimit set in the config file a warning will be displayed during the process Save the raw converted image from the _scan folder as a ZIP archive If selected the raw converted image 8 or 16 bits will be stored as a ZIP archive in the Zooscan_scan _zip folder If a file already e
37. this ratio to define the WHITE point of the 8 bit conversion e OD range 1 8 value is also adapted It means that objects having a 1 8 range 64 times darker than the background of optical density will be well imaged by your system This range and the ratio will be used to define the BLACK point of the 8 bit conversion 83 e Minimum 16 bit scale the grey level value that will correspond to grey level O black in the 8 bit converted image used ONLY if the automatic grey adjustment is not chosen e Maximum 16 bit scale the grey level value that will correspond to grey level 255 white in the 8 bit converted image used ONLY if the automatic grey adjustment is not chosen White Point WP Black Point BP Image median grey level MG Ratio ratio OD range OD WP MG x ratio BP MG log OD x ratio Zooprocess 5 05 allows now processing images acquired with the narrow and the large frame at 2400 dpi as a unique frame canceling the need of checking for duplicates and reducing the processing time and the number of resulting files In order to Keep the same settings for the projects created before this new release of Zooprocess we recommend keeping the default settings of 220 Mo for the splitting limit All new projects created with versions above 6 11 will have a setting of 800 Mo canceling the splitting for the narrow and the large frames scanned at 2400 dpi 84 LUT FILE version 5 05 date 2008 05 14 a xj
38. to 8 bit calibrated image PROCESS Converted Image process one STATS and GRAPHS Manual measurements on selected vignettes Manual measurements on all vignettes VIEW Image with outlines VIEW vignettes Create Learn PID file from a TRAINING SET EXTRACT vignettes in folders according to prediction LOAD identifications from sorted vignettes Extract vignettes for PLANKTON IDENTIFIER CREATE subset of a Learning Set from identified vignettes random EDIT configuration file EDIT LUT file e CHANGE CHECK Zooscan version amp parameters e Create NEW project e CALIBRATION Tools e DIAGNOSTIC tools e OTHER Tools e CLOSE all opened IMAGES e UVP5 main menu These tools are presented in a different order in the following chapters of this manual They have been grouped by main functions 1 Image acquisition Image conversion if necessary Image process single and batch modes Particle process single and batch modes Checking tools Separation tool Measurements tools Plankton Identifier related tools Diagnostic amp Calibration tools SH NAAARWD 10 ZOOPROCESS version 6 11 2009 05 1s x SCAN CONVERT Background Image Da essais_duplicates_sep v The Vuescan installed version is detected automatically and displayed All the existing projects are automatically listed and become available version 6 16 When you have processed one tool the project remains selected and you are asked again to select a tool
39. 00 Median_level 19984 Measured median grey level of the raw image INEO Hardware Perfection4990 Flatbed Software Vuescan Imagelype 4oo0scan Resolution gt 2400 Author GG Sample Sample information from the metadata file SampleId rg_demo Scanop Marc_Picheral Ship Sagitta Scientificprog ptB StationId ptB Date 19990416 0000 Latitude 43 41 Longitude 7 18 Depth 85 CTDref CTDfilename Otherref Other 49 Townb 1 Towtype 3 Nettype RG Netmesh 690 Netsurf 0 7854 Zmax 7 5 Zmin OQ Vol 58 905 Fraction Code A Fracld a Min 200 Max 999 Subsample SubPart 32 SubMethod CellPart 1 Replicates 1 Volini 1 VolPrec i Observation NaN motoda Process General Zooprocess header ConfigFile Biotom_framel_narrow_OD_config txt config file loaded Version 10 03 2006 Picheral CNRS Multiple_image_process NO Date 20060323 1053 Image Process Date 20060323_1053 YES for BATCH mode Process date and time Image process section visl_file PROCESS PROCESS or LOADED if you have not computed the vis file now Processing time for the vis file ConftigqdDatalmo rrolitnorCnRs LOV 2006031441004 0473 2400 0 9704150t270 02570 2 Image_process Zooscan_lasep Version 13 03 2006_Picheral CNRS Path G Zooscan Image rg_demo_1 tif frame 1 Raw_image_depth 8 bits Raw_image_size 42410352 Raw_Image_BackUp_as_ZIP YES Final_Image 8bits Image_width 4006 Ima
40. 16 Graphs amp 15 Histograms Save ALL created graphs Save ALL created histograms Froma sample fincludes frame 2 if exists From all samples ofthe project JK Cancel 11 3 1 PLOT 16 Graphs amp 15 Histograms e Displays graphs with organism number on the X axes and useful parameter on the Y axes e Display a 2D graph with Major as X axes and Minor as Y axes e Display histograms of these parameters plus basic stats e Create a data file with the stats that can be also saved This data file is more complete than the one created in the BASIC stats tool as it include the STD and histogram values for all the parameters e Displays image information onto the graphs e The graphs are not closed at the end of the process Use the Zooprocess Close all opened images tool to close them or start another tool for this purpose e ESD Minor and Major are in mm 11 3 2 PLOT selected graphs amp histograms You can there create your graphs by selecting the available X and Y axes from the pid data section If the organism number is selected as X axes both histogram and graph will be displayed 62 You can also choose the axes limits rg_demo_1_histo_Mayjyor O x 300x240 pixels 86 bit FOF i SS 0 325 Count 86 Min 0 35 Mean 1 74 Max 11 31 Std Dew 1 93 Mode 0 60 6 Bins 256 Bin Width 0 04 Zoopracess Img r demo 1 Major Value 6 16 List Copy Log Count Plot rg_demo_1 Major
41. 3715740772950 467136 84 254 Ie oop 04 Tae ee owe he Oe Oe TO oe LIS Vow eS L00 9542 952 1 So rg demo 1 l0ZZ7 217 057 26 089 2437 147 251 9924 047 10549 3597 4992 107 105949 3 OF Zoos oe JOP LOO2 849746547 30 Ow F450 1 06 LO oO wl 221000 2267 0 6 Q40 LOGS 2984s 1052 731 10 2 Meas txt file description The meas txt file contains a copy of the DATA section of the PID file As for the PID file all measurements are in PIXELS 10 3 Measured variables Grey Level Parameters gt Mean Average grey value within the selection this the sum of the grey values of all the pixels in the selection divided by the number of pixels gt Max Maximum grey value within the selection gt Min Minimum grey value within the selection StdDev Standard deviation of the grey value used to generate the mean grey value gt Mode Modal most frequently occurring grey value within the selection gt Median Median of the grey value used to generate the mean grey value gt Skew The third order moment about the mean It is the measure of lack of symmetry The skewness for a normal distribution is zero Negative values for the skewness indicate data that are skewed left and positive values indicate data that are skewed right 52 gt gt Size Parameters gt Kurt Sum Area The fourth order moment about the mean It 1s a measure of whether the data are peaked or flat relative to a normal distribution Positive kurtos
42. 4 is an alternative to the first separation macro that was available in the main Image process flowchart It can handle images up to 2400 dpi divided in two frames This function is easier to use as the vignettes of the organisms are displayed one by one These vignettes can also be sorted by decreasing size if the option is selected 66 Outlines can be added on the vignettes contrast can be enhanced and the background particle and noise can also be removed Detailed process Select the Project Select the tool Separation using vignettes from the main Zooprocess menu Select the configuration file to be used to reprocess your image after separation of multiple This configuration file should be the same than the one used for the initial image and particle analysis of your sample You can anyhow cancel reprocessing the image after separation and do it afterward using the Batch image process tool Select setting for the vignettes display the vignettes will not be saved o Sort vignettes by decreasing area default the vignettes having the largest width x height value will be displayed first as we observed that most of the multiple objects are large ones o Add outlines default the outlines should be added to better see the contours of the organisms If the outlines are not added the corners of the rounding box are displayed on the image o Clear outside outlines allow to clean the background of the image It should be sel
43. 5000 MEAN MODE MEDIAN grey 16 bits Stddev 16 bits 0 time day 663 Zooprocess narrow frame 200707 13_1050 to 20090506_1459 grey 8 bits Stddev 8 bits MEANOD 50 0 time day 663 0 time day 663 14 2Check normalization with OD discuss previously Process threshold OD disk inter calibration This tool allows user to check the complete inter compatibility of their Zooscans providing that the same image process method are used It uses calibrated disk of Optical Density OD 0 3 and 0 9 that we can provide upon request STEPS e Create a dedicated project for this operation and create ONLY the large 1200 dpi scanning file e Edit Configuration file to change min ESD to 4mm e Scan a background using the large frame e Scan and process a sample of the provided targets The sample name must contain the word targets at least User MUST SET the scan resolution to 300 dpi in VUESCAN prior scanning the targets and let process the image as a normal sample The targets will be re processed in the next step Special car must be applied to avoid superposed and touching disk that would lead to wrong grey measurements Take care of remaining bubbles too The name of the image must include the word targets in order to be selected by the tool 94 e When the image is scanned and processed enter the DIAGNOSTI TOOLS menu and select Check Normalization with OD Discus FOOSCAN DIAGNOSTIC to
44. Close VW UCS CAMs tic ten cssrncn tenia dacs ettaaySuneduen E 24 17 SCAN CONVERT Back CrOu nd ia Ge escini aiaia 25 8 Convert Raw images to 8 bit calibrated images nneseessseoeeeesssssssseerrssssssseerersssssssses 28 8 1 Modu UOM E etuentsdasataaeiualeudineens ecemteucie tains 28 S 2 ZLOOPLOCESS CONV EFSIOM essiri sus Madd siaebesdous a austiigaarsiebnedane hauddendebes 28 9 Image processing and particle analysis single batch cecceecccccccessssseeeeceeeeeeeaeeeeees 30 9 1 Mirod cton and FLOW CAI ssa R A 30 9 2 CONVERT amp PROCESS Images in batch mode s snnneenesssseoeenssssssssseerrssssssseeerres 32 9 3 PROCESS Converted Image process one sssseessssssssseeeerssssssssseeressssssseeeeres 32 9 3 1 ADOUt thelmacel Status Dal einn Dira 32 Sete Option selection and Config Menu for the process cccccscccceceeeesseeseeeeeees 32 9 3 3 Sine le Tila Ge Process areren aa N 33 9 3 4 Background Warning re processing sseesesccccecceeeeeeseeeeeceeeeaeeeeeeecceeeeeeaas 33 93 3 Process parameters image AND particle process ccsccseceeeeeeeeeeeeseeeeees 34 9 3 6 Process parameters for particle re process cccceccessseeseeceeceeeeeaeeeeseeeeeeeeeaas 36 9 3 7 Check or correct metadata information cccceccccccccccceesessseeeeeeeeeeesseeeeeeeeees 36 DAs Process TIO W EMAL eana a a E a dewtereeliseuese sete 36 9 4 1 afi set Win F282 01 20 E Mee meena E Die N to
45. ET gamma 0 4 5 ann CLEAR background SELECT Image fram the project targ ets_1_1 The data from the existing length file if exists and the data file are displayed to help choose the vignette Select a data line and press OK to measure or Cancel to END VIGNETTE SELECTION x Select line and press OK to visualise ESD and Feret diameter are converted in mrm Oo MOT press CANCEL to EMD 0o MOT close any vignette during process Mo Area ESD Feret Fractal Lengthi Lengthe MeanGrey Bs By Width Height The vignette is then created from the vis1 jpg image 71 Press with the roll button onto the vignette to activate the tool and then draw one or two measurement lines using the left button When the measurement lines are drawn the values are displayed in millimeters as they will be saved in the file SSS ti MEASURES Length N71 2 2397 Length N 2 0 9144 Press NO if you want to ABORT and SAVE data GOON MEASURING Mo Cancel Select another data line to process other measurements 12 3 Manual measurements on all vignettes This tool added v 4 00 allows filling a length1 txt file with up to three manual lengths measurements for the organisms of an image If a previous length1 txt file exists you can either keep the previous measurements or reset them to zero The length1 txt file is saved into the vis image folder It contains the following columns Ttem Tag Area Major
46. F image files M Skip allready processed images W ZIP 16 bits RAW image if archive doesnt exist The information from the lut txt file of the project is displayed e Select single or batch process e Select the files you want to convert TIF or ZIP e Select to save the TIF file as a ZIP archive If set to yes the ZIP archive will be saved in the project _raw folder only if the archive doesn t exist yet An image file TIF or ZIP 1s available for conversion only if its corresponding meta and log files in the _raw folder exist in the raw folder If the process ends correctly this message is displayed ti End of image conversion TIF file s created in the Zoo0scan_sean folder meta amp log files copied into the images _work sub folders 29 Note Zooprocess conversion will work only if you have a lut txt file in the project Zooscan_config folder This file can be edited using the tool Edit LUT file in Zooprocess We recommend keeping the default settings of this LUT file as they insure you that your system is fully inter calibrated with others 9 Image processing and particle analysis single batch 9 1 Introduction and flowchart Zooprocess 4 01 allows you both to process a converted TIF image saved in the Zooscan_scan folder of a project and to re process images for particle analysis directly from their visl image folder
