User Manual - AgesGalore
Contents
1. Chapter 7 Grain segmentation For single grain evaluation the user has to define regions of interest ROIs which correspond to what is considered to be a grain Within these ROIs the signal from all pixels will be summed and the grain thus be treated as one entity AGESGALORE2 uses the IMAGEJ ROI format which consists of binary files one for each ROI with extension roi In the common case of multiple ROIS the binary files will be enclosed in a single compressed file with extension zip The detection of grains and definition of ROIs is essentially an image segmentation problem Since a plethora of segmentation algorithms exists for IMAGEJ AGESGALORE uses indirectly or directly not yet the corresponding IMAGEJ plug ins for maximum flexibility 7 1 Using ImageJ The use of IMAGEJ for segmentation is reasonable as long no ROI definition from within AGESGALORE is possible or if new algorithms should be tested The following describes the corresponding workflow 1 First download the latest version of IMAGEJ version 1 x not 2 x here http rsbweb nih gov ij download html We recommend the ver sion bundled with Java to avoid additional installation problems 2 Install IMAGEJ on your machine 3 To read images from a CCD camera in WINSPEC format extension spe you need the SPE plugin which is found here http rsbweb nih gov ij plugins spe html Follow the installation instructions on the page i e copy the files2. sequence sequence of the measurement syntax 13 Number of images for step dose group type dose where needed detector set parameter sets the current detector model implemented at the moment 1 SIMPLE see 4 2 To check if a detector is set use data list parameter for the chosen detec tor 1 SIMPLE a single number giv ing the background eval dose fitType performs a dose response evalu ation i e computes the equiva lent dose its error for all ROIs if defined and all pixels sets type of fit to be used imple mented as at moment 1 Linear see 2 ExpSat see save index saves the results of a dose evalu ation to disk help displays this information 14 protocol set interval useTestDose set protocol to use for dose response implemented at the moment 1 MASS SAR like protocol for pixelized data see 5 1 To check if a protocol is set use data list Two or four numbers will be truncated to integer values sep arated by that indicate the one based index of the time intervals to integrate the sig nal and the background see 5 1 For example interval 1 2 8 10 will integrate the first two images of the shine down curve and subtract the 8 to the 10 image as background If given the photoncounts use within the MASS protocol will be normalized by the corresponding testdo
3. and 25 d Image File Edit Image Process Analyze Plugins Window Help Boeljon Is 41812 gt Stk Sor3 NGS0013 DRT 1512 SPE a sz Adaptive Threshold Preview 102 2x512 pixe The local threshold will be calculated by using the mean y from the pixel block with size 3 gt 76 then substract Y DIE Substract a smaller negative value will result in a higher threshold thus less pixels are thresholded while substract a larger value will give a lower threshold M Output Mask M Preview OK Cancel Figure 7 2 Adaptive threshold user dialog Output Mask and set the values so that grains are reasonably repre sented highlighted in red Irregularities and artifacts e g small high lighted areas outside the grains single highlighted pixels can be adjusted in a later step 8 Click OK If asked whether to process all N images answer no we only need the first one This will create a new black white image called the mask Fig 7 3 9 To transform the mask into ROls choose Analyze gt Analyze Particles which will open the dialog shown in Fig 7 4 Be sure to check Add to Manager and set Show to Overlay Outlines Use the other settings at your convenience e g to get some statistics on the ROIs Adjust the fields Size and Circularity to fine tune the segmentation For example choosing
4. and 8 10 will be the same 7 2 Using AgesGalore The definition of ROls from within AGESGALORE is not implemented yet Status 16 Oct 2013 S Greilich Started document 29 Part II Reference Manual Chapter 8 Class description 8 1 Photon counts 8 2 Growth curve 8 3 Dose reponse 8 4 Evaluation 8 4 1 Dose evaluation 8 5 Protocol 8 6 Detector 8 7 Data Status 25 Oct 2013 S Greilich Started document 31 Chapter 9 Result format Results are stored in the working directory within a uniquely named folder including a time stamp the protocol used etc Within this folder the resulting images from MASS are stored in 32bit TIFF format Additionally an xml file containing the information on the growth curve the doses the equivalent doses the fit parameters etc is saved For the ROIs if requested the data is directly in the xml file while for the image only links to the local image are given to limit the size of the xml file Status 25 Oct 2013 S Greilich Started document 32 Bibliography 1 Steffen Greilich H L Harney Clemens Woda and G nther A Wagner AgesGaloreA software program for evaluating spatially resolved lumines cence data Radiation Measurements 41 6 726 735 July 2006 2 Daniel Richter Andreas Richter and Kay Dornich lexsyga new system for luminescence research Geochronometria pages 1 9 2013 33
