CellBuster 1.0.3 User Manual
Contents
1. el a EES RE EE 2 1 4 Generate the project In this last step pressing the Generate button you generate the project The Wizard verifies that all the images have the same frame that all the images are readable and copy everything in the project folder it also create a raw copy of the images selected and a file named proj cbw Design4Fun CellBuster User Manual TUCO OEE Project created in pee ale uster C hemotasis U Cell E uster Fress this button to launch CelBuster CellBuster then press New and import your sml project H some problem occurs you can step back and try to fix it when everything is OK you can launch CellBuster and open the proj cow pressing CellBuster button the wizard is closed and CellBuster is launched then you have to go to File New menu and select Import Proj Wizard See next section to see how to open the created project New Project x Select Manual to manually specify every parameter Select Import Wizard Proj to use 4 project previously created with the Wizard Select Launch Wizard bo run the wizard to help you in creating a new project Manual 7 Launch Wizard canes 2 2 Using CellBuster application The application CellBuster can be used to open or create a CellBuster project find cell movements and save result in terms of tracks on the screen and or data to be analyzed with Excel CellBuster is not very user friendly there is no help and also the only avail2. raw frameUd A row bramellT row frameO Oran frameOOS row fra ef II rsm w F format Multiframe TIFF 240 frame Size 640 ale g Prev Next gt gt When you click on an image a preview appears on the right side of the form If the file is a multiframe image you can use the two lt and gt button to move between the frame The two lt lt and gt gt button can be used to move to the next or previous file In the bottom left area of the form there are information about the format and the size of the image file This area is important especially for RAW image files where the guessed size could not be the right one you can change the width and the height of the image until you see in the preview area an image that looks to you familiar Regarding the RAW format this version of CellBuster Wizard can handle only RAW files that have O header size and 1 byte per pixel 256 gray level Design4Fun CellBuster User Manual F tandel raw tande 00 raw KU EN Sugml FCS framer raw frameU d raw lt i frame 2 ray framel 1 toua frame UL raw frameOOd raw frame NNA ram an Fk format Multiftame TIFF 240 frame width Bia Height SIE Header Size Prev Next gt gt Bytes per Pixel 4 KI sl zl zl zs farmat i format Multiframe TIFF 240 frame Multitrame TIFF 240 frame width RIESS oz Height width Height d SE
3. Design4Fun CellBuster User Manual CellBuster 1 0 3 User Manual Authors Andrea Giacosi Alessandra Sirri Page 1 of 10 Version 1 0 3 Design4Fun CellBuster User Manual REVISION HISTORY Date Revision Status 15 Jul 2004 0 1 Preliminary draft 1 TIN TRODUGC TION DEER S 1 1 WHATS CEUCEBUS TER Dor ao AE EEEE EREEREER ET AEAEE AE AAEE AE AAAA RTE 1 2 EE EE S 1 3 Ree E E WEE S 2 ee EEN A EEN R 2 1 USING CELEBUSTER WIZARD E S E ee E S E SNA RY 4 IMMUN TT Ee rs SENERE ONRAN EON ORR OIN ONRAN eee et eeeee Tees Tyrer Terr rece Tess eee eer AAEE 4 213 Order Images and select Size Step sis dessssssssssssssssssssssssssdssdsdsdssssdddddddddddddddddsdsdsddassssssscsssccccecscsesgeaceeessageceececeeeeececeees S 22 CREATE ANEW PILE EE f 2 3 Tis POR NAM ee 2 4 ANALYSE A NEW e e I aa LELEII LEERE EAEE ELE EEEE EE EEEE EEEE EREEREER EELEE EREEREER EE EELEE EEEREN A E E AE EE c PAZ AI E E EE R RE E e E d EE 2 4 3 MICHIEL GCQUISTHOW NODE csc ccccccssstllsssssssseererororiiiiiiiiiiiniiiiiiiereriiieiriiiieieeeeneieeecceeeeeccececcecesdemummumuuuuumnuumanuuuuanuugeuren 2 4 4 RE PA E EE EE E automatic track E R E e EE Jr SE E dl TTT EE Jr SN Export data filen Cee reece E RY It Design4Fun CellBuster User Manual 1 Introduction 1 1 What is cellbuster Cellbuster is a software created by Andrea Giacosi to analyse cell movement Chemotaxis esperiments are performec with a Dunn s chamber and migr
4. able image format is RAW one this is why developed the CellBusterWizard To create a new CellBuster project you use the new button on the toolbar or you go to the File New menu then you have to select between three possible modes e Manual to manually create the project e Import Wizard Proj to import a project created with the CellBusterWizard e Launch Wizard to launch the CellBuster wizard and create an empty project The Import Wizard Proj mode is the easiest way to start a new project you have only to browse for the file proj cow you previously created and press OK Design4Fun CellBuster User Manual Import Wizard x OK Cancel proje Browse Open 2h x Lookin Oi J enam Fe raw S proj chw My Recent Documents E Desktop My Documents re g My Computer s e de a Files of type CeliBuster Wizard output cbw DI Cancel o INTE File name Places x Results for CreateProcess After you have imported the Wizard created project you can work on it creating cell tracks as explained in the remaining part of this document When you have finished with your work you can save all your work in a CellBuster project extension is TRK you should save your project in the same folder you created with the Wizard Remember that is you want to restart later your work you have to open the CellBuster project using File Open and not import againg the proj cbw the proj cow does not con
