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1. Check the global registration result using the procedure described in Section 13 4 e Click the Automatch button to perform local registration e Click the Gauss conv button to perform final refinement e Click the Error analysis button to run the outlier detection e Inspect the positions of any points that have failed outlier detection correcting them where necessary using the procedure described in Section 13 8 14 1 Setting the Free Parameters The automatic landmark point placement software depends on multiple free parameters the patch sizes for the local registration the smoothing kernel sizes for both the local and global registrations the convolution kernel size for the final refinement stage and the thresholds used in outlier detection The default values were optimised for micro CT images of rodent skulls with a resolution of 658x658 voxels within each slice and containing 1000 to 1200 slices loaded with the Stride and Downsample factors set to 2 If the software is applied to data that varies significantly from this description then the user may have to optimise the free parameters in order to ensure proper operation of the software This can be done using a leave one out experiment as follows Obtain 10 to 15 representative image volumes and manually identify the desired set of landmarks in all of them Then run the automatic point location software for each image volume using the others
2. 81 References 10 11 12 13 14 15 16 17 18 19 P A Bromiley Performance evaluation of the TINA medical image segmentation algorithm on Brainweb simulated images http www tina vision net docs memos 2008 003 pdf P A Bromiley M Pokri and N A Thacker Computing covariances for Mutual Information coregistration In Proc MIUA 04 pages 77 80 2004 P A Bromiley M Pokri and N A Thacker Emprical evaluation of covariance estimates for Mutual Information coregistration In Proc MICCAI 04 pages 607 614 2004 P A Bromiley and N A Thacker Computing covariances for Mutual Information coregistration 2 http www tina vision net docs memos 2003 002 pdf P A Bromiley and N A Thacker Empirical validation of covariance estimates for Mutual Information coreg istration http www tina vision net docs memos 2004 001 pdf P A Bromiley and N A Thacker Stability testing of the TINA medical image segmentation algorithm http www tina vision net docs memos 2005 013 pdf P A Bromiley and N A Thacker Multi dimensional medical image segmentation with partial volume and gradient modelling Annals of the BMVA 2008 2 1 22 2008 P A Bromiley and N A Thacker Multi dimensional medical image segmentation with partial volume and gradient modelling supplement 1 Mathematical derivations and proofs Annals of the BMVA 2008 2 s1 1 11 2008 R A Drebin L Carpenter and P Hanrahan Volme rend
3. Download the TINA Geometric Morphometrics toolkit max_planck_toolkit 2 0 tar gz from the TINA web site http www tina vision net tarballs manual_landmark_toolkit place it in a convenient directory e g home my_username tina_toolkits then unzip and unpack it A new sub directory will be created cd into this subdirectory and type configure The configure script will search for the external libraries needed to build the toolkit issuing warnings about any it fails to find In particular if it cannot find the tina libs or tina tools libraries and headers then their locations can be passed to the configure command using the following flags with tina includes PATH with tina libraries PATH with tinatool includes PATH with tinatool libraries PATH e g configure with tina libraries usr local Tina6 tina tools lib If the tarballs were unpacked in usr local Tina6 then the correct locations are e g with tina includes usr local Tina6 tina libs 6 0rcbuild008 with tina libraries usr local Tina6 tina libs 6 0rcbuild008 lib with tinatool includes usr local Tina6 tina tools 6 0rcbuild008 with tinatool libraries usr local Tina6 tina tools 6 0rcbuild008 lib Finally type make to build the toolkit The TINA Geometric Morphometrics toolkit needs to find the Volpack header and library On standard Linux systems these should be located in usr local include and usr local lib once Volpack has been insta
4. Up and Down buttons in the Landmark Points dialog box These buttons are also assigned keyboard shortcuts in all TVs of the Manual Landmark tool see Sections 5 2 1 5 2 2 and 9 In addition the 5 and 5 buttons move up and down by five points in the landmark list and the Start and End buttons move to the first and last landmarks respectively Finally the Last outlier and Next outlier buttons move to the previous and subsequent points with type codes greater than 2 if any i e points that have been identified as potential errors in the automatic landmarking process see Section 13 7 These buttons allow the user to scroll through the landmark list quickly when correcting any errors from the automatic landmark point placement software see Section 13 8 In order to avoid having to keep the Landmark Points dialog box open during manual landmark identification a 29 subset of the dialog is also available in the Manual Landmark tool which displays the number name and type code of the current landmark and has copies of the Up Down 5 5 Start End Last outlier and Next outlier buttons However the current landmark is always kept consistent between these two windows so when scrolling through the landmark list using the buttons in the Manual Landmark tool the user will also see the list scroll in the Landmark Points dialog box The Landmark Point