5. OSL experiments The software was at that time not intended for general use and grew rather chaotically and driven by demand AGESGA LORE was written in C using MICROSOFT VISUAL STUDIO 6 0 and ran on WINDOWS machines Before I left the lab an official version 1 0 0 as of May 8th 2006 was released to preserve the ability to analyze spatially resolved OSL data for future in house research and interested colleagues 1 At this time AGESGALORE was closed code There was no actual license but users were asked to registered and after sending the installation ID generated during installation they received an unlock key that would allow to use all features of the software In 2012 with the advent of the CCD equipped LEXSYG reader 2 and the need for a corresponding software AGESGALORE was considered to be a promising candidate but had be made fit for reasonable use by commercial users Within approximately 100 man hours the worst bugs were fixed the design improved and the code changed to compile using VISUAL STUDIO 2010 and to run on WINDOWS 7 It became clear however that the design of the software C MFC with user interface and functionality inseparable knotted was a dead end The last version of AGESGALORE was 1 10 1 build 2 as of Aug 14th 2012 Consequently AGESGALORE2 was started in 2013 as a completely new soft ware designed from scratch e By using JAVA as programming language effective development and cross
6. be used rather than the indices this would also yields true rates For further information on photon counts and how arithmetic operations are done on photon count data by AGESGALORE see section 8 1 AEn ga ee _ Joconde 4 4 At the moment the following detectors are implemented in AGESGALORE 4 2 Simple detector This detector assumes random Poisson governed arrival of photons and detec tion with perfect quantum efficiency Additionally the detector has a spatially and temporally constant background that can simply be subtracted Thus if Z is the observed digital signal the expectation value of the photon counts can be derived via r Jexp 3 6 2 4 5 where is the DC offset derived for example from a measurement with a closed shutter The uncertainty is given by Ad frar 2 V2 vA 4 6 The simple detector is a good approximation for efficient detectors high quantum efficiency at reasonable high photon count rates When the count rate is low shortcomings become obvious first of all the generation of negative photon counts 1 In cases where the MASS protocol see section 5 1 subtract a later part of the shine down curve from an earlier to obtain Ly might be irrelavant 18 Status 25 Oct 2013 S Greilich Started document 4 Nov 2013 S Greilich Added rates 19 Chapter 5 Protocols Protocols determine how photon count data are used to establish a dose response grow
7. of course also for the test dose measurement of the natural signal e g data loadImageSequence file ImageFile sequence ImageSequence Most likely the user wants to evaluate the dose response for each pixel of the images but also for regions of interest ROIs These include a number of pixels and represent for example a single grain To do so users have to load either an externally defined set in zipped IMAGEJ format rois load file ROIfile zip or trigger AGESGALORE to start IMAGEJ plugins for the definition of ROIs rois create NOT YET FUNCTIONAL Then the user has to chose which detector and which protocol they want to use detector set protocol set The first will determine the way in which the raw signal data are converted into estimated photons counts see chapter 4 the latter how these photo count data are used to establish a growth curve dose response see chapter 5 Using data list the user gets an overview on the data and results AGESGALOREholds in its memory at the time of the execution The actual growth curve and equivalence dose computation is then started using 10 eval dose fitType XXX where XXX is the fit to that should describe the relation between dose and photon counts see chapter 6 After the evaulation is done it appears in the list when calling data list 7 The user can then save any of the evaluations they did by eval save index i to disc where it is stored
