Proxan`s manual
Contents
1. Proxan User s Manual Rev 1 0 6 Bendor Research Pty Ltd Proxan User s Manual Table of Contents Acknowledgements and Credits 222222440000002000000000 RR nnnnnnnnnnnnnnnnnnnnnnnnn nn nnnnennnnnn nennen 5 A E EEE E een ee 7 ILL G 110010 Fo 9 NAN 11 V ET lg 15 PPP Pe 13 ARS po AM PP E E E esac aetna eeedse esate 13 V II Opening an IMaQf ccccccccncnnnccnnccnncnoncnnnonnnnnnnnonnnnnnnnnncnnnnnnnnnnnnnnnnnnronnnrnnnnnnnnnnnnnnarinnnns 13 AES Je lla AE AE SEE mA per 15 VI Proxan 0 0 eee 17 PENE NN 18 Vl l a Microscope Type uuesnenseennennnnnennnnnnennnnnnennnnnnnnnnnnennnn nenne nnnnnnnn nennen nenne nnnennnnnen 18 Vl l c Active and Reference COlOUFS ooooonccccnccccccononccnnnnncnononanncnnnnnnnnnnnnnnnnnnnnnnononanens 19 VII Appendix The GNU General Public LiCense cccccccoconccnccccnonoconccononaconcnnnnancnnnnnnos 25 List of Figures FOUS VET Proxan Man WINGON an dota eler REE 13 Figure V II Open file dialoo occcccccococccncocconcnononononcnnnnnnnancnnnononnncnnnononanrnnnnnnnaronnnonnnnos 13 Figure V III Image processing WINOW ccccooocccccoccoccccononccnononononcnnnnnnnnnnnnnnnnnnnnnnnnonannnnnnnnnnns 14 Figure V IV Containment mode control panel cccccccocccnncccccnnncnccnnnnnnnnnnnnnnnnnnonononcnnononnnnos 15 Figure V V ANN TN Hj 15 Figure V VI Section with Curved region of INteresSt ooccc2. Proxan is a software tool to determine if two sets of features on a microscopic image statistically exhibit a spatial relationship or not If you have an image on which two kinds of biological features are marked with different colours you may want to know whether they are related or not For example you have a tissue sample and you label the cell nuclei to green and attach a red fluorescent marker to a particular enzyme Your theory is that the enzyme is somehow related to the nucleus Since with fluorescent methods red and green together make yellow then yellow spots on the image would tell you that that particular enzyme occurs in the nucleus Note that the author of Proxan has no biomedical background so the examples are probably incorrect from a bio scientific point of view Please bear with me If you have a lot of yellow in the nucleus and there is not much red in anywhere else then you can be quite sure that the enzyme is indeed concentrated in the nucleus This is a well know method and various tools exist to analyse colocalisation Scientific papers often show scattergrams and colour distribution histograms to prove that a biological phenomenon is so strongly connected to an other that on images they overlap This kind of analysis however will not show relationships where the biological processes are related but would not spatially coincide Using our previous example if the enzyme in question does not occupy the nucleus but for some
3. conditions for copying distribution and modification follow 23 EEE Bendor Research Pty Ltd gt Proxan User s Manual GNU GENERAL PUBLIC LICENSE TERMS AND CONDITIONS FOR COPYING DISTRIBUTION AND MODIFICATION 0 This License applies to any program or other work which contains a notice placed by the copyright holder saying it may be distributed under the terms of this General Public License The Program below refers to any such program or work and a work based on the Program means either the Program or any derivative work under copyright law that is to say a work containing the Program or a portion of it either verbatim or with modifications and or translated into another language Hereinafter translation is included without limitation in the term modification Each licensee is addressed as you Activities other than copying distribution and modification are not covered by this License they are outside its scope The act of running the Program is not restricted and the output from the Program is covered only if its contents constitute a work based on the Program independent of having been made by running the Program Whether that is true depends on what the Program does 1 You may copy and distribute verbatim copies of the Program s source code as you receive it in any medium provided that you conspicuously and appropriately publish on each copy an appropriate copyright notice and disclaim
4. should execute without saying a single word It extracts Proxan s source code from the file you downloaded and places it in a new folder called proxan 1 0b Then you have to type the following cd proxan 1 0b to go to the new folder When you are there you have to build the program so type make If you everything Is right you will see the following Compi ling cli main c Building cli cli mod lots of lines with compiling and building Compi ling utils dialog c Building utils utils mod Building toplevel mod Linking proxan Done If you see the Done then you managed to build Proxan If you see an error message then there will be an error log file with the name of the last file processed but with an err suffix That is if the process dies while doing the Compiling utils dialog c then there will be a file utils dialog err explaining what the problem was Chances are that you will need somebody to read that file and see what can be done Nevertheless that should not happen and Proxan should compile with no problem If you succeeded there is one more step to do cp proxan usr local bin That command will place the Proxan executable into a place where executables programs are stored that are not strictly part of the system but any user of your machine should be able to use them To execute that last command more than likely you have to be roof the unix term for system administrator lf you do not know how to become root o