47. LEAR outside outlines SAVE image with outlines SELECT Image from the project B03042801 Both outlines and object labels are displayed onto the image 64 lox 3244x10584 pixels 38 bit 33MB The resulting image can be saved as a zoocont jpg image in the same folder than the source visl image If a previous file exists 1t will be replaced 12 Additional tools Zooprocess offers different useful tools allowing performing separation from vignettes and manual measurements and more 12 1 SEPARATION 12 1 1 Separation mandatory flowchart e Scan your sample e Process the image frame s including the doubloons detection Do not select the separation tool available in the main image process menu e Process separation using one of the separation tools in the main Zooprocess menu Always process frame 1 first if working with two frames images e Process again the images either into the separation tool or in batch mode later The second option is more suitable if you have installed version 6 15 12 1 2 Separation from B W msk image This new tool added in version 6 14 is an easy and rapid way to routinely separate touching objects It utilizes the B amp W _msk gif image issued from an initial image and particle process Detailed process e Select the Project e Select the tool Separation using vignettes from the main Zooprocess menu e Select both the Image to separate and the config file If an image has
48. Minor Len1 Len2 Len3 T2 Manual Measurements v 3 14 x Project essais duplicates sep Press ROLL mouse button on the vignette to activate the measure tool lf gamma 1 gt contrast enhanced lf gamma 1 gt image smoothed lf gamma 1 No gamma correction SET gamma 0 6 45 Lan ADD outlines on vignettes M CLEAR background SELECT Image from the project targ ets 1 1 SET Mouse lett fis SET Mouse roll fio SET Mouse right If you have chosen to recover previous measurements and the file doesn t exist you will be advised as below If the file is OK the second message is displayed LENGTH file notfound lt willbe created atthe end ofthe process Mo Cancel The vignettes are displayed and you can activate the measuring tool by pressing the roll button of the mouse on the image A grey square indicates that you can proceed by drawing the measurement line using the left button of the mouse You can 1 Move to next vignette using the SPACE bar of the keyboard or clicking on the NEXT button of the vignette 2 Move to previous vignette on the PREV button of the vignette 3 ERASE all previous measurements displayed by black lines on the vignette 73 You can t modify a previous measurement The solution is to manually remove it from the length1 txt file or disable the Recover previous measurement tool PE lolx 150x113 pixels 6 bit 16K If you have chosen to
49. Note _1 and _2 will be added to your sample name The software checks 1f the file already exists in Zooscan_scan or _ raw folders If it exists the following window will be displayed till you enter a sample name that doesn t exist yet Note all the entered names will be converted to lower case and space character replaced by under score ones Zooprocess will NOW replace the vuescan ini file in the Vuescan folder 6 5 4 Plankton Scanning Procedure Note 1f you leave the sample in your Zooscan for a long time prior to scanning bubbles may appear in the cell This is a problem that may make Zooprocess count bubbles as objects Users should place samples in the Zooscan room and use dilution water that has also been kept at the same temperature Zooprocess will display a list of operations to perform in order to scan your sample If you are not familiar with Zooprocess always KEEP the default parameters that have been automatically set during the installation press OK and do not modify You can now set your parameters 20 e The name of the selected vuescan_zooscan ini file is displayed e The operation performed is indicated scan or scan_process according to the selected menu in the main Zooprocess page e Information is given on the 16 bit raw image according to the content of the vuescan_zooscan ini file NOT SAVED or SAVED SCANNING PROCEDURE version 3 05 Operation pertormed sca
50. abel This tool is helpful to load a complementary manual identification validation made by sorting vignettes extracted using the EXTRACT vignettes in folders prediction or vis folder tool The tool can both work using the default sorted_vignettes folder or a user selected directory READ IDENTIFICATION from folder sorted vignettes version 6 11 x Project essais duplicates sep Defaults sorted vignette folderis Evwooscan_essais duplicates sepiPiD_processiSorted_vignettes Fichier Editon Affichage Favoris Outils Fr c dente gt T a Rechercher IIE Dossiers a oF x ie m Dessiers adiscus_dark oo Fid Its B T a Sdiscus_light P rect_dark E Sorted_vignettes Se inte HI me 40090512 2119 _to_ validate Srect_lignt O discus_dark E Analysis_20090512_b bet C discus_light E Analysis_20090512_b_rectangle_vifr2_a_1_datl tet fm rect dark E Analysis_ 20090512 b_targets_vifr2_a_1_dati bet ee E Extract_to_folders_log txt z Id_from_sorted_vignettes_20090512_2125 tct C rect_light 20090519 _1615_to validate 20090519 _1618_to validate w This macro read the position of all the vignettes contained in the selected Sorted_vignette subfolders first rank only It adds the folder name into a new column of the txt selected file a new column is added to the file each time this routine is used 91 Select sorted vignette folder containing Id folders and
51. aining for the completion of the task alelx File Edt Image Process Analyze Plugine Window Help Modo N4sINAl_ Ne 7 _ mia SAVING FROM ZQOSCAR TO DISK WAT 31 seconds 6 5 6 Fill metadata form When the file 1s completely recorded on your hard drive you have to fill the metadata form If it is the first time you use Zooprocess a default file as in the example below named metadata txt is created in Zooscan_meta folder of your project Each time you press OK in that window this metadata txt file 1s updated with the more recent values 23 Project essais_duplicates_sep Ima x Sample ld fsample_002 2o00stan Operator qualif Ship qualit Scientific program qualit Station Id th a Sampling date vy MMOC HHMM 20050119 11 Latitude DD NM for Morth OD MM for South Longitude ODD MM for West DDD MM for Est oo oo ET Aa Bottom Depth tr CTO reference filename CTO filename Other reference Other_referer Humber of tow in the same sample Tow type Oblique 1 Horizontal 2 Vertical 3 oo oo Mettype WP JB Omori Lia La Net mesh cod end ym co oo Met opening surface m2 oo Maximum Depth imi lt max J im im im Minimum Depth fn 2min Em Lom Lom Filtered volume m3 Fraction Id Fraction min mesh urn oo a jo i Fraction max mesh um Fraction spliting ratio x ih 99 zZ Remark a Subhlethod jmotoda D Can
52. already been separated SEP is added to its file name 65 SEPARATION from MSK image v 614 x Project test rectangles This tool requires a mouse equiped with a ROLL button WORKS ONLY on SINGLE FRAME SAMPLES SEP a separtion mask exists forthe sample SELECT Image to process from the project rectangle_vifre_a_1 The separation mask exists SELECT PROCESS COMFIG FILE from the list SELECT WORKING OPTION process_install_both_config te ADD to existing ADD to existing i OK Cancel If you have selected an image having being separated yet you have the option to Add to existing or to Create a new mask The black and white image is then opened and instructions displayed NEXT OBJECT x FIRST OBJECT x INSTRUCTIONS INSTRUCTIONS Utilize ZOOM amp MOVE tools to go to the objectto split Utilize ZOOM amp MOVE tools to go to the objectto split Press Roll Mouse Button before drawing line Press Roll Mouse Button before drawing line OK to draw another line PRESS OF to start process CANCEL to END drawing lines i Cancel i Cancel If you press Cancel to end separation the mask is saved and a conclusion screen displayed RSS ti i NORMAL END 1 lines added The separation mask is saved Process again images to include mask DESABLE skip process again images option 12 1 3 Separation using vignettes This new tool modified v 3 1
53. and save selected vignettes using different methods e Select vignette from the data section of a datl pid file previously selected e View vignette from any txt file providing image name and position of the organism This function can be used to display vignettes of organisms identified by any software All the information requested is displayed during the process e Select one or more data points on a 2D graph built from a dat1 pid file e From a ident file created by manual identification see above From a 2D graph built from aPIDfle From a TAT file multiple images allowed From a ident file created by manual identification 11 2 1 View vignette from the data section of a pid file IEW VIGNETTES from a PID data section 3 11 x f gamma 1 gt contrast enhanced f gamma 1 image smoothed lf gamma 1 gt Mo gamma correction SET gamma 0 5 5 ADD outlines on vignettes CLEAR outside outlines SAVE thumbnail images of organisms KEEP vignettes OPEN till and of process SELECT Image fram the project Jost 1_50100_large_1 If you have selected this option you will be asked to open a visl jpg file from any project folder In addition you can choose to enhance contrast of the vignette using gamma and save the vignettes If a vignette of the same name already exists in the vis1 jpg image folder it will be replaced Only few data fields are displayed during this process It doesn t mea
54. article analysis writes the _meas txt file in the sample sub folder It also writes some temporary files in the Zooscan_temp folder We consider that the parameters computed by the ImageJ Particle Analysis plug in are basic and insufficient New parameters should be added in near future fractal dimension was added in version 2 01 Writes the outlines file as a _outl gif file in the sample subfolder and a temporary copy in the Zooscan_temp folder Allows the creation of a separation mask to help separating connected objects see remarks below and the new Separation using vignettes tool It can also record an image file named _zoocont jpg file composed from the _visl jpg file and the outlines All the process parameters are recorded in the LOG file that will be included in the PID file The image process is critical for the object s measurements The critical values are e Background subtract method keep the default background image subtract method e Grey level reference in the config file if the systems are correctly inter calibrated we should be able to set the same value for all Zooscans See the calibration section A change in the setting will directly influence the size and the values of the measurements If the MANUAL color balance is used and the default LUT TXT file not modified there is no need to adjust the grey level as the 16 bit to 8 bit conversion will do it much better e Threshold value for the segmentation it
55. cel For successive use the software checks if a _meta txt file already exists in the subfolder named as the sample e Incase of new image there is no existing _meta txt file therefore the content of the default metadata txt stored in zooscan_meta 1s displayed and you have only to fill the information that changed since the previous scan e If you are post processing the image the _meta txt file already exists and its content is displayed The information entered now is not used by Zooprocess This information is only stored for future use in both log and pid data files They will be included in the _log txt and in the datl pid file 6 5 7 Close Vuescan When you press OK in the previous window Zooprocess asks you to quit Vuescan This procedure will record the scanning parameters from Vuescan ini file 24 SCAN PROCEDURE x QUIT YVUESCAN BEFORE pressing OK Cancel The scanning parameters the one that were used in Vuescan are now red and stored in the sample log file 7 SCAN CONVERT Background Image Zooprocess allows to select the background subtract method between rolling ball and different Image selection and method see EDIT configuration file below This last option is recommended only for images scanned using the none or the manual color balance see above The Image RECOVER is the default option The Image subtract methods are absolutely recommended
56. co tT a 0 050 0 100 D 4 in o 4 2 co 4 m 4 is 5 4 OK Cancel This image shows the default parameters from the file created during the installation Below is an example of the file content and some information about the values User should use the Zooprocess editor and must be careful when editing and modifying the fields manually The order of the fields is critical and the syntax too The accepted syntax is name value A space must be included after the symbol and this symbol must be connected to the name All the values used during the image and particle processes are recorded in the _log txt and the _dat1l pid files Background_process recover set to roll if the Rolling Ball Subtract Background is to be used see value to be set below set to select if 81 enhance_thumbnail yes calibration CNRS LOV_ 20060314 jpeg 100 Zip 0 greycor 3 resolution 2400 greytaux 0 9 yminref 0 doyofset 150 doxpos 2 xdimref_inch 0 025 ydimref_inch 0 25 dostd 2 doecart 20 subimgx 0 method 0 upper 245 greyref 124 voxelwidth voxelheigth 1 voveldepth 1 voxelunit pixel backval 300 minsizeesd_mm 0 3 maxsizeesd_mm 10 longline_mm doxabspos_inch 0 3100 you want to manually select the background image set to recover if you want to recover previous background when re processing an image set to last if the c