8. real detector and thus to infer the actual number of photons and the uncertainty AA that impinged on the detector from the raw digital signal Z that has been recorded and optionally other parameters In this document we follow the nomenclature used in 1 A fexp Z 4 1 AX frar 2 2 4 2 The computation of photon counts from signal data always happens before AGESGALORE uses the data e g to evaluate the dose response of a sample Usually the raw signal data are recorded in a number of time intervals t In conventional luminescence dating these are often referred to as channels in 17 AGESGALORE they correspond to a series of images taked during the shine down of the sample Z can therefore be obtained from a single or multiple subsequent time intervals channels images by simply summing up the raw signal data before the conversion to photon counts ere gt 4 4 3 J j1 J2 If not stated otherwise Z will in the following always refer to the raw signal data from either a single or multiple images In contrast 21 3 for example ex plicitly says that the raw signal data were summed from image 1 to 3 In order to make photon counts from different time spans comparable they have to be converted into count rates weht j2 j 1 This operation assumes a constant time of exposure for all images channels which might not be always the case So for future releases of AGESGALORE2 the actual time span might
9. sa ca o A A dow a das 14 Sits Datars tek a on ete ene ek A E Be Dan O 9 Result format 30 31 31 31 31 31 31 31 31 31 32 Part I User Manual Chapter 1 Introduction 1 1 Summary AGESGALORE is a software designed to evaluate spatially resolved luminescence data especially in the frame work of luminescence dating It allows the user to display the raw signal data which are preferably obtained by CCD detec tors infer the actual photon counts from the raw signal data For this purpose it features a series of detector models i e mathematical representations of the photon to signal conversion processes in a low light detector see chapter 4 establish growth curves dose responses or shine down curves time de pendencies using the photon count data employing protocols that reflect the measurement sequence such as a single aliquot regeneration etc see chapter 5 use curve fitting procedures see chapter 6 to compute the equivalent or paleo dose s for the sample both for each pixel of an image and predefined regions of interest ROIS analyze the results in a way adapted for spatially resolved luminescence data i e using additional spatial filtering Please note that these features were all covered by AGESGALORE1 whereas its successor AGESGALOREZ2 is still not including all of them 1 2 History I started to write AGESGALORE during my PhD thesis in order to analyze the data from my
10. AGESGALORE2 User and Reference Manual Steffen Greilich December 16 2013 Contents I 1 User Manual Introduction LL SUMMALY EE Duel lio da Sint id id dod botana 12 HIStory 2 0 ado des e A SPS A e E Using AgesGalore 2s Installation 2d is a BA ur DEA 2 2 Running the software 2 2 1 Interactive mode 2 2 2 lt Seript Mode Liu ses dus AR ee dates aus DIS Server Mode 20 id a ee D a elle 2 3 General workflow with AGESGALORE List of commands Detector models AT General y 4 wae e sch a es Ae IAA Sn Eee aan 4 2 Simple detector cna ce di 4 e a nern ad Protocols 5 12 MASS pete aldo ds IE er A aed Rh me DLI Growth cutv 60 222 24 AN A 5 1 2 Equivalent dose 5 1 3 Recycling rations fading rates Fitting routines 6 1 A E Ponant 6 2 Exponential saturation Grain segmentation A Using IMAGES 22 222 ee au PEPE SEE Gage pese 7 2 Using AGESGALORE ee 00 12 17 17 18 20 20 20 21 21 22 22 22 II Reference Manual 8 Class description 8 1 Photon counts 22 co ve BAe 2 8 we er dame rt 8 2 Growth Curve oe he rn eee de ran 8 3 Dose reponse 8 4 Evaluation 222 2 ts A A eh A qu 8 4 1 Dose evaluation 25 Protocol Lits Ss a A ALE 8 0 Detectors
11. Size 0 Infinity will result in too many small ROIs Fig 7 5 while a setting like Size 10 Infinity will yield the result seen in Fig 7 6 i e 23 ROIs corresponding to grains the very dim ones were not caught 10 At the same time the ROI Manager window Fig 7 7 will open and display the ROIs In this window choose More gt Save to save the 26 Me 102 ts ES 51x57 2 pixels 8 hit nye 256K Figure 7 3 Mask d Analyze Particles Size pixel 2 0 Infinity Circularity 0 00 1 00 Show Kercem IEE ba Display results Exclude on edges Clear results Include holes Summarize Record starts Y Addto Manager J Insitu Show OK Cance Help Figure 7 4 Mask 27 Mask 102 cre fit 512x512 p Figure 7 5 Due to inproper settings for the Particle Analyzer too many artifacts were defined as ROIs that does not correspond to actual grains Mask 102 EE 512x512 pixel ep ya LC 250K Figure 7 6 Useful ROI definition 28 0089 0178 0090 0220 0091 0237 0092 0244 0093 0254 Rename 0094 0269 Measure 0095 0264 0096 0276 0097 0283 0098 0301 0099 0302 0100 0302 0101 0325 0102 0325 Deselect Figure 7 7 ROI Manager ROI definitions as a file Use a reasonable file name e g your sample ID as you will have to later enter it into AGESGALORE When using another plug in for segmentation steps 6