5. In order to select a bone threshold intensity e Start the volume renderer as described above Using the Zoom mouse mode rotate the image such that a point with a solid bone surface is clearly visible Start the Imcalc tool from the top level tinaTool e Click Graph in the TV choice list of the Imcalc tool start a new TV tool and click Install in the TV tool to assign it to the Imcalc Graph TV Select Pick mode for the 3D TV mouse e Move the mouse pointer to a location within the 3D TV that has a clearly visible bone surface e Right click with the mouse a grey level profile through the image volume along the vector under the mouse pointer will be displayed in the Imcalc Graph TV The x axis of this graph represents depth through the 3D image with 0 being the back of the image and 1 being the front of the image from the current viewpoint The y axis represents the intensity grey level of the data along the profile e Identify the step change in the intensity profile that represents the transition from soft tissue to bone Several bone surfaces may be visible in the profile also be sure to clearly identify soft tissue from background e Calculate an intensity value half way between the mean intensity of bone and the mean intensity of soft tissue e Enter the calculated intensity value into the Bone threshold field of the Manual Landmark tool As with the 2D TVs see Section 5 2 1
6. i e changing the selection of the Rotate option has no effect whilst Lock Rotation is selected When Lock Rotation is deselected the images in the 2D TVs will be re drawn either using the rotation currently applied to the image in the 3D TV if Rotate is selected or with no rotation i e aligned to the axes of the original image volume if Rotate is not selected When the Rotate or Lock rotation options are selected the increment and decrement buttons i e the arrow buttons in the Manual Landmark Tool window still move the position of the 3D cursor along the major axes of the original image volume However when the keyboard shortcuts are used in the 2D TVs the cursor is moved along the rotated axes of the image displayed in the TV not along the axes of the original volume This allows the user to rotate the volume to a preferred orientation and then select extremal points on structures displayed in the 2D TVs relative to that rotation The user should be aware that the definitions of landmark points identified in this way is relative to the orientation they select Therefore although the orientation can be selected manually by rotating the image displayed in the 3D TV it may be preferable to use a more rigorous definition Section 8 1 describes methods to achieve this by identifying an arbitrary plane and axis using landmark points and then rotating the image displayed in the 3D TV to a fixed orientati
7. is associated with e g click on 3D in the TV list of the Manual Landmark tool Start the View tool by clicking the View button in the tinaTool window The View tool will start and will display the name of the currently selected TV in the Current Tv field Click the Dump button in the View tool This will start the Dump tool Enter a relative or absolute pathname relative pathnames are relative to the directory containing the tinaTool executable into the Filename field and select the colour depth using the Dump Type field B amp W will give a greyscale image Colour will give a full colour image and lbpp will give a black and white image which can be useful for saving graphs displayed in the Imcalc Graph TV Click on either the TIFF or EPS button to save the image from the currently selected TV to file in TIFF or Encapsulated Postscript format 36 11 Using the Manual Landmark Tool A complete set of steps required to specify a set of landmarks is as follows Prepare a landmark names file specifying the points that you intend to identify see Section 7 Optionally prepare the linklines file Start TINA start the Manual Landmark tool Start the Sequence tool assign a TV tool to the sequence tool TV optional see Section 3 and load a 3D medical image volume see Section 4 Start the volume renderer using the 3D Tv switch in the Manual L