57. copying optionfile tt into the config folder of the previous project Process also new measurements added in version 6 03 7 You can select the Image process default method for the project See Image Process and Edit configuration chapters about the different options If you have selected one file at least you will be asked to check the scanning positions The displayed values are read from the files created in the main Zooscan folder when you first installed Zooprocess T1 Scanning area for plankton imaging x fooscan selected system is Biotam VUESCAN installed version is 6 3 23 Default values are read from the ooprocess installation settings Change them according to calibration sheet if necessary or Change them according to the needs of your project position Y position xOfset 10600 Yofset 1140 xSize 21290 Ysize 48640 OK Cancel Zooprocess will read the position of the scanning area from the e vuescan_zooscan_calibration OD_level_cadrel ini e vuescan_zooscan_calibration OD_level_cadre2 ini and copy them in two scanning files for your project e vuescan_zooscan_2400dpi_frame1_narrow ini e vuescan_zooscan_1200dpi_frame2_large ini e vuescan_zooscan_2400dpi_frame2_large ini lolx Fichier Edition Affichage Favoris Outils G Pr cedente gt pi Rechercher fe Dossiers wes EB x e m Dossiers E i Zooscan_az luk tet Document texte El E PIC_process E proc
58. cts folders in a separate large capacity drive There must be at least 1 Gb of space on the drive that will contain the Zooscan root folder O x File Edit Image Process Analyze Plugins Window Help E oleoj jaloa elal zul Zooprocess v 6 16 If it is the first time you start Zooprocess you will get this screen F E y WARNING fooprocess is notinstalled correctly in the root of a drive Press CANCEL to abort Press YES to install ooprocess and create a project Mo Cancel Press YES to install Zooprocess and select the Zooscan root folder No data will be saved there except some configuration files and some temporary files Please allow anyhow 500 Mo of memory on the installation drive for temporary files saving INSTALL MANAGER version 5 02 VUESCARN installed version is 6 3 23 Select drive to install the ooscan root folder favoid selecting CD drive Clo Select ZOOSCAN model Biotom i Cancel Zooprocess will now create the necessary folders on the selected drive and ask you for some initial parameters for your Zooscan The parameters of the new Hydroptic systems will be delivered with the machines The parameters of the first Biotom Zooscans have been defined when we calibrated the Zooscans are available at this URL http www obs vitr fr LO V ZooPart ZooScan article php3 id_article 81 Select the Zooscan model from the list The installed Vuescan version is detec
59. d file Its content may vary depending on the process parameters and the choices made by user It may also change depending on the new releases of Zooprocess The comments are written in italic The section that is essential for image post processing is written in blue This section should always be available in the log file too The Image Process section is written in green It is loaded from the LOG file when the vis1 jpg file is loaded for particle process All the measurements in the DATA section are in pixels 47 PID Image Scanning_time YYYYMMDD_HHMM VueScan Input Perfection4990 Flatbed Media 1 Quality 4 BitsPerPixel 2 MakeGrayFrom 2 PreviewResolution 6 ScanResolution 3 Rotation 3 Mirror 1 Input Options 2 Crop Perfection4990 Flatbed AutoOffset 0 AutoRotate 0 CropsSize 0 XOffset 9112 XSize 12056 YSize 48064 PreviewArea 0 PreviewX Offset 10455 Preview Y Offset 1393 PreviewXSize 21173 Preview YSize 48067 Crop Options 2 Filter Options 2 Color Perfection4990 Flatbed ColorBalance 2 WhitePoint 0 WhitePointRed 236 WhitePointGreen 236 WhitePointBlue 236 Brightness 900 Color PixelColors l1 Options 2 Output Perfection4990 Flatbed PrintedUnits 0 Output We named this file pid in order to avoid confusion with the ZI1 file in the data section delimitation Image section begins there This is a copy of the vuescan ini file stored when you quitted Vuescan after
60. de by Plankton Identifier The tool can create the best quality thumbnail images It includes possibility to clear background possibility to add outlines possibility to enhance the vignette quality volume range selection for the particles from Major and Minor random extraction of a fixed number of objects per image possibility to create new extraction sub folders resolution checking Zooprocess reads a text file a csv file tabs and semicolon separator accepted but semicolumns are preferred for large files or all the _datl txt files issued from Plankton Identifier and placed into the PID_results folder The read files are copied into the Pid_results Dat1l_extracted directory and in the sorted vignettes selected folder see below Vignettes are extracted into subfolders of Zooscan_projectname PID_process Sorted_vignettes folder These folders are named according to the identification contained in the file column beginning by pred_ The file must contain at least the columns e Item Label Bx By Width Height pred_ where is the name of the identification method This pred_ column is only need for the Prediction folder extraction The data files will be read in the Zooscan_projectname PID_process Pid_results folder txt and csv files allowed only and the VIS images must be in their respective subfolders As the large frame images cannot be opened by imageJ you can set the maximum size of allo
61. e 6 Launch VUESCAN H Start light tempo press green button on cover af Click on PREVIEVY button in YUESCANM D Check your image Mo white strip no dust onthe OD ref 10 Wait more than 30 seconds 11 Click 5 times on SCAN 5 times for 5 scans D button in YUESCAN DO MOT CHANGE ANY SETTING window size in VUESCAN PRESS OF just after pressing SCAN iirst time in VUESCAN 7 Cancel The procedure is then detailed step by step Please press SCAN first in Vuescan before pressing OK If you ve first pressed OK you can still launch Vuescan and scan anyhow Each of the scanned images is then processed converted using the lut txt file 1f necessary and averaged to create a final smoothed image at 300 dpi You must absolutely perform this operation carefully as any dust or scratch contained in the background will modify the results of all following samples process YOU MUST ABOLUTLY WAIT 30 seconds between the time a scan ends and the next starts to help the Zooscan calibration light to keep warming up If you correct your sample images using the blank background you must process the image completely before removing the sample from the Zooscan It would allow you to scan it again if the quality of the sample background is not homogeneous with the selected background blank You should also KEEP all the YY YYMMDD_HHMM_background_narrow tif blanks in your Zooscan_back folder to be
62. e CLEAR Greytaux 0 9 Greylimitright 206 Right_limit 3140 Greylimitrleft 220 Left_limit 0 Greylimittop 193 Upper_limit 139 Greylimitbottom 210 Bottom_limit 10485 Vis_image_save YES Particules_ Process Version 25 01 2006 Picheral CNRS image process method CRITICAL if greycor set to 3 in config new image width after suppression if greytaux defined above 0 01 ratio of the mean grey level defined to find the transparent frame limits greytaux x meangreylevel measured in the right reference rectangle computed right limit image is compressed and saved as Viet aie particle section Zooscan_separation_analysis txt and son macros ConiigDataP roll tnotCNRS LOV 200603147 1L00 0734240040 97F0FLo0 24 0 02570 25 Resolution 2400 Calibration CNRS _LOV_20060314 Threshold 245 PixSize O 0106 MinSize 0 3 MaxSize 10 Method default 0 3000 10 Measures_unit pixel Min Area 631 Max Area 701207 Separation_Mask INCLUDE Separation_Mask CREATE 23 UFZ OFU FUT A 72 01 24titleit pixel 300 OFO 3FLOFL 0 0 lt 2144 02716 0410 074 070 OAP USFO s LFLO FNAC row The data from the chosen config file for the particle process are written there and separated by from the Image section from config file CRITICAL in mm from resolution above for Zoolmage set in the config file ECD in mm CRITICAL set in the config file ECD in mm CRITICAL from above for ZooImage
63. ected if you choose to enhance the contrast of the vignette o Gamma SEPARATION from VIGNETTES Wiad x Project essais_duplicates_sep This tool requires a mouse equiped with a ROLL button lf gamma 1 contrast enhanced lf gamma 1 image smoothed lf gamma 1 No gamma correction SET gamma 0 5 5 ADD outlines on vignettes W CLEAR outside outlines SELECT Image to process from the project targ ets_1_1 E OK Cancel Select the image to be processed The first vignette is displayed and the progress displayed in the ImageJ status bar 67 a vie 150x119 pixels 58 bit 17K 1 ERASE You can now activate the drawing line tool by pressing the ROLL button anywhere on the vignette A grey disk pops up to indicate that you can draw a line using the left button of the mouse PEZE oxi 150x122 pixels 8 bit 17K You can close this vignette and display the next one by pressing the SPACE bar or clicking on the NEXT button on the image You can close vignette and see the previous one using the PREV button You can erase a previous separation line by pressing the ERASE button The black separation line will be displayed in grey The button will erase all separations made on the selected vignette e When you end drawing a line on the vignette or when you close a vignette this menu is displayed allowing you to o Display next vignette o End and save the separation information o Cancel
64. elected YES or RESET OK Cancel You can now EDIT again the separation mask and go on drawing lines You can accept the mask shown as a black image with white lines and press YES or RESET the whole mask In that later case the vis image will be computed again and you will restart the separation process from the beginning Important note you can actually delete a separation line only manually in the _sep gif file with ImageJ outside of Zooprocess Press YES to enter TAG function if selected in the config menu for the whole process The white lines of the separation mask are added to the original vis image as you can see below The image is analyzed again and a new number of objects displayed in the log window This number should normally be higher than the previous one but some splitted parts may be too small to be considered as particle because they are below the size limit rg_demo_1_ isi jpg 700 loj x 323910584 pixels 85 bit 33MB 41 The final measurement window is then computed and stored in the Zooscan_temp folder as measurementsB txt file 9 6 Tag function If you have decided to work with tags Zooprocess loads again the vis image from the subfolder or from the Zooscan_temp folder if you ve just computed this image It displays the TAG PROCESS MENU window which explains how to process the Cancel button action will be corrected in future release TAG PROCESS MENU x BLACE circle Objects to be
65. ension if the option is selected The tool can also be used to change the default OD and frame positions in the root Zooscan folder configuration files These parameters will then be used when user will create new project These parameters must also be checked before the Grey Level calibration 85 Zooscan selector version 5 02 x Hydroptic v2 ml AU W Update Yuescan configuration file an config folder The previous scanning files are saved and named according to the previous model name If the selected Vuescan version doesn t fit with the Zooscan system this message will remind you the proper choices WARNING EO x VUESCAN 8 3 23 is OK for Biotom and Hydraptic w1 Zo0scans Hydroptic v2 Zooscan requires VUESCAN 8 4 57 VUESCANsx exe installers are available in the Zooprocess install archive from on the Zooscan website 13 Procedure for automatic identification prediction 13 1 Flow chart The Pid_folders are created to help placing the proper files for Plankton Identifier http www obs vlir fr gaspari Plankton_Identifier index php According to its needs and following the Plankton identifier manual user can decide to work using other folders locations but the Zooprocess tools work ONLY in this folder arrangement e The first step is to create a learning set In order to get the vignettes and the PID files we recommend use the Extract vignettes for PLANKTON IDENTIFIER tool to extract vignettes in
66. er JPG 23 03 2006 11 05 J Olnovi2st435 l rg_demo_1_83 jpg Ko Fichier JPG 23 03 2006 11 05 fa O1sept1 1st230 2 rg_demo_1_82 jpg 6Ko Fichier JPG 23 03 2006 11 05 ao O1septi 1st330 2 rg_demo_1_81 jpg 7 Ko Fichier JPG 23 03 2006 11 05 fa O1sept1 1st435 2 rg_demo_1_80 jpg Ko Fichier JPG 23 03 2006 11 05 rq demo 1 lra demn 1 _A inn 189 Kn Fichier 1PG 23inalznnA 11 05 tal te eaan z E E 97 objet s Espace disque disponible 26 0 Go 51 17 Mo Poste de travail e zoocont jpg optional visl image combined with outlines zool jpg optional visl image combined with identifications visl zip corrected image zipped tif image e _sep gif optional separation mask white lines on black background outl gif outlines issued from last particle analysis mskl gif segmented image issued from last particle analysis _meta txt metadata file written during the scan process and maybe corrected during other processes o meas txt data file issued from the last calculated Results window log txt log information on the whole process _ident txt optional list of identifications datl pid text file containing the log file plus the measurement file some additional derived and normalized parameters will be added in the data section in future releases _length txt optional text file containing the manual measurements done on the organisms 10 1 Pid file description We describe here the typical pi