5. you to use this feature of the program efficiently we explain you how Proxan identifies colours On a two dimensional surface like your screen or a sheet of paper you can not represent a three dimensional colour space You have to choose two of the dimensions to be mapped onto a planar object and the third dimension must be treated separately Customarily the HSV colour scheme is represented by a disc The circumference of the disc represents the possible H values with usually red being at the top Thus a H value is actually an angle measured from the vertical 0 degrees means red 120 degrees is green 240 degrees is blue As you increase the angle further you will get up to 260 degrees where you are again at red Along the radius changes the saturation the centre of the disc being zero saturation no colour at all and on the circumference lay pure colours The disc is painted with maximum intensity or value so the centre of the disc is actually white Therefore the V component of the HSV colour is not represented on the disc and is shown in a separate way You are already familiar with the colour selection window Figure VI IV shows you the left hand side of the dialogue box Figure VI IV Colour selection with HSV representation On the figure you can see the HS circle and the V bar The H value is the angle measured in degrees anticlockwise from 12 o clock The little circle in the HS circle represents the point selected by the actu
6. Close f you move the cursor over the image instead of the pointer you will see a circle that is semi transparent Now move the pointer to the bottom left of the image somewhere about the 200um ruler and press the left mouse Brush size Er Small 4 Medium x Large button While pressing the button move the mouse You will see that it leaves a semitransparent trail Doodle with the mouse until you completely cover the ruler and the text but not much around it something like on Figure V V Clear all Restore The semi transparent area represents an area on The image that Proxan should ignore for any further activity Objects are not searched for within those areas and when Proxan creates random images those areas are off limit No objects can be in them not even partially You can have as many such areas as you wish On the control panel you can select from three different brush sizes to allow you to mark large areas as well as small areas with ease re Ml de Figure V IV Containment mode control panel Figure V V Containment area If you want to undo some containment marking you have several choices If you press the right mouse button and move the mouse it then will delete containment areas instead of creating them So doodling over an already marked containment area while pressing the right button you can get rid of that area If you want to delete all containment areas you
7. ED BY APPLICABLE LAW OR AGREED TO IN WRITING WILL ANY COPYRIGHT HOLDER OR ANY OTHER PARTY WHO MAY MODIFY AND OR REDISTRIBUTE THE PROGRAM AS PERMITTED ABOVE BE LIABLE TO YOU FOR DAMAGES INCLUDING ANY GENERAL SPECIAL INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE USE OR INABILITY TO USE THE PROGRAM INCLUDING BUT NOT LIMITED TO LOSS OF DATA OR DATA BEING RENDERED INACCURATE OR LOSSES SUSTAINED BY YOU OR THIRD PARTIES OR A FAILURE OF THE PROGRAM TO OPERATE WITH ANY OTHER PROGRAMS EVEN IF SUCH HOLDER OR OTHER PARTY HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES END OF TERMS AND CONDITIONS 27
8. al H and S values and the line across the V bar indicates the actual V value for the colour 20 VI Proxan Options When you define the reference and The active colours to Proxan if determines their position on the HS circle If draws two radii through these points Then it halves the angular distance between them and that will be the acceptance angle for the particular colour If sounds complicated but a simple diagram explains it easily On Figure VI V you see the colour circle with R and A representing the reference and active colours respectively Figure VI VI shows the same colours with the radii passing through them The dashed line is the colour midway between them and a is half of the angle between R and A Figure VI VII shows the circumference of the circle mapped to a line The R and A markers represent the points where their respective radii intersect the circumference The shaded trapezoids below show the acceptance level of any particular H value for reference or active colour or to use fuzzy set terminology the membership functions for R and A As you can see as colours change from green to yellowish green the colour more and more starts to belong to the reference From yellowish green to orange the colours all considered to be reference colours From orange to red all colours do still belong to the reference but they slowly start to belong to the active objects as well Red is both reference and active in the same t
9. and the button changes back fo Show Whether the options window is visible or not has no effect on Proxan The hiding feature is merely an aide to minimise the cluttering of your desktop Proxan has lots of options and you should be familiar with them if you want to achieve good results 17 Bendor Research Pty Ltd Proxan User s Manual VII Pre processing Options VI l a Microscope Type First you have To select whether the image you process was taken by a light Microscope or with a fluorescent one If might sound unreasonable All in all an image is an image and it is none of Proxan ss business to know how you got it If is not so f you use fluorescent microscopy you use a fluorescent marker on the biological phenomena The marker will emit light when the microscopes UV light source excites it Consequently if an area of your slide is stained with two markers both will get excited and emit light with their characteristic colours For example if one marker glows green and the other glows red on the colocalisation areas you will see yellow Fluorescent microscopy uses additive colour Mixing Light microscopy uses stains that absorb parts of the visible spectra If you have a purple stain it means that it absorbs the middle of the spectra that is the green If you have a stain that looks yellow it will absorb the short wavelength end of the spectra that is blue If you then have a slide where these