8. registration results Scroll downwards through the list of landmark points in the current automatch database entry Select an image patch from those stored in the database X the stored y z patch around the current point in the current database entry Y the stored x z patch around the current point in the current database entry Z the stored x y patch around the current point in the current database entry DX the smoothed derivative images for the y z patch around the current point in the current database entry DY the smoothed derivative images for the y z patch around the current point in the current database entry DZ the smoothed derivative images for the y z patch around the current point in the current database entry RX the full sized y z patch through the centre of the volume from the current database entry plus the corresponding patch from the current volume transformed using the global registration result if available RY the full sized x z patch through the centre of the volume from the current database entry plus the corresponding patch from the current volume transformed using the global registration result if available RZ the full sized x y patch through the centre of the volume from the current database entry plus the corresponding patch from the current volume transformed using the global registration result if available Push the selected image patch es onto the Imcalc stack
9. scroll up to the previous landmark in the landmark list see Section 7 e Page Down scroll down to the next landmark in the landmark list see Section 7 The size of the increment for cursor movement is selected using the Resolution choice list in the Manual Landmark tool by default it is set to one voxel Note that in order for the TVs to receive the keyboard inputs the window manager focus must be on the TV In many window managers the window that has the current focus is displayed with a coloured title bar all other windows have a grey title bar Most window managers provide two modes for focus selection click to focus in which the user must left click with the mouse inside a window in order to switch focus to that window and focus follows pointer in which the focus in on whichever window the mouse pointer is inside If click to focus is selected then the user will have to left click on a specific TV tool in order to send subsequent keyboard instructions to it Therefore if significant use is made of keyboard shortcuts it may be easier to change the window manager focus policy to focus follows pointer so that keyboard instructions are sent to whichever TV the mouse pointer is inside In order to switch to focus follows pointer mode in Suse 10 3 e Click on the start button in the bottom left corner of the screen select Configure desktop Select the Desktop tab from the menu on the left Select the W
10. we can show how scattered the points are in the plot If they are scattered inside the statistical allowable limits then the Monte Carlo test validates the results Otherwise one reason would be the presence of outliers in the data which may be dealt with by removing few samples up to 5 based on the list of potential outliers provided in the Tina dialog box There might be other reasons for the points falling outside the allowable limits For instance if the linear model do not fit the data due to non linearity of the data or due to setting the number of model components to a value which is either too small or too large Figs 21 22 show two example plots of the Monte Carlo tests performed on 3D Apodemus mandible data see 16 for more details In this case we compute the statistical allowable range using the formula ce max 1 2 K 1 where K is the number of shape samples and emaz is the maximum value of the horizontal axis from the error plot Hence to draw the two dashed lines one point is the origin and the second point is emaz 2 806 61 W L2 L1 covariances D gt R2 R1 covariances Figure 20 Fly wing 2D data 200 samples 15 landmarks the x DoF ratios are plotted versus the landmark number when the Likelihood method is applied to two sets of repeat fly wing data FL2 left wings and FR2 right wings using a 3 component model and fixed covariances estimated earlier from the FL1 and FR1 data sets respectively t
11. 2 in the Fill G reg points function Specify a landmark point number for G reg point 3 in the Fill G reg points function Specify a landmark point number for G reg point 4 in the Fill G reg points function Copy the locations of the four landmark list points specified by their point numbers in Greg 1 Greg 2 Greg 3 and Greg 4 to the four G reg points Run the global registration stage of automatic point location Specify options for the approximate landmark projection algorithm All replace all current landmark point coordinates even if they have already been identified for point based global registration Snap move projected point locations to the closest surface as defined by the Bone threshold field of the Manual Landmark tool Run the approximate landmark projection algorithm Size of image patches to use in the first stage of local registration Size of the smoothing kernel applied to the images during the first stage of local registration Size of image patches to use in the second stage of local registration Size of the smoothing kernel applied to the images during the second stage of local registration Size of image patches to use in the third stage of local registration Size of the smoothing kernel applied to the images during the third stage of local registration Run the local registration stage of the automatic point location algorithm Specify whether to move