67. ess will process Image AND Particles anyhow 32 i PROCESS Converted Image v 4 03 i x Your configuration filename contains config and either narrow or large words SELECT CONFIGURATION FILE process_install_narrow_config tet 9 3 3 Single Image Process Select the image to be treated from the Zooscan_scan folder SINGLE IMAGE PROCESS x Select image to processin EMzooscan_pointh_rg 9 3 4 Background Warning re processing If you are re processing an image using the recover background option default setting and the background initially used for the initial process doesn t still exist you are asked to manually select another background L BACKGROUND SELECTION MENU x The recovered t20070903_1057_background_arge_manual tit image doesnt exist Select a background image size narrowilarge adapted to the frame size used when the images to be processed was scanned Background image will automatically be splitted if necessary SELECT Background file in EtwZooscan_essais duplicates seplizooscan_backitt20070903_ 1057 _background_large_manual tit 20070903_1057_background_large_manual_tif 33 9 3 5 Process parameters image AND particle process This menu simplified for the BATCH process will then be displayed It indicates the existing file already computed or not in the sample subfolder of the _work folder Keep the default settings targets_1_1 tif PARAMETRES ve
68. ess_install_narrow config txt Document texte C Learning set yuescan_zooscan_2400dpi_framet_narrows ini Parametres de confi 3 Pid_results yuescan_zooscan_2400dpi_frame2_large ini Parametres de confi 9 Sorted_vignettes 9 Fooscan_results E Fooscan_scan O raw 9 work You can modify the settings manually according to the documents delivered with your system or you can do it manually using Vuescan 78 12 5 2 Remove an existing project To remove an existing project delete or move its folder using Windows Explorer It will be removed from the Zooprocess list of projects when you will try to select it 12 6 Edit metadata from raw folder This tool modified in v 5 03 allows you to edit safely the metadata files from the raw folder It allows you to modify the data originally written just after the scanning process The new version will check if meta log and pid files exist in the subfolders of the selected sample In that later case the data are corrected in all files and the list of modified files is displayed at the end of the process 79 12 7 EDIT configuration file Zooprocess offers an editor for the process configuration files The files to be edited must be placed in their project config folder prior their edition This editor is secured and all the entered values are checked The original configuration filename will be automatically renamed in order to end by both_config narrow_confi
69. fig folder An optionfile txt is written if you select to process the new shape and texture parameters added in version 5 05 Previous Zooprocess users must be aware that the pid files containing these new parameters will not be compatible with the previous ones in Plankton Identifier as the number of columns must be the same in all files If necessary you will reprocess all the previous samples and replace the previous PID files in the learning sets already created Zooprocess will read the OD position from the process_install_narrow_config txt configuration file of the root folder possibly adjusted using the OD calibration tool and write a process_install_narrow_config txt in the config folder of the project You are asked to create the scanning files for your project We recommend creating ONLY ONE scanning file per project to avoid errors during the scanning procedures 76 PROJECT MANAGER version 5 03 x Project ad fooscan selected system is Biotam VUESCAN installed version is 8 3 23 W Create scanning file for NARR OW frame at 2400 dpi Create scanning file for LARGE frame at1200 dpi Create scanning file for LARGE frame at 2400 dpi WARNING LARGE 2400 dpi images willbe ARCHIVED at 2400 dpi and CONVERTED and PROCESSED at 1200 dpi WARNING New PID files containing new measurements are not compatible in Plankton Identifier with the previous ones Process again previous files if necessary after
70. g or large_config according to the selected frame size A default file is created during the installation This file cannot be edited by this tool as it is not in a project but it is used by the Find OD position tool Another default file 1s created in each project and includes the OD position parameters from the default one that is in the root folder We recommend keeping ONLY ONE process configuration file in a project configuration folder It will ensure a safer image process The reference to the narrow or large frame is not necessary as all the position parameters are read in the LOG file and Zooprocess 5 03 includes now the both option It may only be useful to distinguish the configuration files if you have noticed a different average grey level for the two sizes of frames SELECT PROCESS COMFIG FILE to be EDITED x Configuration files trom the project config folder can be edited here SELECT PROCESS CONFIG FILE from the list process_install_both_config tet 80 CONFIGURATION FILE x Project folder is Elfoosctan_aa Opened configuration fileis process_install_both_contig tt Frame selected for calibration of grey level Enter information on calibration JPEG quality 100 should be used aw images Hrom scan folder ZIF compression allowed f option selected during process Background Subtract Method Rolling ball method may create artefacts on large organisms IMAGE PROCESS
71. ge_heigth 10584 Grey_Level_Adjustement YES Perfection4990 Flatbed 3929 Hardware ODxminref SFZ 020 0 F0 0 247 0 124 1 1l l pixel 30 O 0F0 371071 040 5174 a N N 0 0 024 0 05 0 14 10 tnarrow The data from the chosen config file are written and separated by Zooscan_lasep txt macro 1 or 2 if the image have been splitted this raw image is the converted one from the Zooscan_scan folder It may be 16 bits if neutral image from Vuescan Careful with file size in octet Yes if selected in the Zooprocess menu Images are converted for the process Raw image dimension Raw image dimension depends on your choice menu and config file Scanning hardware from Image section Position pixel of the upper left corner of the OD measuring area config file and resolution computed CRITICAL 50 ODyminref 4760 ODxdimref 60 ODydimref 600 OD_measured 126 Max min_grey_difference_in_OD 28 STD 2 2489 OD_Settings 120 OD_corrected 120 measuring area width in pixels measuring area height in pixels average grey level measured in the area CRITICAL difference max min std of grey level greyref in the config file CRITICAL measured value after image correction Rolling_ball_background_subtrack_value 300 from config file CRITICAL Background_correct_using 20070903_1057_background_large_manual tif Image_treatment log gamma2 Right_side Automatic_supress Right_side_Limit 3245 Transparent_fram
72. ges processed before the version 4 03 were grey level modified using a log and gamma process if any Image background was selected The rolling ball treated ones were not modified leading to the need to use thresholds of 250 and 243 respectively These default modifications of the images made them incompatible different training sets to be used Zooprocess can now force the image process to natural equivalent to previous rolling ball method or log gamma equivalent to previous Background subtract If set to keep as before v4 the previous different methods will be applied as before See Edit configuration file GREY CORRECTION WARNING Gg x Grey level correction above 15 grey levels 8 bits press CANCEL to abort Cancel 37 Zooprocess 2 09 includes a new feature to reduce the number of false objects in the images A Clear function finds the limits of the transparent frame draw a black line and clears the outside area The tool measures the mean grey level of a reference rectangle close to the border and successively checks the grey level in moving lines When the grey level of the line reach a value of a define ratio of the reference level the limit position is defined and written in the log file in addition to the reference grey level This process may be biased by some big Opaque organisms that should be placed elsewhere in the cell lol xi File Edit 9 4 2 Particle process
73. ic closest background selection e The second tool which requires much time is to visualize the global image with outlines e The third tool is to process stats and graphs to check the results on the measurements It can be completed using the View vignettes tools e Vignettes can also be extracted randomly using the Extract Vignettes for plankton Identifier to visually detect problems using an image browser such as Plankton Identifier or Xnview 11 1 CHECK process by viewing segmented images As we used to scan samples and process them later during night or week ends we were looking for a rapid and simple method to check that the image process background subtract was properly done The method would be simple and methodic not requiring any manual operation The best way to check this process is to visualize the segmented images issued from the first particle analysis and named _msk1 gig This new tool allows to select the first segmented image from a project and to visualize all the successive ones till the end or the list or the operator decision The following image is opened when the active one is closed by operator The process ends at the end of the list or by pressing the SPACE bar of the keyboard We recommend checking all the processed images using this method to detect the ones showing a bad background suppression too many noise See above for example of segmented images 54 11 2 VIEW vignettes You can view
74. ine CANCEL to END drawing lines i Cancel 39 When you press OK the combined image is displayed at 100 magnification and the scrolling tool activated If you want to change magnification press the ZOOM tool in ImageJ and again the SCROLLING TOOL to move into the image window The mouse position displays a hand till you press the ROLL button to activate the tool You can enlarge the image for better overall visualization of the organisms When you ve drawn a line between two objects you get this image and the NEXT OBJECT window Be careful if you want to go on splitting objects press OK and Cancel if you want to end Note that if you ve pressed OK and you have nothing left to split you can draw a line anywhere outside of the objects without any influence on the next particle analysis NEXT OBJECT x ZOOM MOWE to the object to split Fress Roll Mouse Button before drawing line OK to draw NEW line CANCEL to END drawing lines i Cancel The image shows black separation lines now lox 3239x10584 pixels 58 bit 33M When you press the cancel button you get the same SEPARATION CONTROL that you would have got if you had chosen the END process in the FIRST SEPARATION MASK CONTROL 40 SEPARATION MASK CONTROL x Choosing RESET will lead you to a TIME CONSUMING PROCESS YES accept the mask EDIT to complete EXISTING mask RESET to erase all and compute NEW mast Press OK when you have s
75. influences directly the size and shape of the objects The critical values for the particle analysis are e Min and max size for the objects The values are set in mm for equivalent circular diameter of the particles The values used are recorded in the log file and converted to pixel area using the image resolution stored with the scanning parameters of the log file 31 In addition the user can 1 Tag objects on the image The Tag column contains the status of the object It is included in both _meas txt and the PID files version 3 11 Objects to be removed will be set to O while the conserved objects will be set to 1 Note that doubloons will be tagged 2 in the frame 2 measurement file 2 Make a manual identification on the image 3 Extract all thumbnail images from the corrected _vis1 jpg file The steps 2 and 3 are not recommended It is much easier to extract vignettes using the Extract vignettes tool and sort them using Plankton Identifier or Xnview 9 2 CONVERT amp PROCESS Images in batch mode Zooprocess 4 03 improves the batch mode tool that was previously available It includes now the RAW image conversion the image processing and the particle process The main interesting features are the possibility to skip already processed images allowing to launch the tool on a working project and to process only the new scanned images Most of the menus are identical to the ones of the single image process tool excep
76. is indicates a peaked distribution and negative kurtosis indicates a flat distribution Integrated density IntDen This is the sum of the grey values of the pixels in the selection 1 e Area GLmean Surface of the selection in square pixel gt Area_exc Surface of the selection excluding holes in square pixel gt gt Perim Feret The length of the outside boundary of the selection The maximum Feret s diameter 1 e the longest distance between any two points along the selection boundary Shape Parameters gt gt gt gt gt gt Circ Major Minor Angle Fractal Circularity 4 Pi Area Perim2 a value of 1 indicates a perfect circle a value approaching 0 indicates an increasingly elongated polygon it is the reverse of compactness Primary axis of the best fitting ellipse to the selection Secondary axis of the best fitting ellipse to the selection Angle between the primary axis and a line parallel to the x axis of the image fractal dimension AreaSkeleton Area pixel of the skeleton Position Parameters Centroid coordinates XM YM BX BY Width Height X position of the centre of gravity of the selection y position of the centre of gravity of the selection Centroid of mass coordinates X position of the centre of gravity of the grey level in the selection y position of the centre of gravity of the grey level in the selection x coordinate of the t