10. atically receives a license from the original licensor to copy distribute or modify the Program subject to these terms and conditions You may not impose any further restrictions on the recipients exercise of the rights granted herein You are not responsible for enforcing compliance by third parties to this License 7 If as a consequence of a court judgment or allegation of patent infringement or for any other reason not limited to patent issues conditions are imposed on you whether by court order agreement or otherwise that contradict the conditions of this License they do not excuse you from the conditions of this License lf you cannot distribute so as to satisfy simultaneously your obligations under this License and any other pertinent obligations then as a consequence you may not distribute the Program at all For example if a patent license would not permit royalty free redistribution of the Program by all those who receive copies directly or indirectly through you then the only way you could safisfy both it and this License would be to refrain entirely from distribution of the Program If any portion of this section is held invalid or unenforceable under any particular circumstance the balance of the section is intended to apply and the section as a whole is intended to apply in other circumstances It is not the purpose of this section to induce you to infringe any patents or other property right claims or to contest validity of any s
11. ave the option of following the terms and conditions either of that version or of any later version published by the Free Software Foundation If the Program does not specify a version number of this License you may choose any version ever published by the Free Software Foundation 10 If you wish to incorporate parts of the Program into other free programs whose distribution conditions are different write to the author to ask for permission For software which is copyrighted by the Free Software Foundation write to the Free Software Foundation we sometimes make exceptions for this Our decision will be guided by the two goals of preserving the free status of all derivatives of our free software and of promoting the sharing and reuse of software generally 26 VII Appendix The GNU General Public License Pr OAX MTT NO WARRANIY 11 BECAUSE THE PROGRAM IS LICENSED FREE OF CHARGE THERE IS NO WARRANTY FOR THE PROGRAM TO THE EXTENT PERMITTED BY APPLICABLE LAW EXCEPT WHEN OTHERWISE STATED IN WRITING THE COPYRIGHT HOLDERS AND OR OTHER PARTIES PROVIDE THE PROGRAM AS IS WITHOUT WARRANTY OF ANY KIND EITHER EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE THE ENTIRE RISK AS TO THE QUALITY AND PERFORMANCE OF THE PROGRAM IS WITH YOU SHOULD THE PROGRAM PROVE DEFECTIVE YOU ASSUME THE COST OF ALL NECESSARY SERVICING REPAIR OR CORRECTION 12 IN NO EVENT UNLESS REQUIR
12. can press the Clear all button If you want to revert to the containment areas that were active when you selected the containment mode you can click on the Restore button Your containment area markings will be committed when on the Main mode panel you change the mode from Containment to Processing 15 Bendor Research Pty Ltd Figure V VI Section with curved region of interest Proxan User s Manual You can use the containment to exclude artefacts like you did with the ruler on the image sometimes the image itself is too large it contains biological sections that are of no use for you If the useful section is more or less rectangular you might crop the image but more offen than not microscopic sections are not like that Figure V VI shows an example Let us assume that what you are interested in is the blue and red labelled region in the middle You can not simply crop the image because the area of interest is not rectangular Instead you can exclude all the rest of the image and leave only that little section active using the containment mode The resulting image should look something like Figure V VII When Proxan processes the image it will ignore everything except the blue and red labelled curved stripe in the middle Fa gps Main mode mM Selected Containment 4 Close Save Brush size vw Small v Medium Clear all Restore Fiqure V VII Curv
13. cccnnonccoccnnccnoncncncnnnnnonancnnnnnnnonononos 16 Figure V VII Curved area of interest after containment rrrrnrnrnnnnnnnvnrnnnnnnnvvnnrnnnnnnernnnnnnenn 16 Figure VI I Processing OPtIONS oocccccocnnccocococonononocnnnnnnonnrononononnonannnnnnnrnnnnnnrnnonannnonnnrnnnnnnnnss 17 Figure VI Il Additive and subtractive colour MIXINO occcccccncncoconnnnocancnnnnoncononancnnononcnnononos 18 Figure VI III Colour selection dialoJ cccccccocncconccocononcnnnccnnnoncnnnnonononenonnncnnnnnnononcnnnnnnos 19 Figure VI IV Colour selection with HSV representatiON cccooooncccccccononcncccononncononnnnnconononanonoss 20 Figure VI V HS circle with selected CO lOUES ccccoconncocccoconnnccononnnncnnnoncnnonononcnnnonnnncnononnnnos 21 Figure VI VI HS circle with acceptance Angle cccccsccssssscceceeceeeseeeeeececsesseeeeeesseneaseees 21 Figure VI VII Colour acceptance fTUNCIIONS ccccooocccnccccconconnconennnonnnnnnnnnnnnnnnonnnnnnnnnoncnnnnnnnnnos 21 Figure VI VIII Classification of various CO OUFS coonccncccoccnncccnoconcconaconcnnoncnnnnonanonononaronnonanos 22 Figure VI IX Colour classification resSultS ooccccccoccnnnnncccononcnnnncononnnoncnnnnnncnnnnnonanconnnnnnnnnos 22 I Acknowledgements and Credits ACKNOWLEDGEMENTS AND CREDITS Proxan would never have been written without the following people Dr Krisztina Valter whose research work gave rise of the original version of a statist
14. cts if there is no such correlation would be evenly distributed throughout the image or parts of it that you did not exclude from the analysis It is extremely important to understand that if the above assumption is not true then you can not use Proxan for analysis because you would get false positive results If The assumption however is true Then Proxan can tell you if there is a spatial relationship between the reference and active objects In particular Proxan examines the distances between the reference and active objects and statistically analyses if the distances correspond to a random distribution of active objects or the locations of the active objects seem to show a correlation to the locations of the reference objects The rest of this manual will explain you how IV Installation Dr OAX MTT IV INSTALLATION Installing Proxan requires that you are somewhat familiar with Linux it is not a click and go affair as yet If you are not comfortable with terms like shell directories command line being root and alike it might be a better idea to ask someone in your IT department to install Proxan for you If you decide to do it yourself here is what you have to do First of all you need to download the source code It will be a file called proxan 1 0b tgz You save this file in some folder or directory Then in a shell window you should go into that directory and type the following tar xfz proxan 1 0b tgz This command