12. 2x2 or 3x3 covariance matrix for each landmark 2 From each covariance matrix compute the corresponding error eigenvalues These correspond to repeata bility errors 3 Set the number of model components to a minimum e g 5 for mouse mandibles 4 Obtain the error eigenvalues estimated when the Likelihood method is applied to one of the two data sets using the steps outlined in Box A 5 Plot the major eigenvalues corresponding to the errors estimated versus those corresponding to repeata bility errors see Section 2 4 and Fig 24 6 For models with 50 landmarks if more than two of the points fall under the 2 8 S D limit decrease the number of model components otherwise increase the number then go back to step 3 and repeat the process until the optimum number of components is found i e that which gives the best predictive accuracy without exceeding the known repeatability of the measurements 66 Part 4 Quick Reference Guides P A Bromiley Imaging Science and Biomedical Engineering Division Medical School University of Manchester Stopford Building Oxford Road Manchester M13 9PT 67 21 Quick Reference 21 1 Sequence Tool DICOM re scale Tv sequence File Image Type Start Stride Downsample Cur frame End Image File Scan Scales x y z t Load Save First lt 2 End Jumpto Stride average Del Seq Del Ins Selects the type of scaling tags in a
13. 3D cursor position shown in red such that the image shown in the x axis TV is generated from a plane perpendicular to the x axis top right the image shown in the y axis TV is generated from a plane perpendicular to the y axis bottom left and the image shown in the z axis TV is generated from a plane perpendicular to the z axis bottom right down sampling factors from affecting any subsequent Procrustes analysis of the landmark points 5 2 Image Display The Manual Landmark tool provides four TVs see Section 3 Three of these provide 2D views of orthogonal slices through the volume centred on the 3D cursor The fourth provides a 3D volume rendering of the volume The TVs can also display 3D cursor landmark axis and plane points Figure 5 shows the four TVs in a recommended arrangement such that the axes are consistent across the three 2D TVs 5 2 1 2D Image Display The three 2D TVs display orthogonal slices through the sequence centred on the current 3D cursor position which is displayed in each TV as a large red cross hair Whenever the 3D cursor is moved the images displayed in these TVs will be updated to reflect this change The TVs are named according to the axis that is perpendicular to the image displayed in the TV see Section 5 1 as shown in Fig 4 Therefore the x axis TV displays an image in 14 X 3 o x X Y Axis lolx Size Mouse ROI Proj Size Mouse ROI Proj install clone init repaint install clon
14. DICOM image rarely used The Sequence tool TV Selects the file format AIFF TINA s standard image file format ANLZ ANALYZE medical image format RAD RAD format NEMA Older pre DICOM format PGM Portable grey map DICOM DICOM format Displays the variable type of the images in the sequence and can also be used to cast the current sequence to a different type by selecting the desired type after loading the sequence bin binary chr short int integer flt floating point The number of the first image in the sequence The number of images to skip whilst loading the sequence e g entering 2 means that every other image will be loaded Down sampling factor used during image loading e g entering 2 means that each image will be down sampled by a factor of 2 on the x and y axes during loading The current image in the sequence i e the one displayed in the Sequence tool TV The number of the last image in the sequence The absolute or relative pathname of the file s to load save Starts a file browser for the Image File field The dimensions of each voxel in the sequence Load data from the file s specified in the the Image File field Save the current sequence to the file s specified in the the Image File field Set the first image in the sequence as the current image Move backwards through the sequence i e decrement the current image Move forwards through the sequence i e
15. Greg 2 Greg 3 Greg 4 Fill G reg points Global Reg Projection LM projection Loc patch size 1 Loc sig 1 Loc patch size 2 Loc sig 2 Loc patch size 3 Loc sig 3 Automatch Snap points Launch the Automatch Database Inspector dialog box The size of the local image patches around each landmark point stored in the au tomatch database on clicking Landmarks gt database The size of the border added around the full sized image patches through the centre point of the image volume stored in the database on clicking Landmarks gt database Create a new entry in the automatch database from the current image volume and landmark list Free any currently loaded automatch database Free the current record of the automatch database i e the one displayed in the Automatch Database Inspector dialog box Specify which stages of global registration to run Point based run the point based stage of global registration Image based run the image based stage of global registration The size of the smoothing kernel applied to the images during image based global registration Specify the source of the points used in the point based stage of the global registration LM based use any available points in the current landmark list G reg based use the four G reg points Specify a landmark point number for G reg point 1 in the Fill G reg points function Specify a landmark point number for G reg point