77. is method is to be well controlled Zooscan cell transparent frame and cover must be perfectly cleaned prior to this operation Zooprocess will display a list of operations to perform in order to scan your sample You can now set your parameters e The name of the selected vuescan_zooscan ini file is displayed e The operation performed is indicated background according to the selected menu in the main Zooprocess page e Information is given on the 16 bit raw image according to the content of the vuescan_zooscan ini file NOT SAVED or SAVED Operation perormed background Actual settings Process 8 bits image using MANUAL color balance 16 bits RAY image SAVED Process a bits image using MANUAL color balance M Save 16 bits RAN image Select the operation to perform according to the way you want to scan and process the images in this project You cannot modify the scanning file there as it can only be done in the SCAN sample menus As this Scan background tool is recommended for Manual color balance processed images this option is set as the default one Note user should NOT modify any of the default settings 26 PROCEDURE for the large frame x D Switch zooscan ON df not done yet 2 Clean zooscan Cell 3 Fill the cell with fresh water 0 6 to 1 cm thick au e Place transparent large frame in normal position comer reference J1 27 Clean transparent frame top side OD zan
78. jpg 2 O x 291x391 pixels 58 bit 111K You can visualize these vignette using different tools such as the Xnview viewer or Picassa or work with Plankton Identifier Notes e Users can also use the different VIEW vignettes tools to extract and save vignettes or the Extract vignette tool e We recommend using the Extract vignettes for Plankton Identifier tool to extract selected vignettes 9 8 What to do if you experiment process problems If a similar message is displayed do not be too frightened It can happen every time you process an image that has been scanned and not yet processed with the tool SCAN Sample with Zooscan for archive no process It means that the log file of the image you want to process does not contain all the necessary lines It thus considers that if it exists the VIS image has not been treated correctly In most cases the log file still contain the Image section which is the minimum data requested to process again the image Press YES and continue processing este x WARNING rg_demo_i_log txt file may not contain all information needed The Vis_image_ save YES or PID line not found A vis1jpg file may exist while the log file doesn t indicate how it was processed or the log file contains only the Image section Check the log file lf the Image section is complete press YES to compute again the VIS image and fil the lf PID first line is missing CANCEL
79. losest background is to be selected CRITICAL set to yes if you want to enhance contrast of each thumbnail image vignette extracted just after the process of the image not applicable for all other vignette saving tools text information to be written in the log compression quality value for all the jpg files stored set to 1 if you let the user chose in the menu to store or not the raw image as a ZIP compressed file define the way Zooscan_lasep image process defines where to measure the OD reference grey level Set to 1 for the Zooscan2003 Villefranche prototype set gt 1 for Biotom machines to 3 if the right OD area of the image is to be removed to save space and to 4 if the OD value is to be measured ONLY default value if not defined during the scanning not used except for old CNRS prototype image process ratio used to compute the greylevel used to find the frame limits not used used if greyco used if greycor width of the OD reference measuring area heigth of the OD reference measuring area limit for the OD grey level STD not used in this version limit for the OD grey level max min difference not used in this version not used 0 if the image process method is to be kept as before version 4 of Zooprocess set to neutral natural or log gamma according to user requirements CRITICAL threshold value for segmentation CRITICAL reference OD grey level value CRITICAL in some cases
80. me must be inside the data file Only tabs or space separators are allowed in files tabs separated files can size up to 1500 rows while ones can size more than 60 000 rows Extract vignettes from all dat1 tt files in Pid_results folder MV Save all data as a unique datafile named data 20090524 2210 t4 if Extract vignettes from all dat1 tt files in Pid results folder is selected Image resolution dpi 2400 0 sA M Add outlines MV Clear outside outlines If gamma gt 1 gt contrast enhanced lf gamma lt 1 gt image smoothed lf gamma 1 gt No gamma correction Gamma value 0 5 5 100 V Fast extraction images not displayed Remove legend and corners Keep only selected object and clean outside threshold 243 I Flip image vertically Replace Gamma info by Zooscan CNRS text Vignette output format peg requested forPKid jpeg v OK Cancel You can visualize the vignettes using XnView or any image browser You can also move them to another folder to manually correct or detail the prediction Plankton Identifier can be utilized too If you have selected the vignette range option enter the minimum and maximum vignette number to be created 13 4LOAD identifications from sorted vignettes This tool ads a pred_valid_datetime column to a TXT file CSV file or a _datl txt file issued from Plankton Identifier containing at least the columns e Item 90 e L
81. method intuentes all the measurements Rolling ball value used when background method is setto rall Grey level setting to clean the transparent frame borders 0 9 recommended amp 0 01 to cancel 5 Grey Level process option Reference grey level ofthe OD isee calibration Maximum grey level correction allowed 6 bits Maximum std range inthe OD area measured Maximum range of OD grey levels in the area measured Width of the OD area finch Height ofthe OD area finch Distance of the OD center from the right side ofthe zo0scan scanning area finch see calibration Distance ofthe OD center from the top side ofthe o0scan scanning area finch see calibration Threshold for segmentation upper ESD min for organisms to be analyzed immi note 0 3 should be a minimum ESD max for organisms to be analyzed frm Tolerance on the position for the detection of doubloons finch Tolerance on the area ofthe organisms forthe detection of doubloons 5 Enhance contrast of vignettes Gamma for vignette display 0 5 5 if setto yes above Length ofthe scale line to be displayed in vignettes mim Value read when the LEFT mouse button is pressed Value read when the ROLL mouse button is pressed Value read when the RIGHT mouse button is pressed both oo created 200 Image CLOSEST background r natural 100 0 0 900 Measure OD and remove right side ofimage 4 5 4 4 0 025 0 340 4 040 0 300 10
82. n predictiONn ccccccccccceceseeeeseeceeeceeaaeeseeeeeess 86 OL SOW ACIVAN Us aes aa cuctccetvean erence cs esa edevasanuecourancdey Mauss ong uuanedeadsansacavecuctievesdseomuaneossaseneuae ees 86 13 2 Extract vignettes for PLANKTON IDENTIFIER ccccccceceeeeeeeeeeeeeeeeeees 87 13 3 EXTRACT vignettes into folders according to prediction cccceeeseeeeeeeeeeeeeees 89 13 4 LOAD identifications from sorted vignettes cccccccccecssseeeeeeceeceeeeaeeeeeeeeeeeeeaaas 90 13 5 CREATE subset of a Learning Set from identified vignettes random 93 14 ZOOSCAMN diad NOSUC nc EAT 94 14 1 Analyse background 1mages ceeeeeesccccecceeeeeseeecececeeaaeeeseeeceeseeeeaueaseeeeeeeeeeaaas 94 14 2 Check normalization with OD discuss previously Process threshold OD disk inter eO O a E E E 94 lo CROCE MO e 061 0 16 aaa Senne pe eae ee ea ee 97 15 Listo add Onin he NEW VECSIONS eenia aes a E Mebane 98 MN Verona Os oae E E acwanansatansanncetaes 98 M2 Veron AO oean tesa tba cu wea areata eanctecai tesa labs eumeravactuceaeaneteet 98 oe Vernors Oseere da aw caacetecniad ne cncteaceats fava T E E dea craeeteanaadeace 98 13A Verona O arr mere ere er tere mene ee er ere meee re eee eee eer ee 99 sts WD o c 01k Os Mere reer pe nn E E E E E ener E Tae een E E 99 V On T 14 Of anmpeee a testa a hore PT eee erates E 99 I Ver ORNS ce ccs ow srec ie es cerns ec grecae aece eeeeeeee esee 99 528 Version Oe lOe Qeactascs
83. n that the others are missing in the data file as these data are displayed only to help vignette selection 55 d VIGNETTE SELECTION x Project pointh_rg Image rg_demo_1 Select data line and press OF to visualise ESD and Feret diameter are converted in mm Press CANCEL to END Mo Area ESD Feret Fractal MeanGrey Bx By Width Height 164 45 1214 When you have selected the first vignette to display its data is displayed on top of the selection area The previously opened vignette is automatically removed when a new one is opened VIGNETTE SELECTION x Project pointh_ rg Image rg_demo_ Select data line and press OF to visualise ESD and Feret diameter are converted in mm Press CANCEL to END Mo Area ESD Feret Fractal MeanGrey Bx By Width Height 4 27898 1 9946 3 58893 1 27 164 45 1214 545 320 241 Mo Area ESD Feret Fractal MeanGrey Bx By Width Height 1 G47 0 7415 Press Cancel to end process The last vignette is closed and you are advised to close the source image if you want 56 11 2 2 View vignette from a text file This function is dedicated to the check of identified organisms from as many images as you want and sorted in a single text file The image files must be in their respective subfolders according to the project selected in the main Zooprocess menu even if the text file can be opened from any other folder The file must contain the column names in the first raw and all data must be
84. n_2400dpi_frarne1_narrow ini Cancel When activating one of the two scanning menus a CONFIG file for SCAN should be chosen from the above popup menu These are the vuescan_zooscan ini files created in the Zooscan_config menu of your project during the Create New Project operation The method to create or modify these files is described in the CALIBRATION procedure Reminder you have to choose the file adapted to your precise task because it contains all the parameters that will be copied to Vuescan before scanning We advise user to manually remove the unused files from Zooscan_config folder and place them in a subfolder as archive to avoid any risk of confusion Any change in these files will modify the result of the image and particle analysis made by Zooprocess This point is CRITICAL The selected file will be edited by Vuescan The correct filename that you will enter will be recorded and some parameters checked before storing the file as vuescan ini in the Vuescan folder Note that the described procedures were checked ONLY with the version 8 3 23 of Vuescan 6 5 2 Scan procedure warning SCAN PROCEDURE x GUIT YJESCAN before proceeding Cancel 19 Vuescan must be closed before proceeding 6 5 3 Scan procedure Turn Zooscan ON place the selected frame and set the sample SCAN PROCEDURE x Enter Sample ID Mo extension or space character allowed OK Cancel Name your sample
85. n_lasep txt 1f selected or in the case that there are no treated file The function of this macro a Save an archive of the converted image as a ZIP file in the _zip folder if selected b Adjust the grey level measured in the selected area of the image to the reference value stored in the selected config file not necessary for manual color balance and if you have selected the adjust median grey level c Reduce image size by removing the Optical Density area d Homogenize background level this operation is critical for the memory in case of large images This operation can be done either by the integrated Rolling Ball Subtract plugin or by the subtraction of a background image v2 09 e Create and save the _visl tif zipped image in the sample sub folder of Zooscan_work f Save atemporary temp_vis1 tif file in the Zooscan_temp folder 30 4 Particle process macro Zooscan_separation_analysis txt and Zooscan_particle_analysis txt uses the _visl jpg or the temp_vis1 tif file to analyze particles a Thresholds the image to the value set in the selected config file and creates the _msk1 gif file of the segmented image in the subfolder and writes a copy as a temp_msk1 tif file in the Zooscan_temp folder The B W ratio of the segmented image is written in the log section of the pid file Analyze particle using the ImageJ plug in on the whole image This process is critical for the ImageJ memory version above 1 02 are OK P
86. n_process Actual settings Process 6 bits image using MANUAL color balance 16 bits RAN image SAVED W Save 16 bits RAW image Save changes to vwuescan zooscan 2400dpi framed narrow ini OK Cancel Select the operation to perform e You can now select to save the 16 bit image e You can also save the changes in the vuescan_zooscan ini file to avoid having to re enter the settings next time 1f you have modified something in this menu Note If you perform SCAN and Process we do recommend to use the MANUAL color balance and to save the RAW 16 bit image default If you perform SCAN only keep using SCAN 16 bits raw image only default option Note user should NOT modify any of the default settings 21 PLANKTON SCANNING PROCEDURE v 3 14 x Do MOT CHANGE ANY SETTING window size in YUESCAN Mote ifyour image is too large it will be automatically splitted D Switch zooscan ON tif not done yet 2 Check that you have placed the large transparent frame 3 Place sample into the zooscan cell 4 Launch YUESCARN fit must be closed 5 Start light tempo ipress green button on cover Bi Click on PREVIEW button in VUESCAN T s Check your image Mo white strip no bubble no organism on the OD ref a WAN more than 30 seconds after the PREVIEVY 4 Click on SCAN button in VUESCAM 10 Press OF in this menu e The