15. d blue reaches the eye Due to the colour of the dyes it is called the CMY colour scheme 19 Bendor Research Pty Ltd Proxan User s Manual While those colour schemes are good for technical purposes they have not much in common the way humans feel about colours It is very hard to interpret a colour description like this you know that greenish yellow more greenish a bit actually and kind of dull like not that harsh and it was sort of dark if you see what I mean To handle colours in that more intuitive fashion an other colour description was invented It is called HSV from Hue Saturation and Value Hue is the actual perceived colour and indeed the H value of greenish yellow is between the H values of green and yellow If it is more greenish then its H value is closer to that of green than yellow S is the saturation which tells you how pure or dull the colour is from the absolute pure colours of the rainbow down to grey where there is no colour any more at all Finally V for value tells you how light or dark the colour is between black and the maximum intensity The RGB CMY and HSV colour representations are equivalent they can be transformed to each other However the HSV representation has certain properties that the other two lacks Proxan uses HSV when mapping your image to the reference and active colour Colour extraction is the first step in Proxan s operation and a very important one To help
16. ed area of interest after containment If is important that when you process an image you have no empty areas To understand why you should know how Proxan processes the statistical data of your image It will be explained later For now if is enough to remember that empty areas of the image should be excluded using the confinement tool 16 VI Proxan Options Pr OA MIE VI Proxan OPTIONS On the main Proxan panel in the Options column press the Show button The button will change to Hide and Proxan will present you an other window like the one on Figure VI I Options Preprocessing Reference extraction Active extraction Microscope Algorithrn Algorithirrr Light 4 Large objects 4 Large objects E Reference colour Minimum seed value Minimum seed value Change 0 53 FEE ERE 0 51 Active colour Minimum acceptance Minimum acceptance ms Change 0 24 0 42 Filter kernel radius Minimum object size Minimum object size rn 4 5 50 Co 25 Gamma correction Fill holes in objects F Fill holes in objects SEE 0 59 F Equalise intensity Object pairing Statistics Display colours Maximum distance Number of samples Reference objects To EEN mM Many pairs for references Active objects Change Distance segments change Confinement area EST Many pairs for actives Figure VI I Processing options If you press the Hide button in the main window the options window will disappear
17. ent itself accompanies the executable If distribution of executable or object code is made by offering access to copy from a designated place then offering equivalent access to copy The source code from the same place counts as distribution of the source code even though third parties are not compelled to copy the source along with the object code A You may not copy modify sublicense or distribute the Program except as expressly provided under this License Any attempt otherwise to copy modify sublicense or distribute the Program is void and will automatically terminate your rights under this License However parties who have received coples or rights from you under this License will not have their licenses terminated so long as such parties remain in full compliance 5 You are not required to accept this License since you have not signed it However nothing else grants you permission to modify or distribute the Program or its derivative works These actions are prohibited by law if you do not accept this License Therefore by modifying or distributing the Program or any work based on the Program you indicate your acceptance of this License to do so and all its terms and conditions for copying distributing or modifying the Program or works based on it 25 SS Bendor Research Pty Ltd a Proxan User s Manual 6 Each time you redistribute the Program or any work based on the Program the recipient autom
18. er of warranty keep intact all the notices that refer to this License and to the absence of any warranty and give any other recipients of the Program a copy of this License along with the Program You may charge a fee for the physical act of transferring a copy and you may at your option offer warranty protection in exchange for a fee 2 You may modify your copy or copies of the Program or any portion of it thus forming a work based on the Program and copy and distribute such modifications or work under the terms of section above provided that you also meet all of these conditions a You must cause the modified files to carry prominent notices stating that you changed the files and the date of any change b You must cause any work that you distribute or publish that in whole or in part contains or is derived from the Program or any part thereof to be licensed as a whole at no charge to all third parties under the terms of this License c If the modified program normally reads commands interactively when run you must cause it when started running for such interactive use in the most ordinary way fo print or display an announcement including an appropriate copyright notice and a notice that there is no warranty or else saying that you provide a warranty and that users may redistribute the program under these conditions and telling the user how to view a copy of this License Exception if the Program itself is interactive but does