12. erver System exit 1 FileReader scriptFile new FileReader Script txt BufferedReader inFile new BufferedReader scriptFile String line read while line inFile readLine null If line successfully read in out println line System out println GUI sent gt line read in readLine System out println GUI received gt read out close in close inFile close socket close System exit 0 2 3 General workflow with AgesGalore A typical workflow will look similar to the series of commands given as a script example in 2 2 2 The commands and their structure are documented in chapter 3 1 First of all the user will point AGESGALORE to a working directory where preferably the original data is located and where the results will be stored data setWD path PathToDir If the directory not exists it will be created given sufficient user right correct path name etc Then the user will load images with the raw digital signal data individually or in a sequence standard for the LEXSYG system and attribute each image or image series a dose group i e natural signal regenerated signal recovery signal fading test signal see chapter 5 on protocols for more information as well as a dose if known not needed for the natural signal and if applicable an image that represents the corresponding test dose measurement In this case again a dose value has be given in the case
13. ile RoiSet zip detector set type Simple parameter 600 protocol set type MASS interval 1 2 8 10 useTestDose data list eval dose fitType Linear data list eval dose fitType ExpSat data list eval save index 1 eval save index 2 exit Tf the last command exit is skipped AGESGALORE will enter in interactive mode Script mode can facilitate the routine use of AGESGALORE 2 2 3 Server mode AGESGALOREdoes also support running as server Commands can be send using Port 9090 from a user client To start AGESGALORE in server mode type java jar AgesGalore jar server The client can be a simple telnet program or more likely a custom made soft ware The following code is an example client that reads a script Script txt and sends it to AGESGALORE line by line package agesgaloregui import java io BufferedReader import java io FileReader import java io I0Exception import java io InputStreamReader import java io PrintWriter import java net Socket public class AgesGaloreGUI public static void main String args throws IOException InterruptedException Socket socket null PrintWriter out null BufferedReader in null tryt socket new Socket localhost 9090 out new PrintWriter socket getDutputStream true in new BufferedReader new InputStreamReader socket getInputStream catch IOException e System err println Couldn t get 1 0 for the connection to AgesGalore s
14. in the working directory in the format described in Status 25 Oct 2013 S Greilich Started document 11 Chapter 3 List of commands Commands in AGESGALORE are completely text based They consist of a main command that represents the area of application data rois etc subcom mands that refer to the actual action loadImage save and if applicable parameters consisting of a keyword starting with and a value Parame ters can be given in any order If a command or subcommand is unknown or a parameter is missing AGESGALORE will prompt the user accordingly The follwing table summarizes all commands available at the moment in AGES GALORE 12 Command Subcommand Parameters Function data clear none removes all data list none lists all data set WD sets working directory path path of working directory can be relative with respect to location of AgesGalore2 jar or absolute Will be created if does not exist loadImage loads an image tif png jpg WinSpec and attributes it to a dose group file file name fileTestDose file name for test dose image op tional type dose group type NATural RE Generated RECycled or FAD ingtest dose dose given to sample in arbi trary units not necessary for NAT testdose test dose given to sample a u loadImageSequence loads an image esp WinSpec from lexsyg system containing several or all steps of a mea surement file file name