87. ner mer E etre rer emu ane A eee a vee A S 36 9 4 2 PCIE 0 er ops mae ers ae cer ere eee en eee Ree ene 38 9 5 NEParatlonm PLOCES Sapena tie acca ee ee See ane ae eee 38 9 5 1 Important note for 2400 dpi images cc eeeccccccceccceesesseececeeeeeeeeeeseeeeeeeeeaaas 38 9 5 2 Process COSC OM aain cath nay aeeiaeh ded naseenidabenanes 39 90 Taz TUNCH ON ec cetecessees E E EE E E 42 9 7 IVS SEES CxracHON one n cea bes tee a ue stad na cael ga eaweddendcmd sn eesin as neameoueas 44 9 8 What to do if you experiment process problems ccccccsssseeseeeeeeeeeeesseeseeeeeees 45 10 Fee a E E oat ir ee rte 46 POST Pid Tes CS Crt Oren E E a a Geevie 47 102 Meas txi fiie desc APUORI 32 WOS MICAS UREA yariabDlES cercen a 32 11 CHECKING Ta OS senres eset ce E E ad 53 11 1 CHECK process by viewing segmented images ccccesseeseeccecceeeeeeeeeeeeeeeeeeeaaas 54 E2 VIEW VIG CTC Sossen a e A sadedananuh seties uadedae tah netindaliamsne tab atiat nosis 55 11 2 1 View vignette from the data section of a pid file ee eccccccccceeeeesseeeeeeeees 55 U22 NIC W vienete THOMA eT TC ses tecct tia a teean Oonertds Sears aawaneredetuasautenedia tania anwabes 57 T23 View viznette omn 2 D Cap erste sonscca Seen e a Ra Ae 58 t24 View yienetie from an ident Mle ccorsernao n a eidesseeeetas 6l IES SIA IS and GRA PTS e E a aa dcavaee 6l Ud PROT dG Graphs ae 1S THS raS a 62 11 3 2 PLOT selected graphs amp histOgraMs
88. of an image scanned with an insufficient quantity of water leading to meniscus effect on the borders targets_c_1_msk1 gif 33 3 E 15 x 2975x1872 pixels 38 bit 5 3MB 16 5 6 Separate touching objects Zooprocess 6 14 and 6 15 introduce a new tool to routinely separate remaining touching objects and reprocess automatically the separated images It is not mandatory to separate objects on the images if you have been careful during the scan procedure 5 7 Process again images If you have separated the objects using the dedicated tool you must reprocess it again using the dedicated option in the SCAN and Convert tool 6 Acquire images from Zooscan 6 1 Introduction This chapter describes all the options available in Zooprocess It can be read for better understanding reference document of the Standard Scanning Procedure presented above Users do not need to read it before using Zooprocess Select SCAN CONVERT and PROCESS Sample with Zooscan or SCAN Sample with Zooscan for archive no process Both menus will use Vuescan to acquire images from the Zooscan These menus can be used ONLY if you have installed Vuescan as described in the Zooprocess installation guide The SCAN CONVERT and PROCESS Sample with Zooscan menu will carry out some image processing conversion grey level background particle analysis It can be used for any resolution up to 2400 dpi with the large surface frame
89. oject folders Do not forget to copy the configuration files that you intend to use for this project The old Zooscan _raw and _work folders will never be available through Zooprocess versions above 2 01 12 5 1 2 Normal procedure During the first installation of Zooprocess Zooprocess will create a new project in the main menu All the necessary folders will be automatically created on the selected drive The _back folder will be created during the first Scan background operation Do not include Zooscan in your project name Zooprocess will do it for you To Import folder selectits drive and enter its name Select drive avoid selecting CD drive Enter project name project name The projects folders are e Zooscan_projectname Zooscan_config contains at least o Vuescan ini files used to set the scanning parametres to Vuescan exe o config txt files used to set the image treatment parametres e Zooscan Zooscan_scan contains o Zooscan Zooscan_scan _raw all raw ZIP compressed images will be stored there 75 o Zooscan Zooscan_scan _work the subfolders containing images vignettes and data files for each treated image will be created here o Zooscan Zooscan_scan _zip the TIF converted images from the Zooscan_scan folder will be compressed there if the option is selected during the image process e Zooscan Zooscan_meta contains the temporary metadata files e Zooscan Zooscan_results contains
90. ol is improved gt The EDIT metadata tool improved to update metadata in the files datl pid amp dat1 txt of the pid_results subfolders 15 15 Version 7 00 gt A new Zooprocess version which is now fully compatible with UVP5 and Flowcam images gt Nothing noticeable modified in the Zooscan process tools 101
91. older tool The extracted _dat1 txt files are copied into the datl_extracted folder Remove the extracted files from the pid_results folder Manually correct or detail the identification by displacing the vignettes between the Sorted_vignettes sub Id folders Load the new identification position in vignettes in subfolders into the _dat1 pid files using the LOAD identifications from sorted vignettes menu The _datl txt files contains now a new column containing the validated Ids This file is automatically copied into the datl_validated directory A summary is displayed and saved helping you to analyze the efficiency of the prediction The Create_Learn_PID_file tool should not be used Plankton Identifier does the job much better Note most of these tools can work using the _datl pid and _dat1 txt files issued from Zooprocess and Plankton Identifier If the option to process these files is selected in the menus ALL the files remaining in the folders will be processed User can select files by displacing some of them in a separate temp folder for example 13 2 Extract vignettes for PLANKTON IDENTIFIER This tool helps extracting vignettes for a training set It reads the _dat1 pid files that have been copied into the Unsorted_vignettes_pid folder It creates vignettes using one of the three available methods Select vignette number range Random extraction of a selected number of vignettes pe
92. older in case of divided images End of SCAN menu clarified Bug fixed in the Extract Vignettes in folders Resolution checked in the Extract vignettes for Plankton Identifier Date is better checked in the metadata form Image process can now be set either to natural or log gamma2 to better adjust the image quality It also permits to get same results with both rolling ball and image background subtract methods Change Check Zooscan version and parameters added 98 Test added in the image process macro to check if the dimensions of the background match with those of the processed image gt Test added in the particle process macro to check that the surface covered by the organisms does not exceed 25 of the image area 15 4 Version 5 02 gt Zooprocess is now compatible with both Vuescan 8 3 23 Biotom and Hydroptic V1 models and Vuescan 8 4 57 Hydroptic V2 gt Zooprocess now detects the Vuescan installed version and checks its compatibility with the Zooscan installed model gt Bug fixed in the background dimension check in image process gt Median grey level of each image added in the background log file gt The Black White ratio of the segmented image is checked lt 25 and written into the suivi file and the PID files for better control of the background gt Liability improved in the batch image process gt Metadata latitude and longitude fields better described in the form 15 5 Version 5 05 gt
93. ols Target diametre immi E s00 Threshold minimum 240 Threshold maximum 250 Theoritical grey values should not be modified Wean grey for OD 0 3 fi qq Mean grey forOO 0 9 fi 13 OK Cancel The default values are automatically adjusted depending on the image process method that was used for the initial target image processing You should keep defaults settings Select the image from the project the one that you had scanned with the disks Adjust the theoretical disk diameter 1f necessary Select the threshold range for the measurements Select the theoretical grey values for OD 0 3 and OD 0 9 If you have any doubt on these values check the data section of the PID file 95 menage Sis S01 BS pels Bebo Me The image is then opened and the diameter of the disks computed at different threshold giving you this figure You can check the accuracy of the diameter 20090524 _2226_targets4_1_1_OD jpg ESD mm 230 Threshold AQOPROCESS targets4_1_1 natural Median grey level bits g median 0 3 148 136 159 median 0 4 72 66 79 N Top Bottom List save Copy The resulting figure gives you the average median grey levels and the range for the O 3 OD disks and the 0 9 ones that should be close to 73 and 150 respectively This figure shows also 96 that the special background heterogeneity is perfectly removed as the disks from the top of the image first disks from the lef
94. ooscan fooscan_contig a gt gt OK Dossiers Taille E 2 Zooscan E process _install_narrow config txt i Document texte Zooscan_calib yuescan_zooscan_calibration_OD_level_cadret Jini Parametres de co Zooscan_config vuescan_zooscan_calibration_OD_level_cadre2 ini Parametres de co Zooscan_download Byuescan_zooscan_calibration_OD _pos44 ini Parame tres de co C Fooscan_temp Zooprocess offers then to create a working project see below Warning Do not proceed to the creation of any project if you are not confident with the above X and Y values for your system 4 2 Create a first project You are asked to create a working project when you install Zooprocess or you can select the Create NEW project menu in Zooprocess PROJECT MANAGER version 5 02 fooscan selected system is Biotorm VUESCAN installed versionis 8 3 23 To Import folder selectits drive and enter its name Select drive favoid selecting CD drive El Enter project name ftrial See the Create NEW project or remove project menu below for more information about this menu 4 3 List of TOOLS SCAN CONVERT Background Image SCAN Sample with Zooscan for archive no process CONVERT amp PROCESS Images in batch mode CHECK process by viewing segmented images SEPARATION from B W msk image SEPARATION using vignettes EDIT metadata SCAN CONVERT and PROCESS Sample with Zooscan CONVERT RAW image
95. op left point of the smallest rectangle enclosing the selection y coordinate of the top left point of the smallest rectangle enclosing the selection of the smallest rectangle enclosing the selection of the smallest rectangle enclosing the selection These parameters are described in the following application note http www obs vlfr fr LO V ZooPart ZooScan article php3 id_article 5 1 They are also published in a JPR paper Other variable description will be available in a paper being written Some information in this presentation http www obs vlifr fr LO V ZooPart ZooScan article php3 id_article 119 11 CHECKING methods Users must be aware that image processing must be controlled by user before using the measurements saved in the PID and the MEAS files Zooprocess offers different method for 53 these tests The first step is to control the image process and the background removal from the original converted image We assume that the conversion is very stable and safe if you keep the default parameters The second step is to check the data themselves according to what you expect to measure Both steps are closely linked anyhow There are different tools to check the background process and the threshold limit for particle detection e The first and fastest method is to visualize all the segmented images chapter below It should be performed on ALL the processed images mainly if they have been processed in batch mode using the automat
96. ou will get the resulting files in the Zooscan_scan _work subfolder of your project This standard scanning procedure will give you the resulting PID file and the background corrected plankton zipped image vis1 ZIP The resulting data can be checked using the Stats and Graph tools The background removal can be checked using the CHECK process by viewing segmented images or VIEW Image with outlines tool and vignettes can be checked using the View vignettes or extracted using the dedicated Extract vignettes for PLANKTON IDENTIFIER or the EXTRACT vignettes in folders prediction or vis folder tools 14 This is an example of a normal sample image The borders of the transparent frame are visible on the four sides of the image There are no bubbles below the OD reference on the right side You cannot see the meniscus on the sides because you have filled it above the first step of the frame 5 5 Check process Utilize the dedicated tool to check that the image has been correctly processed This is an example of a well scanned and processed image The borders of the transparent frame have been automatically removed on the four sides of the image You cannot see the meniscus on the sides because you have filled it above the first step of the frame The noise Small targets is rare and randomly spread 15 targets_b_1_msk1 gif 33 3 Oj x 2913x1872 pixels 8 bit 5 2MB This is an example
97. r image Extract all vignettes from all dat1 pid file 87 1 Zooscan vignettes for train 6 11 q x Vignettes will be extracted from the PID files placed into the folder E Zooscan_essais_duplicates_sep PID_process WUnsorted_vignettes_pid PRESS OK when done Select method Extract all vignettes x Resolution dpi 2400 0 M Add outlines M Clear outside outlines lf gamma gt 1 gt contrast enhanced lf gamma 1 gt image smoothed lf gamma 1 gt No gamma correction Gamma value 0 5 5 IV Fast extraction images not displayed Remove legend and corners Keep only selected object and clean outside threshold 243 I Flip image vertically l Replace Gamma info by Zooscan CNRS text Vignette output format jpeg requested for PKId lipeg As usual it is possible to add outlines not recommended and clear outside outlines The default gamma of 1 1 is recommended New vignette options have been implemented in version 6 11 A log file is recorded into the Zooscan_temp folder ioa T File Edit Font Heooscan targets marc Zooscan scan workitargets_1targets_1_is1 zip OPEM Clear background TOTAL extracted vignettes S0 Log saved as Extract to folders log tet in the 400scan_temp folder Note Plankton Identifier reads ONLY JPEG vignettes 88 13 3EXTRACT vignettes into folders according to prediction This tool has been created to allow visual check of predictions ma