19. hese things To protect your rights we need to make restrictions that forbid anyone to deny you these rights or to ask you to surrender the rights These restrictions translate to certain responsibilities for you if you distribute copies of the software or if you modify it For example if you distribute copies of such a program whether gratis or for a fee you must give the recipients all the rights that you have You must make sure that they too receive or can get the source code And you must show them these terms so they know their rights We protect your rights with two steps 1 copyright the software and 2 offer you this license which gives you legal permission to copy distribute and or modify the software Also for each authors protection and ours we want to make certain that everyone understands that there is no warranty for this free software If the software is modified by someone else and passed on we want its recipients to know that what they have is not the original so that any problems introduced by others will not reflect on the original authors reputations Finally any free program is threatened constantly by software patents We wish to avoid the danger that redistributors of a free program will individually obtain patent licenses in effect making the program proprietary lo prevent this we have made it clear that any patent must be licensed for everyone s free use or not licensed at all The precise terms and
20. ical proximity analysis program for biological features on microscopic images Prof Jonathan Stone who pursued me to make Proxan general enough so that it could be released publicly Dr Karen Cullen whose research work called for an extension of the original program ultimately resulting in Proxan as if is NOW All my teachers who put real effort into teaching me those morsels of knowledge that ultimately made the creation of Proxan possible All the programmers whose often unrewarded effort created a high quality open and free development environment where knowledge is shared for the common good rather than exploited for the benefit of the selected few My children who patiently accepted that while writing Proxan I spent a lot less time with them than either they or wished Thank you all The microscopic images used in this document are the courtesy of the Department of Anatomy and Histology of The University of Sydney and the Research School of Biological Sciences of The Australian National University and used with permission The Proxan project was funded entirely by Bendor Research Pfy Ltd Proxan II License Pr OAX MTT ll License Proxan is released under the Free Software Foundations GNU General Public License version 2 0 or any later revisions at your own discretion or GPL for short The entire text of the license can be found in the Appendix Still we explicitly spell it out that there is no guarantee or warran
21. ime From red to reddish magenta the colour keeps to mark an active object while in a lesser and lesser degree if still also indicates a reference object From reddish magenta to pure magenta all colours belong solely to the active objects and from magenta to blue the colours decreasingly indicate the presence of an active object Colours from blue to green do not indicate either active or reference objects Prox ait Figure VI V HS circle with selected colours Figure VI VI HS circle with acceptance angle Figure VI VII Colour acceptance functions 21 Proxan User s Manual Bendor Research Pty Ltd On Figure VI VIII you can see the colour circle again with 4 colours marked on it numbered from 1 fo 4 as well as the active and reference colours When Proxan classifies the samples 1 to 4 it maps Them to the linear representation of the circumference of the colour circle and calculates their membership value in the active and reference colours This is shown on Figure VI IX In our example colour 1 will 100 belong to the reference objects colour 2 will be 100 active and in the same time about 35 reference Colour 3 indicates an about 45 presence of an active object Colour 4 does not indicate either active or passive objects and is simply ignored by Proxan Figure VI VIII Classification of various vi These percentages do not fully classify a pixel For each pixel Proxan calculates an inte
22. indow that looks like Figure V II The buttons Create Dir Delete File and Rename File do create a new directory delete The file marked in the Files window or rename the marked file respectively The Home and Desktop buttons go to your home directory or to your desktop folder if you are using a desktop system such as KDE or GNOME The Directories window show the directories within the one you are currently in By double clicking on a directory you can enter into that directory or open that Create Dir Delete File i eat cli control folder if you like the folder docs notation better The ie RE directory called means 28 options the directory or folder that 0 K contains the current one that is it takes you one level closer to the root of the directory tree Since the directories are usually represented as an upside down tree this is customarily called traversing up on the selection rusrelocakwork Bendor Prosan Rename File rustlocakwork Bendor Prosan A proc tiff A stat tiff ti B tiff tif O tiff Makefile Makefile inc hdakefile inc hal afila OK Cancel J Figure V II Open file dialog directory hierarchy The list selection bar above the two windows contains a list of directories above the current one using That list you can fraverse up as many levels as you want in one hit instead of double clicking the directory many times The in the Directories wi