15. in your ImageJ plug in directory and restart the pro gram 4 In this example we use for segmentation an adaptive threshold algorithm which is found here https sites google com site qingzongtseng 24 a Image Lo Am File Edit Image Process Analyze Plugins Window Help Bolco s sl AJa m I 4 Magnifying glass or use and keys A Dev Stk Sor3_NGS0013 DRT_r512 SPE 1 132 512x512 pixels 32 bit 132MB 486 00 4118 87 ll gt Minimum A gt Maximum EN HA Brightness Ku a Contrast Auto Reset Set Apply gt lt gt Figure 7 1 Loaded CCD image in IMAGEJ and adjustment of brightness and contrast adaptivethreshold Download the version for your operating system Windows 32 64 bit Linux etc and install following the instructions The page also features a description on how to use the plug in 5 Now restart ImageJ and read in your image s Assuming the sample has not been shifted you can use any frame image for segmentation preferably one with bright signal For better contrast use the adjustment in IMAGEJ Menu Image gt Adjust gt Brightness Contrast Fig 7 1 6 Convert your image to 8 bit format by choosing Image gt Type gt 8 bit 7 Start the plug in by choosing Plugins gt adaptiveThr A new dialog Adaptive Threshold Fig 7 2 will open Check both Preview
16. l usually be 1 and ja gt j has to be fulfilled More often the a background signal obtained from later channels images in the shine down curve will be used in addition so that Lx becomes Lx D Areg Di 21 32 Areg Di 255 54 5 3 with ja gt j3 gt ja If the corresponding test dose data Afes Dies 2 exist reg and the user requested it MASS can use the very same procedure for Tx Tx Di Ates Di Zi ja Ates Di Zjs ja 5 4 reg reg In this case Lx Tx is then used to establish the growth curve In this case of course all Dit have to be the same 5 1 2 Equivalent dose To compute the equivalent dose Deg AGESGALORE performs curve fitting to the Lx Tx D values using a function fit D pi that depends on dose and a set of parameters p see chapter 6 When the best set of parameters p is found the natural signal is computed in the very same way as Lx according to Eq 5 2 Lx Dea Anat De t 5 5 or Eqs 5 3 5 4 resp Then Deg can be found using the reverse of far Dea f Ex Dea Bi 5 6 The error of Deg ADeq computed by Gaussian error propagation from the AA s of all A s involved 5 1 3 Recycling rations fading rates Should be computed according to Eqs 5 2 and 5 4 BUT ARE NOT IMPLE MENTED YET Status 25 Oct 2013 S Greilich Started document 4 Nov 2013 S Greilich Added rates 21 Chapter 6 Fitting routines Fits are parametrized analytical func
17. plattform compatibility could be assured As both JAVA and C are object oriented languages successful structures that reflect entities or pro cesses in luminescence dating could be transferred directly e JAVA allowed to use the NIH hosted software IMAGEJ the public domain gold standard for post processing of microscopy data as a class library Thus AGESGALORE2 could be freed from unnecessary code e g for han dling image data regions of interest I O which made the software and its development leaner and faster e Additionally this approach allowed to access a plethora of IMAGEJ plug ins e g for single grain segmentation image processing etc e To provide a tiered structure AGESGALORE2 was designed as a shell to be controlled with text commands like data loadImage file test SPE This allows to implement graphical user interfaces without intermingling with the functionality e AGESGALORE2 was designed from the beginning to generically deal with single grain and rock slice samples while its predecessor was mainly limited to rock slices In the following document AGESGALORE always refers to AGESGALORE2 if not stated otherwise Status 25 Oct 2013 S Greilich Started document Chapter 2 Using AgesGalore 2 1 Installation AGESGALORE comes as a java archive file extension jar that contains both its own classes and third party classes needed such as IMAGEJ To run AGESGA LORE you have to have JAVA
18. se Tx For the testdose signal the integration rules given using interval apply as well rois Status load saveSignals file loads ROIs in ImageJ format zip filename saves average raw signal within defined ROIs to xml output file These data can be used in other single grain evaluation tool e g after import by the correspond ing function of the AGESGALORE R package 15 25 Oct 2013 S Greilich Started document 4 Nov 2013 S Greilich Added new syntax for eval dose 16 Dec 2013 S Greilich Added saveSignals subcommand for ROIs 16 Chapter 4 Detector models 4 1 General Detectors for luminescence measurements in the real world convert impinging photons into a measurable signal For the detection of light there is a large vari ety of detectors such as photomultiplier tubes PMTs Silicon photomultipliers Si PMTs avalanche photodiodes or charge coupled devices CCDs Each detector comes with its own mechanism and hence properties for the conversion Among the parameters describing this conversion are e the quantum efficiency i e the average probability to convert a photon e the background the base signal that is measured in the absence or additionally to the photon signal e various kind of noise that is created during the conversion or due to the random arrival of photons e etc A detector in AGESGALORE is a set of functions f that tries to mimic the properties of a