98. rame according to filename 12 8EDIT LUT file The default lut txt file is created with your project It insures a full inter calibration of all Zooscans Modifying this original LUT file will make your converted images and the measured parameters on the organisms different from the ones of the other systems You should not modify these parameters Note if you reset the original parameters and re process the real RAW images you will recover inter calibrated 8 bit images If you click on the tool in the main Zooprocess menu the LUT txt file from the selected project configuration folder is displayed e Select Gamma option the gamma conversion can be done on the 16 bit raw image BEFORE 8 bit conversion using the min max range or AFTER this 8 bit conversion A conversion made before will require more memory and take more time but it will keep a better grey level resolution in the resulting 8 bit image e Gamma 0 5 5 0 Gamma to be used for image conversion The value must remain between 0 5 and 5 If set to 1 no gamma will be performed It must be set to 1 for a perfect background subtraction e Select the automatic median grey adjustment to fully inter calibrate your system Selecting this option cancels the need to later adjust OD level e Ratio the 1 15 value is adapted to the Zooscans we have tested This ratio allows including the background heterogeneity The median grey level of the image will be multiplied by
99. roject tool implemented to create now some additional default directories in the Pid_process folder 15 10 Version 6 12 gt VV VY Rolling ball background subtract method works now bug fixed with all size of images acquired at 2400dpi The Dat1l_extracted folder is created now in the PID_process pid_results directory The Extract_vignettes_to_folder tool saves also the extracted files in the Datl_extracted directory The Dat1_validated folder is created now in the PID_process pid_results directory The Load_vignettes_from_folder tool saves also the validated files and the summary in the Datl_validated directory 15 11 Version 6 13 gt gt The change check Zooscan parameters tool displays now the updated values Bug fixed in the particle analysis routine which prevented processing the images containing three times more objects in the left side of the image 100 15 12 Version 6 14 gt New separation tool added SEPARATION from B W msk image 15 13 Version 6 15 gt New process function to re process only newly separated images 15 14 Version 6 16 gt Zooprocess checks now all the projects of all connected drives when it starts You do not need to import projects when you connect an external drive containing projects gt The Extract Vignettes to folder tool has been improved It checks now that the vignettes of the samples have never been extracted yet gt The Extract sub learning set to
100. rsion 3 14 x INFORMATION on EXISTING FILES METADATA FILE YES CORRECTED IMAGE YIS FILE YES LOG FILE exists and complete YES ZIP FILE N SEPARATION MASK YES TAG DATA YES IDENT DATA NO PAR METADATA FILE NO SELECT IMAGE TREATMENT PARAMETRES OD process option Measure OD and remove right side of image Border CLEAR Background subtract 20070903_1057_background_large_manual tif recovered V OD process ZIF image from scan folder into zip folder SELECT PARTICLE TREATMENT PARAMETRES Threshold 244 0 Min ESD mimi 1 5 Max ESD mm 100 SAVE thumbnail images of organisms SAVE also FALSE tagged object thumbnail images W Force doubloon check for frame 2 images Work with Separation mask CREATE MODIFY INCLUDE Work with TAG fonction for bad objetes CREATE MODIFY INCLUDE Process Manual Identification CRESTE MODIFY INCLUDE 7 CHMRS 2003 import parametre M INCLUDE the metadata info from the PAR file TO the new META file i Cancel The title of the window displays the name of the image to be processed The first section is absent in case of BATCH image process It indicates the files that already exist in the sample subfolder It also informs 1f the file ZIP archive already exists e Metadata file inform about the fact that a metadata file exists or not in the sample subfolder e Corrected vis file inform about the existence of a corrected image file in the
101. s acquire a PROFESSIONNAL licence of Vuescan see Vusecan installation above We do recommend now to scan real RAW images ONLY and use the Zooprocess included image conversion tool to compute the 8 bit images This is the only methods that allows full inter calibration of different Zooscans and the perfect background subtraction using a background blank Keep default scanning settings to follow this recommendation Operation perormed scan_process Actual settings Process 8 bits image using MANUAL color balance 16 bits RAY image SAVED M Save 16 bits RAV image Save changes to wuescan zooscan 2400dpi tamel narrow ini OK Cancel Saving real RAW images is the ONLY option that absolutely requires saving 16 bits images before the conversion These images will have to be later converted in Zooprocess to apply both the proper histogram adjustment and the best gamma correction to get good quality reproducible 8 bits images Zooprocess 3 13 allows you to scan and save RAW images in the _raw folder For very early users of Zooscan you can keep processing neutral balanced images and save the raw file in addition for later processing It allows you also to convert these raw images to 8 bits ones that will be used for the organism analysis This operation will be performed according to the settings of the default lut txt file that has been created with your project You will find some detail on this procedure in the
102. s of the particles in order to define the outlines of the connected object to be displayed for visual control The vis image and the outlines are displayed and you can decide to keep a copy of this combined image SEPARATION CONTROL SAVE image wit CONTOURS before process If previous image exists twill be replaced i Cancel If you press YES an _zoocont jpg file is stored in the sample subfolder If not or after the image recording you will get the measurements table the combined image zoom and the next separation window SEPARATION MASK CONTROL x Separation mask operation EDIT add separation lines to the mask END accept the existing mask SELECT Homie Press OF when you have selected YES or RESET Mever press CANCEL Ok Cancel You can now choose to edit the separation mask or END if you accept the mask or you don t want to work on it NEVER press CANCEL here You will abort the whole process and get error messages to be modified in future release The next window EDIT is named FIRST OBJECT It explains how to draw and record the separation lines in the _sep gif file The drawing tool is activated when you press the ROLL mouse button You then click right at the line start position and again at the end If you want to end drawing lines press Cancel button FIRST OBJECT my x ZOOM MOVE to the object to split Press Roll Mouse Button before drawing line OK to draw NEW l
103. se V Process detailed statistics slower process THE OPENED FILE WILL BE COMPLETED WITH VALIDATED IDENTIFICATIONS TABS or SPACE char will be replaced by SEMICOLUMN Valid identifiaction column is added to the file 92 Note Plankton Identifier saves the data separated by tabs In that case Zooprocess can only handle files containing less than about 1500 objects New Releases of Plankton Identifier will give the option to save dotcoma separated files An option allows user to create the _ident txt file which can be loaded in the Manual Identification function to place organisms names onto the image When the job is completed a final message indicates the number of vignettes that have been moved from the prediction folder to a corrected one error rate In addition a file named validation_summary_20090524 2211 txt containing detailed stats is saved in the sorted vignette directory All updated files are also copied into the Pid_results Dat1_validated directory 13 5 CREATE subset of a Learning Set from identified vignettes random We have added this tool to help people create a Learning set from validated vignettes or from an existing learning set The tool will randomly select a defined number of vignettes from each folder and create a new learning set that user will have to check using Plankton Identifier The resulting Learning set will be equilibrated if the source folder contained a sufficient number
104. separated by space character or dot coma csv files from excel works well The file must at least contain these columns e Item organism number in the dat1 pid file e Label visl jpg image name the image must be available in the project sub folder to be opened Bx Upper left corner X position By Upper left corner Y position Width width of the rounding box Height height of the rounding box At least and additional column with some identifications If this column is named Ident it will automatically be selected Otherwise you will be asked to select it yourself Most of the information is displayed in the next window The resolution must be selected by the user and should be the same for all the images You can keep all the vignettes opened till the end of the process You can sort the file on any column name to allow easier selection of the vignette to be displayed Due to limitations of ImageJ and JPEG image format the whole source image of a vignette has to be opened before the vignette extraction We thus recommend to view vignettes from the same image consecutively to accelerate the process Important note if the file contains tabs separators the maximum number of rows allowed is about 1500 rows only If semicolons are used EXCEL CSV files more than 60 000 rows can be read 57 VIS image is to be found in its project Zo0scan_scanwork subfolder Macro will abort if an image is not found Label
105. t of the graph have the same values than the others 14 3 Check mouse buttons This process is used to control the value read by ImageJ when you use the mouse You may have to modify the corresponding fields in all the config files Note that the process ends if you do not press any roll button during more than 8 seconds The method is described hereby Check MOUSE ROLL Button X Click on image with ROLL mouse button READ Value in LOG window Wait 8 seconds without any click to abort Write the value in the broll field in the contig files i Cancel When the OK button is pressed this blank image is displayed LTT inix 500x400 pixels 8 bit 195K Check the LOG window to define the values to be entered in all the config files Normal values are e 16 for the LEFT button e 10 for the ROLL one or 8 or 12 e 4 for the Right one These values may be different for other computers 97 15 List of add on in the new versions 15 1 Version 4 01 7 VYY A Y VVV VW VV V 1 VVVVV VVVV VV VO VV WV v Bugs fixed in Extract vignettes to folders tool Bugs fixed in Separation using vignettes which is now fully operational and improved using a flag into the image New naming of the Scan and Process tools Manual measurements improved using also a flag in the image Automatic re use of the initial background during image reprocessing default setting now Metadata form secured date position and fraction
106. t that it adds the batch conversion option Zooprocess 5 03 makes the images not visible during the batch process allowing a 80 reduction of the processing time Important note a record of the batch process is stored in the Zooscan_temp folder and in the Zooscan_check folder of the project as a Suivi_Y YYYMMDD_HHMM_log txt file This file should indicate the starting date and time for each image and two numbers of processed particles 1 amp 3 If these two numbers are indicated and the values are the same the related image has been analyzed properly 9 3 PROCESS Converted Image process one 9 3 1 About the ImageJ status bar The user can control all the processes by checking the STATUS BAR of ImageJ and the LOG window 9 3 2 Option selection and Config Menu for the process This window will be displayed every time you will process an image even if remaining in the same Zooprocess session We recommend removing unused config files from the Zooscan_config menu and store them in a subfolder to avoid confusion in selecting the config file Nevertheless the configuration file name will be written in the log file of the images The same window is displayed for both the single and the batch image process gt Select the config file to be used for the image s you want to process gt Image AND Particle Process or Particle re process option If you have just scanned a sample the Choose process option will not be displayed as Zooproc
107. ted and listed These parameters can be later checked and adjusted optional using the Change Check Zooscan version tool INITIAL SETTINGS x fooscan selected system is Biotom VUESCAM installed version is 8 3 23 Check carefully the default values below Change them according to calibration sheet if necessary These settings will be re used for each of the created project position 0 340 Y position 4040 MARROW FRAME 22 2 offset 10600 YOtts eat 1140 Size 21290 YSize 45640 CEGC LARGE FRAME sOffset BATT Offset qq wSize 30171 Coa CEN CN YSize 5640 Zooprocess 6 11 indicates the chosen Zooscan system and Vuescan installed version Check the parameters from the qualification sheet provided with the Hydroptic systems or from the Zooscan website for the Biotom ones The X and Y positions are OK if the borders and the OD reference can be seen in the images as below 20081210_1056_background_large_manual tif 33 3 10 09x6 24 inches 3027x1872 8 bit 5 4MB The following folders are created and the default files saved in the Zooscan_config folder Mt Zooscanconfig eee 10 x Fichier Edition Affichage Favoris utils KJ Pr c dente k PAP L Rechercher gt Dossiers a y x ie E Adresse E HiiZ