23. ite and the right hand side shows the colour represented by the sliders lf you click on Cancel then the window will be closed and nothing else will happen If you press OK then the colour set by the sliders will be used for selecting the active or reference colours Since colour selection is the first step of identifying the features on your image we give you a detailed explanation of how Proxan decides if a pixel belongs to an active object a reference object simultaneously both or none at all Colours can be described in many ways Proxan uses three of them At the end of the day it all goes back to the fact that the human eye has three kinds of colour receptors Each kind is most sensitive to a particular wavelength or colour Namely you have red green and blue sensitive receptors Not everything but almost all colours that you can see can be synthesised by mixing light of that three colours Therefore when using additive colour mixing if is most convenient to describe the colour or light by giving the intensities of the red green and blue light sources That notation is known as RGB The most obvious example is the colour TV If you use subtractive mixing such in photography or in printing you use three kinds of ink that absorb one of red green or blue The resulting dye colours are cyan magenta and yellow respectively By mixing these you can control what is left from white light and at the end determine how much red green an
24. lled images to Proxan If you want to analyse them use an image manipulation program to eliminate the third label 22 VII Appendix The GNU General Public License PrOXLEN VII Arrenoix THE GNU GENERAL Pustic License GNU GENERAL PUBLIC LICENSE Version 2 June 1991 Copyright C 1989 1991 Free Software Foundation Inc 59 Temple Place Suite 330 Boston MA 02111 1307 USA Everyone is permitted to copy and distribute verbatim copies of this license document but changing it is not allowed Preamble The licenses for most software are designed fo take away your freedom fo share and change it By contrast the GNU General Public License is intended to guarantee your freedom to share and change free software to make sure the software is free for all its users This General Public License applies to most of the Free Software Foundation s software and to any other program whose authors commit to using it Some other Free Software Foundation software is covered by the GNU Library General Public License instead You can apply it to your programs too When we speak of free software we are referring to freedom not price Our General Public Licenses are designed to make sure that you have the freedom to distribute copies of free software and charge for this service if you wish that you receive source code or can get if if you want It that you can change the software or use pieces of It in new free programs and that you know you can do t
25. n the image you are going to process HM SS O O E i gt Overlap of red and green fluorescent markers Overlap or red and green absorbent stains Fiqure VI II Additive and subtractive colour mixing 18 VI Proxan Options Pr OX MIE VI l c Active and Reference Colours These select the colours of the active and the reference objects on your image Colour selection is a very important part of Proxans operation When looking at a pixel Proxan has To decide how much that pixel belongs to a reference object and how much it belongs to an active object The only information she has is the colour of the pixel and the two colours you nominate to be representative for the two kinds of objects To nominate a colour you have to click on the Change button for the reference or active colour in the Options window A new window will be opened where you can change the colour for the relevant object RASO ETER Hue PL saturation FESTER Value MR Red eee OK Cancel Figure VI III Colour selection dialog Figure VI III shows the colour setting dialogue box You can set the desired colour by moving the sliders The Red Green and Blue sliders change the given component of the colour The Hue Saturation and Value sliders express the same colour in a different form If you move a slider in either group the sliders in the other group will move correspondingly In the bar below the colour circle the left hand side is always wh
26. n your machine then ask the person who administers your machine to do that last step for you He or she can also assign Proxan to a menu entry or a desktop icon for you if you dont know how to do It 11 V Getting Started V GETTING STARTED V I Starting Proxan Proxan You can start Proxan by simply typing the word proxan in a shell window Alternatively if you created a desktop item or a menu association for it you just click on the relevant icon or menu item When you start Proxan you will see a window like the one on Figure V I The actual shape of the buttons might be different from what you see on the picture depending on what GTK version your Machine is using and what theme you chose for your desktop but that should not be a concern The operation of Proxan is not affected by the button theme Under the Proxan logo you can see the progress bar That will inform you about the progress of each operation that Proxan does Currently Proxan is not doing anything so it is not showing any activity Below the progress bar you find the actions The leftmost column the image buttons if what you have to be concerned about first V II Opening an Image Proxan Progress Actions Image Process Options Misc Open Colour Load Guit Gamma Save Oo all d Extract Default JOptlock Fairing Show _ Stats Fiqure V I Proxan main window If you click on the Open button Proxan will open a new w
27. ndow refers to the current 13 Bendor Research Pty Ltd nl Proxan User s Manual directory double clicking that will have no noficeable effect Finally the Files selection window itself presents you the files in the directory sorted in alphabetical order Please note that all capital letters come before any of the lower case letters thus Z tiff is before a tiff If you single click on a filename it will be copied to the filename line under the Selection line If you click on OK then whatever filename is in the bottom filename line will be passed to Proxan as your image file You can edit that line by hand by clicking on it if you know the name of the file If the name of the file contains a slash it will be interpreted as a file in a directory in the current directory For example if you are currently in usr local images and you type the filename mystuff foo bar tiff then click OK then Proxan will try to open the file usr local images mystuff foo bar tiff On the other hand if the name in the file selection line starts with a slash then it will be interpreted as the full filename regardless of where you are That is if you are in usr local images and you type home col league experiment nice tiff then Proxan will look for home colleague experiment nice tiff and ignore your current directory usr local images If you click on Cancel nothing happens Proxan just closes the file selection window If you selected a file that is