19. th curve Up to now the following protocols are implemented 5 1 MASS 5 1 1 Growth curve MASS is an extension of the well known SAR protocol single aliquot regener ation to multi pixel images assuming that each pixel represents its own inde pendent source of light and dose age information respectively Thus MASS performs a SAR protocol on each pixel yielding new images of spatial equiva lent dose distribution goodness of fit etc and on groups of pixels that have been binned on the level of the raw signal data i e regions of interest ROIs e g for single grains MASS use the photon count rates from multiple n gt 1 regeneration mea surements Areg Di Zin je 5 1 where D are the regeneration doses with i 1 n and j and ja the indices of the first and the last subsequent image channel the raw signal was summed over Some D can have the same value but not all of them j and ja have to be the same for all D Thus the simplest way to obtain the entries for the growth curve Lx is Lx D Areg Di Zi 52 5 2 1This means especially that the summed ROI raw signal data are converted into photon counts and then used to establish a growth curve This is an important difference between AGESGALORE 1 and AGESGALORE 2 With the first MASS computed equivalent doses for each pixel first and THEN binned averaged them to a ROI equivalent dose This is a minute but important difference 20 where j wil
20. tions fa amp X p that are applied to find hypothetical relation behind data The depend on a variable X e g dose D or time t For example the dose response of a sample can be interpreted as a straight line or as an exponential saturation The user has the freedom to chose and always has to chose the model they think is most appropriate AGESGALOREuses fits for example to compute the equivalent dose from a growth curve which always has a limited number of data points only see section 5 1 2 To compare the growth curve to the natural signal and infer the equivalent dose the growth curve data have to be interpolated This is in most cases best done by fitting one of the following functions to the data point The set of parameters for which the fa describes the data best is found in AGESGALOREusing the minimization of the squared sum of the residuals 6 1 Linear The is a simple straight line with slope p and offset po Y DP1 T P2 6 1 6 2 Exponential saturation Starting from an offset for x 0 this function approaches it upper limit expo nentially The approach is determined by the parameter pa y p 1 e P2 pg 6 2 For growth curves more often another nomenclature of this equation is used S Smax 1 e7 P Po Sres 6 3 22 where the increase from a residual signal Spes at dose D 0 to Smax at D is governed by the parameter Do in units of dose The two versions are of course fully equivalent 23
21. version 7 or later installed AGESGALORE runs on all platforms that can run JAVA You can download JAVA here http www java com 2 2 Running the software 2 2 1 Interactive mode To start AGESGALOREin interactive mode open a new terminal or command line window and type java jar AgesGalore jar This will start the AGESGALORE shell where you will be prompted to enter commands see chapter 3 and the software will respond immediately To end AGESGALORE enter quit In case your are processing lage data sets you might experience that the Java Virtual Machine runs out of memory Then add an additional Xms flag in between java and jar to increase the memory allocated for Java java Xms6000m jar AgesGalore jar Here 6 GB 6000 MB are allocated Of course this only works if your PC has a corresponding amount of RAM available 2 2 2 Script mode AGESGALORE can also be run in script mode i e driven by an external file To do so open a new terminal or command line window and type java jar AgesGalore jar Script txt where Script txt is a plain text file containing AGESGALORE commands with a single command per line for example This is a comment TestScript for reading ImageSequence from lexsyg data setWD path Sor3_NGS0013 data loadImageSequence file Sor3_NGS0013_DRT_r512 SPE sequence 11 NAT 11 TD 50 11 REG 50 11 TD 50 11 REG 100 11 TD 50 11 REG 150 11 TD 50 11 REG 0 11 TD 50 11 REC 50 11 TD 50 rois load f
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