5. ating cells are observed using a time lapse microscope This program written fo Microsoft Windows calculates the exact position of a cell through a stack of images It also calculates distance direction and speed of a cell from the start position Cell tracking can be done manually or automatically In the first case you have to follow a cell with your mousepointer and the position that is recorded is the one indicated through the mouse In the second case you have to select lt filter and click on the first frame on the cells you want to track The software will automatically calculate the pathways of the cells you have indicated 1 2 CellBusterWizard Starting from this version the CellBuster is distributed with an additional utility that helps the user in the creation of the CellBuster project this utility is called CellBuster Wizard This application has been developed using the Net framework and so it is required to install it in order to use 1 3 Technical support For any problem regarding the use of this software contact Andrea Giacosi giacosi libero it 2 Procedures 2 1 Using CellBuster Wizard In order to create from scratch a new CellBuster project you can launch the CellBusterWizard from your StartMenu JOK CellBuster The wizard is composed of 5 steps Welcome General Info Select Images Order images and select size Generate the project The output of the Wizard is a folder containing all the images and the additional
6. ave drawn Cell position relative to the origin setting the starting point as 0 0 coordinates Cell speed calculated on the shift of the cell center beetwen two following frames Vector distance module and direction of the displacement of a cell from the starting position Straightened distance total length of the pathway of a cell Average speed module and direction calculated on the vector distance Indicate a file name and four different file will be created
7. ce you have clicked on the cell you want to track 2 5 4 Automatic acquisition mode For this kind of acquisition you have to select a filter to distinguish a cell from the background At the moment the filter that best approximates a cell is the threshold Once you have set the threshold select the cells you are going to follow and the software will calculate their pathways through all the sequence 2 5 5 Select a filter Select the threshold filter from the tool select filter command To visualize the image with the threshold vo have created press on the cell icon on the toolbar You can adjust the threshold through the too filte option on the main menu Choose a threshold as stringent as possible On the dialog box you will see jus a part of the image but using the mouse you can move on different part of it Press the arrows button t see how the cell looks like through all the sequence 2 5 6 Create an automatic track Press on the 1pt icon on the toolbar and click on the cell you are going to follow just on the first frame Select the tool refine track command from the main menu At this point the software will follow automatically the cell and a tract will be created The position that is recoded rappresents the center of the cell area that is over the threshold in eact frame You can also set some parameters in order to help the software to recognise and follow a cell Press the tools refiner option command from the main menu A dialog bow wi
8. information required by the CellBuster in order to properly work The user can jump from one wizard step to the next previous using the Prev and Next button on the bottom of each step lt lt Prey Herst gt 2 1 1 General Info Step In the Description area you can write any information helps you to describe your experiment The name text field will be the name of the folder that will contain all the data describing the CellBuster project we are making The location is the parent folder that will contain the project folder we are creating the Browse button can be used to easily select the folder without typing it Design4Fun CellBuster User Manual Create New Project Wizard Step 2 of 5 Description Experiment number 3 July the 20th Chermotasis migration Mame Chemotaxis O04 Location C personalCellBuster Browse 2 1 2 Select Image step In this step you select all the images that you want to analyze with CellBuster On the left panel you will see the file you have selected and are currently part of the project On the right panel you see the files available in the folder you have selected using the Browse button On the right panel you select one or more images and you add to the selected images using the arrow buttons in the middle To add to the selected images list use the first two buttons To remove images from the selected images list you have to use the other two buttons Create New Projec