28. ng a Accompany it with the complete corresponding machine readable source code which must be distributed under the terms of Sections 1 and 2 above on a medium customarily used for software interchange or b Accompany It with a written offer valid for at least three years to give any third party for a charge no more than your cost of physically performing source distribution a complete machine readable copy of the corresponding source code to be distributed under the terms of Sections and 2 above on a medium customarily used for software interchange or c Accompany it with the information you received as to the offer to distribute corresponding source code This alternative is allowed only for noncommercial distribution and only if you received the program in object code or executable form with such an offer in accord with Subsection b above The source code for a work means the preferred form of the work for making modifications to it For an executable work complete source code means all the source code for all modules it contains plus any associated interface definition files plus the scripts used to control compilation and installation of the executable However as a special exception the source code distributed need not include anything that is normally distributed in either source or binary form with the major components compiler kernel and so on of the operating system on which the executable runs unless that compon
29. not a TIFF image Proxan will send you an error message in a window and do nothing else If you selected a TIFF image Proxan will open it and you will get a new window as shown on Figure V III Main mode r Selected Processing 4 Close Save gt Background v Blank u ET S A ard p y gt ay A YE u RE v Filtered u 9 A e A Original Ba AR y Equalised v 3 Foreground _ Objects 4 Distances 1 Overlaps 4 Confinement J Hatch objects Figure V III I mage processing window This is the image processing window As you can see most of it is occupied by the image itself Proxan tries not to open a window larger than about of your display area If the image is 14 V Getting Started IPPON MIL larger than that then the scrollbars under and to the right of if can be used to pan around the image Alternatively you can resize the window Note that if you shrink the window you may hide the control panes as well The control panels are not scrollable V I Containment At the moment the various display selections are not too useful for you mainly because there is not much To select from you only have the original image There is one tool however that can be useful If you move the mouse over the button that says Processing on the Main Mode panel press it and select Containment you will notice that the control panel changes to what is shown on Figure V IV Also Containment i
30. not normally print such an announcement your work based on the Program is not required to print an announcement These requirements apply to the modified work as a whole If identifiable sections of that work are not derived from the Program and can be reasonably considered independent and separate works in themselves then this License and its terms do not apply to those sections when you distribute them as separate works But when you distribute The same sections as part of a whole which is a work based on the Program the distribution of the whole must be on the terms of this License whose permissions for other licensees extend to the entire whole and thus to each and every part regardless of who wrote it 24 VII Appendix The GNU General Public License Pr OX MTT Thus It is not the intent of this section to claim rights or contest your rights to work written entirely by you rather the intent is to exercise the right to control the distribution of derivative or collective works based on the Program In addition mere aggregation of another work not based on the Program with the Program or with a work based on the Program on a volume of a storage or distribution medium does not bring the other work under the scope of this License 3 You may copy and distribute the Program or a work based on it under Section 2 in object code or executable form under the terms of Sections I and 2 above provided that you also do one of the followi
31. nsity value that is dependent on the selected microscope type the actual brightness of the pixel and the saturation of the colour Then according to the classification results the pixel will receive its reference and active value l R 2 A 3 Figure VI IX Colour classification results After the above described process Proxan ends up with two values for each pixel These values indicate the calculated active intensity and reference intensity of the pixel In effect Proxan ended up with a new image using only two colours First if the reference and active colours are close to each other then the acceptance region will be very narrow Consequently you have to set the colours very precisely if you want Proxan to recognise your objects Secondly Proxan s colour classification works on the colour similarity basis and not by component values Consequently if a pixel has a colour that by human perception does not resemble either the active or the reference colours Proxan will not assign that pixel to either set This might be a problem if you use triple labelled images Say you label your images with red green and blue Your reference colours are red and green If blue is not present in a pixel Proxan works fine However if there is a lot of blue and not that much red and green then Proxan will simply discard the pixel Its hue will be outside the acceptance region even though it contains both red and green Do not give triple labe