108. th your project to ensure that it is fully compatible with other Zooscans The conversion is done following these steps Optional compression into a ZIP archive Measurement of the median grey level of the image inside the transparent frame A median filter is applied by default on image acquired with Zooscan Hydroptic V2 If Background median grey adjustment is set to YES the difference between this median and the Median grey level setting is calculated and the Minimum 16 bit scale and Maximum 16 bit scale values corrected using this difference 5 If Select gamma option set to BEFORE the Gamma correction is now made on the 16 bit image using the Gamma 0 5 5 0 setting The gamma correction is made between the Minimum 16 bit scale and Maximum 16 bit scale 8 bit linear conversion between the Minimum and Maximum settings 7 If Select gamma option set to AFTER the Gamma correction is now made on the 8 bit image using the Gamma 0 5 5 0 setting The image is Rotated 90 clockwise 9 The image is flipped vertically oe A 28 Settings from the LUT TXT file ofthe project Grey_mode_auto yes Ratio 1 15 OD range 1 8 Gamma correction before Gamma setting 1 Min grey 0 Wax grey 65536 BATCH Image Process Process only TIF uncompressed image files Process only TIF uncompressed image files Process only ZI
109. the Unsorted_vignettes_pid folder An existing learning set can also be utilized Check Zooscan website for existing learning sets http www obs vifr fr LO V ZooPart ZooScan rubrique php3 id_rubrique 33 lang en We recommend using the following prefix naming conventions to help grouping categories and compute statistics later app clad chaet cop crust gel moll ostr pte rad mult det_ nt_found e When all the necessary vignettes are extracted use Plankton Identifier to create the Learning Set picking the vignettes from the Unsorted_vignettes_pid folder You should use the Learning_set folder to place your training set e When you have created and checked your identification procedure place the PID files to be analyzed into the Pid_results folder and process identification An analysis 86 file and optionally some _datl txt files will be created in the user selected folder enable SAVE detailed results for each sample option We recommend saving to be selected in Plankton Identifier when you START ANALYSYS the results of the prediction in the prediction sub directory of the pid_process folder The data section of these files now contains a pred_ column If you want to proceed a complementary manual sorting extract the vignettes from the dat1 txt files into the Sorted_vignettes folder using the EXTRACT vignettes in folders prediction or vis f
110. thumbnail images of organisms SAVE also FALSE tagged object thumbnail images i Force doubloon check for frame 2 images Work with Separation mask CREATE MODIFY INCLUDE M work with TAG fonction for bad objetes CREATE MODIFY INCLUDE Process Manual Identification CREATE MODIFY INCLUDE i Cancel 9 3 7 Check or correct metadata information The metadata window is displayed again in order to check that you are processing the right image You can there modify the fields Refer to the FILL METADATA form section if necessary 9 4 Process flowchart 9 4 1 Vis Image process The raw image from the _scan folder is zipped as a compressed archive if option selected and saved in the _zip folder version 3 02 This operation is not requested if you have saved and zipped the real RAW image which will remain the source of all the others The image from the Zooscan_scan folder is opened and corrected according to the config file parameters and the image process setting set above grey level adjustment background crop and saved as a _vis1 zip file 36 You can control the process in the log window where the operations are written in the Image Process section of the log file The comments of the log file will be described in a different section of this document The different steps are a b c d e f Open image the converted image which is in the Zooscan_scan
111. tion lines for an image The only solution is to remove the _sep gif files from the image folder s and process again the visl image or to select the CREATE new option in the separation tool menu 12 1 5 Process newly separated images Zooprocess 6 15 includes a new tool to automatically re process all images having being separated and not yet processed This new tool is only available in the CONVERT amp PROCESS Images in batch mode function It checks the date of the separation mask and compares it with the date of the VISI image If the SEP file is more recent the image is automatically re processed including the separation mask This tool needs the TIF image of the sample from the Zooscan_scan directory PROCESS Converted Image v 6 15 SELECT PROCESS OPTION Convert AND Process Image AND particles images in RAVY folder Convert AND Process Image AND particles images in RAWY folder Process again particles from processed images images in WORK sub folders Process again particles from processed images to include SEPARATION MASK images in WORK sub folders OK Cancel 69 BATCH IMAGE PROCESS version 6 15 j EJ SELECT IMAGE TREATMENT PARAMETRES ALL recently separated images will be reprocessed OD process option Measure OD and remove right side of image Border CLEAR Background subtract closest Image process natural MV OD process ZIP image from scan folder into zip folder
112. treatment and restaure a log file before processing note a temporary backup log file may be found inthe Zooscan_temp folder Mo Cancel 45 In other cases first check that your Image folder exists and contains at least the LOG and the META files and that these files are not corrupted If they are OK and you are still unable to process your image check the configuration file in your project folder If you want to compute again your data from the first steps of image process remove at least the VIS1 jpg or the VIS1 ZIP image archive and process again your image Note if you remove the MEAS file all the manual TAG and the DOUBLOONS will be removed If you remove a LENGTH file all your manual measurements will be lost If you remove an IDENT file all your manual identifications will be lost If you remove a SEP file all your manual separations will be lost The Zooscan forum can be very useful to exchange information and solutions http zooscan forumakers com If necessary send me an email Picheral obs vlfr fr describing your problem Do not forget to include a copy of the LOG window that you can save using the SAVE AS TEXT function of the FILE menu from ImageJ 10 Resulting files This image shows the content of the Zooscan Zooscan_scan _work rg_demo_1 folder in the case you ve perform all the operation available in Zooprocess The RAW LOG and META files should never be deleted 46 ree i 15 x
113. ucted following the safety regulations It is adapted to liquid samples recovery It has bottom illumination for sample disposal It has a special lighting system It has an integrated Optical Density reference It has special transparent frame to achieve good quality image borders It uses special calibration procedures We have developed the Zooscan following our experience in different imaging devices for oceanography We have imaged thousands of samples and stored in numerical form since 2003 with our prototype and the Biotom models We did not loose any sample when filling or recovering the sample The system is well adapted to liquid samples procedures Samples were however lost later due to accidents We consider that the numerical storage is an important point 2 2 Why using dedicated software for imaging plankton with scanner or Zooscan We are imaging Zooplankton for enumeration sizing and identification The results are directly dependant of the imaging parameters that should be homogenous for all the sample series In order to insure that your images and the data derived are compatible with other images or other devices it is necessary to keep a record of the imaging procedure for each image This is also the only mean to confirm that a change in sample composition size spectra is not a result of a change in the imaging procedure For this purpose we have developed a specific scanning procedure at LOV We do think that any imaging soft
114. ware for the Zooscan or a scanner must be evaluated in regard to the possibility to keep a record of the scanning and image pre process parameters in a separate file You can configure different scanning tasks in EPSON SCAN or SILVER FAST and other scanning software but the parameters are not easily accessible for users and may be modified without any control or record We have chosen VUESCAN because it uses the vuescan ini text file that contains all configurations and parameters It allows users to load save special configuration files for different tasks see calibration procedures below Vuescan also saves this information to the same vuescan ini file when you quit the application Vuescan is cheap even in its professional edition Zooprocess do not pilot Vuescan itself but if the procedures are respected it writes the correct parameters into vuescan ini prior to image acquisition with Zooscan It also reads the parameters used by Vuescan after each scan and saves them in the header of the image analysis data file _dat1l PID and in the log file The creation of these files is the only procedure that can certify the quality of the images and the data acquired with the Zooscan 3 Install Zooscan drivers and Vuescan 3 1 Install Zooscan drivers Check first the recommendations from the USER MANUAL provided with your instrument e If you already installed the Zooscan software you can skip this installation If you only use ZooProcess for
115. wed visl jpg images to be opened As usual you can adjust the gamma add outlines and remove background More options added in version 6 11 Some warnings can be displayed if you have already used this tool in this selected project some Identification folders exists in EVWZooscan_phyto_lama iPlD_process sSorted_vignettes it doenst matter if you wantto extractto VIS image folder Press Cancel to abort Press YES to go on 89 Mo valid results file in Hiwooscan_targets marciWPID_processiiPid_ results Mo Cancel If some too large images haven t been opened a warning message will inform you at the end of the process EXTRACT VIGNETTES into folders version 6 11 date 2009 05 19 Project essais_duplicates_sep VIS image is to be found in its project Zooscan_scanwork subfolder The datafile can include as many additional identification columns File header must include the column names pred_ column is requested only for extraction to the PREDICTION folder Vignettes can be extracted either in the VIS folder unsorted or in the Sorted_viqnettes folder sorted according to prediction if exists Extract vignettes to Prediction folder Select method Extract all vignettes Y l Size selection V Select new sub folder prediction option selected above MV Convert all predicted Id to lowercase Open TEXT or CSV file or select UPDATE all dat1 tt files in Pid_results folder option Image na
116. xists the default value is false If you have kept a real RAW image in the _raw folder you do not need to keep the raw converted image from the Zooscan_scan folder as a ZIP archive as you will be able to convert again the RAW image from the _raw folder The SELECT PARTICLE TREATMENT PARAMETRES section allows user to define the additional operations to perform Defaults options are automatically displayed according to the files and data already processed Save thumbnail images of organisms vignettes If selected all the vignettes of the objects not tagged or set to 1 will be extracted in the sample subfolder Save also false tagged objects for the extraction of the tagged object s vignettes of the 0 false or 2 doubloons in case of frame 2 images Force doubloon check for image 2 The doubloon duplicates from the overlapping area of both frames of a 2400 dpi scanned sample check is done normally during the process which follows the scan In case of treatment failure or post processing of a frame 2 image you can force the comparison of the objects contained in the meas txt and 2 meas txt Work with separation mask allow user to separate objects If a separation mask already exists it will be used and edited if you say YES If you choose to skip using an existing mask the vis image will be automatically computed again You have there the possibility to save an image of the organisms with the outlines _
117. zoocont jpg file Tag function if some objects or doubloons have already be tagged in your _meas txt file they will anyhow be recovered and kept even if you haven t chosen the TAG function Note that if you have divided an object with the previous function the new computed objects will not keep the tag value of the source one Process manual identification allow user to manually set name to the objects in the image using an existing reference list of names This list can be upgraded during the process If you have automatically identified the objects you only have to create the right format identification file for the image and the names and labels will be displayed in your image Image with names can be stored Include PAR data is used only for images processed with the CNRS prototype 35 9 3 6 Process parameters for particle re process This menu simplified for the BATCH process will then be displayed It indicates the existing file already computed or not in the sample subfolder of the _work folder Keep the default settings targets_1_1 zip PARAMETRES version 3 14 x INFORMATION on EXISTING FILES METADATA FILE YES CORRECTED IMAGE WIS FILE YES LOG FILE exists and complete YES fIP FILE NO SEPARATION MASK YES TAG DATA YES IDENT DATA NO PAR METADATA FILE NO PROCESS PARTICLES ONLY SELECT PARTICLE TREATMENT PARAMETRES Threshold 244 0 Min ESD mm 1 5 Wax ESD mmi 100 SAVE

Zooprocess for ZOOSCAN MANUAL - Observatoire Océanologique

Contents

Download Pdf Manuals

Related Search

Related Contents

Zooprocess for ZOOSCAN MANUAL - Observatoire Oc&eacute;anologique

Contents

Download Pdf Manuals

Related Search

Related Contents

Zooprocess for ZOOSCAN MANUAL - Observatoire Océanologique