9. ll be displayed You can indicate the minumum anc maximum area of the cell in pixel and the maximum shift of the cell center beetween two following frames search zone radius You can analyse one cell at a time or many cells simultaneously In this case after pressing the 7 pt icon select all the cells you are going to follow Design4Fun CellBuster User Manual 2 5 7 Visualize a cell track To visualize a cell track click on one cell and press the single line icon on the toolbar Otherwise press the right buttor of the mouse and select the show track command To visualize all tracks on the movie select the three lines icon forn the toolbar Automatically created tracks are in blue while the others are in red Selected track appears in green Cell tracks are also recorded on the tracks editor Select the tool tracks editor command from the main menu to shov it 2 5 8 Visualize cell numbers There is a number for each cell track You can show them by pressing the 7 2 3 icon on the toolbar Automatically created tracks are numbered as P 1 P 2 P 3 Otherwise you can show cell numbers pressing the right button of the mouse and selecting show numbers 2 5 9 Export data file At the end of your analysis you can export the x y coordinates of a cell in each frame into an excel file Select the file export command from the main menu and a dialog box will be displayed Select the data you want to export Cell position relative to the axes you h
10. p Pro to convert your stack of images into the raw format 2 5 Analyse a new file 2 5 1 Create axis If your images are not in the correct orientation you can insert axes so that the source of chemoattractant is in the direction of one of the two axis The software calculates the positions of your cells respect to the axes you have drawn To rotate the axes press on the blue button on the toolbar and adjust axes orientation with the mouse Otherwise select the axes edit command on the main menu 2 5 2 Create cell tracks To follow the track of a cell you have two options a manual acquisition mode and an automatic acquisition mode 2 5 3 Manual acquisition mode Press on the noose command on the toolbar a fore sight will appear Point it on the cell you are going to follow anc click on the left button of your mouse Keep the button pressed through all the sequence until the end When you release the mouse button the movie will go on but acquisition will stop You can visualize the cell track by pressing or the single line icon on the toolbar A number is assigned to this track and you can visualize it by pressing on the 7 3 icon This kind of acquisition let you to know the position of your mouse pointer in each frame If a cell is moving very fas and you can not follow it in this way evevn changing the aimation speed you can use the click by click mode In thi case you visualize just one frame ata time and you can go to the next frame on
11. t Wizard Step 3 of 5 Selected Images Browse ftandeOO1 raw H andrea Projects CellsFi x tandcOU raw ee GE FCS 12 ore Bel aac Fain Gr ee ee frameU04 raw famel 3 Ge frame005 raw o S ramel UE ray E frame00 raw frameQ 1 ra Der ITER H framell rau EE EES 2 ramell raw frame009 raw DEE ane framed 0 rau tame eara frameO1 1 raw A4 frameO 2 raw framed 2 rau frame A rau type A rau You can browse for your image anywhere the selected images are automatically copied to the project folder Design4Fun CellBuster User Manual The supported image formats are GIF JPEG TIFF also multiframe Bitmap RAW A project can contain images of different formats 2 1 3 Order Images and select Size Step In this step you can perform several actions e Preview your images e Select the size of the RAW images e You can re order the images The image reordering is performed selecting one image and pressing the up down V button until you reach the desired position if in the previous step you select the images in the desired order you can save this step If a file is a multiframe image like a tiff image you cannot change the internal order of the frames to reorder frames in a multiframe image you have to use an external tool Create New Project Wizard Step 4 of 5 File 3 Frame 1 240 Land raw tande raw KO EN Sugml FCS fameli raw frameU A raw fameli
12. tain tracks info and everything you changed after the import You can have more then one cellbuster file analysis1 trk analysis2 irk in the same project folder that they can differs from tracks and analysis performed on the original empty project only images imported You can also restart from the empty project re importing the proj cbw file How to create manually a project is described in the next chapter 2 3 Create a new CellBuster project To create a new file choose the file new command from the main menu A dialog box will be displayed You have to fil this box indicating the length in pixel of the bridge where you can see the cells width and height in pixel of your frames time in minutes between two frames animation speed number of the last frame of the stack number of digits the folder containing your stack At this point press the command create and you will visualize your movie You can move through the sequence witt the arrows commands or using the arrows on your keyboard Otherwise you can see the entire movie pressing the filn icon on the toolbar To change the animation speed press the As command on the toolbar 2 4 File format Design4Fun CellBuster User Manual The movie should be composed of a stack of images saved as separate files Each file should be any size but i needs to be grayscale 8 bit per pixel color depth 256 gray level The files should be saved in raw format You can use Paint Sho
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