32. reason it has a high concentration around It then you will not have yellow on your image so scattergrams will not prove your theory about the relationship even though you are right The enzyme is indeed related to the nucleus it just doesnt get in The reason for the failure to detect the relationship is that colour analysis examines a pixel at a time and does not even try to locate features on the image lo discover spatial relationships that do not manifest in actual overlap you need to employ some other method Proxan is a tool for the above mentioned kind of problems She identifies two kinds of objects on an image One set that Proxan calls the reference objects is supposedly something that is inherent in the biological matrix of your image It can be for example blood vessels cells in a Tissue the nuclei from the previous example or even some bacteria The location of these objects is considered given that is determined by the tissue or sample itself and whether your theory is right or wrong has no effect on it The other kind of objects the active objects in Proxan s terminology represent some other biological phenomenon that you marked by staining or with a fluorescent marker That can be blood plaques or debris from dead cells or some specific enzyme or phagocytes congregating around some nasty thing What you want Proxan to do is to tell you if there is some spatial correlation between These objects Proxan assumes that the active obje
33. stains colocalise the overall image will have a reddish colour because that is the only part of the spectra that is not absorbed by either stain We can also use the fluorescent example s red and green colours If your stain is red it means that it absorbs blue and green If it is green then it absorbs red and blue So if you apply both together they absorb all red green and blue Thus you are left with the lack of any light that is black This method of eliminating certain components of the spectra is called somewhat mistakenly subtractive colour mixing If is not subtraction what really happens If you have a stain that eliminates 80 of the red and the green part of the spectra and an other one that eliminates 80 of the green and blue part of the spectra when they are co localised together they do not absorb 160 of the green light What the stains do is not subtraction but attenuation multiplication by a number between 1 and 0 Nevertheless the method is called subtractive and thanking to the logarithmic nature of human vision if you treat it as If it was subtraction you get quite good results That is what printers and photographers do and for that matter that is what Proxan does Figure VI Il shows the result of red and green fluorescent markers and stains overlapping in an area It clearly shows how different the end result is That should explain why it is Proxan s business to know if you used fluorescent or light microscopy to obtai
34. ty associated with Proxan Proxan and its documentation are Copyright O 2004 Bendor Research Pty Ltd All rights reserved This program is free software you Can redistribute it and or modify it under the terms of the GNU General Public License as published by the Free Software Foundation either version 2 of the License or at your option any later version This program is distributed in the hope that it will be useful but WITHOUT ANY WARRANTY without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE See the GNU General Public License for more details You should have received a copy of the GNU General Public License along with this program if not write to the Free Software Foundation Inc 59 Temple Place Suite 330 Boston MA 02111 1307 USA IN NO EVENT UNLESS REQUIRED BY APPLICABLE LAW OR AGREED TO IN WRITING WILL ANY COPYRIGHT HOLDER OR ANY OTHER PARTY WHO MAY MODIFY AND OR REDISTRIBUTE THE PROGRAM AS PERMITTED ABOVE BE LIABLE TO YOU FOR DAMAGES INCLUDING ANY GENERAL SPECIAL INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE USE OR INABILITY TO USE THE PROGRAM INCLUDING BUT NOT LIMITED TO LOSS OF DATA OR DATA BEING RENDERED INACCURATE OR LOSSES SUSTAINED BY YOU OR THIRD PARTIES OR A FAILURE OF THE PROGRAM TO OPERATE WITH ANY OTHER PROGRAMS EVEN IF SUCH HOLDER OR OTHER PARTY HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES III Introduction Dr OAX MTT Ill INTRODUCTION
35. uch claims this section has the sole purpose of protecting the integrity of the free software distribution system which is implemented by public license practices Many people have made generous contributions to the wide range of software distributed through that system in reliance on consistent application of that system it is up to the author donor to decide if he or she is willing to distribute software through any other system and a licensee cannot impose that choice This section is intended to make thoroughly clear what is believed to be a consequence of the rest of this License 8 If the distribution and or use of the Program is restricted in certain countries either by patents or by copyrighted interfaces the original copyright holder who places the Program under this License may add an explicit geographical distribution limitation excluding those countries so that distribution is permitted only in or among countries not thus excluded In such case this License incorporates the limitation as if written in the body of this License 9 The Free Software Foundation may publish revised and or new versions of the General Public License from time to time Such new versions will be similar in spirit to the present version but may differ in detail to address new problems or concerns Each version is given a distinguishing version number If the Program specifies a version number of this License which applies to it and any later version you h
Download Pdf Manuals
Related Search