16. Type 1 Link no 1 Down 5 Next outlier End AIP G point Ax 1 Ax 2 PI1 Pl2 PlL3 G1 62 63 G4 Name NULL x 1 0 y 1 0 z 1 0 TPS NTSYS D NOT SPECIFIED Field no 1 Clear axis Clear plane ClearG_reg Clear links Clear LM Input format Names Raw TPS NTSYS Input pathname Scan Load Output format Names Raw TPS NTSYS Output pathname Scan Save Figure 14 The Landmark Points dialog box The TINA Manual Landmark tool was developed to allow the generation of lists of landmark point positions for use in Procrustes analysis In this type of analysis it is essential that each list of landmark points contains the same set of physiological locations in the same order Therefore rather than allowing the free form generation of lists of points the Manual Landmark tool requires the user to prepare a text file containing a list of point names and point numbers referred to as a names file which must be loaded into the tool before point locations can be specified An example is shown below The file can be prepared in any text editor and on each line should contain a point number at the start of the line followed by whitespace one or more spaces or tab characters then the text description of the point In many geometric morphometric studies some or all of the landmarks will occur in pairs arranged symmetrically on either side of the plane of bilateral symmetry The Linklines option in the 3D TV see Section 5 2 2 d
17. a number of keyboard shortcuts are available in the 2D and 3D TVs enabling commonly used functions to be applied without moving the mouse cursor away from the TV tools These can be reassigned to any key with the exception of a few keys reserved by the window manager such as the print screen key using the TV Keyboard Controls dialog box This dialog box is started by clicking the Keyboard Controls button at the top of the Manual Landmark tool and is shown in Fig 15 In order to reassign a key left click with the mouse inside the field displaying the keyboard control you want to reassign and then press the key you want to specify The name of the key will appear in the entry field The same set of keys are used across all TVs 34 10 The View Tool xK View Tool O X Help Current TV 3D dump Bkgrnd white light grey dark grey black blue Line 0 1 2 3 Color black blue white red Colormap Standard Greyscale Anaglyph False colour default colors o Default Font 6x1 0 _ endian Show cmap Show tv s cmap_lookup Push tv screen X dump too 10x Movie init add one make seq show random Beit tft Rot x 0 0 Roty 0 5 Steps 10 Filename Timer 0 01 sec 100 Count 4 Pump Type Baw colour tbpp Figure 16 The View tool and Dump tool The View and Dump tools provide additional interaction with graphics displayed in TV tools See the T
18. a sequence is loaded and so if the image volumes for individual specimens are stored in individual directories with meaningful names reflecting the name or code for the specimen then this process will be handled automatically NTSYS files can hold multiple lists of landmark points If landmark points are saved to a NTSYS file that already exists then the new set of landmark points will be appended to the existing file The contents of the TPS NTSYS ID field will be saved in the file as a comment The landmark point names will be saved to file as a list immediately preceeding the data matrix however NTSYS only supports labels of up to 16 characters and so the names will be abbreviated if necessary Users should be aware of this when preparing lists of landmark point names if they intend to use the NTSYS file format The landmark point coordinates will be saved to the data matrix of the NTSYS file as columns i e each column of the matrix represents a point e Click the Save button in the Landmark Points dialog box Landmark point information can also be loaded from any of these file formats The Field no field allows the user to specify which record to load from a TPS or NTSYS file that contains more than one set of landmark points Since the software can only maintain one list of landmark points at a time a conflict may arise in situations where the user attempts to load a file when some landmark informatio
19. a single point could lead to systematic error through marking up all points in a given dataset using the single database entry with the most similar shape thus transferring any errors on the point locations in the database entry directly onto the dataset being marked up This could exaggerate any shape differences observed in subsequent Procrustes analysis Instead a procedure is required that uses a logical OR process i e finds the best single estimate based on the consensus of the estimates contained in the database The final refinement achieves this by constructing a 3D space large enough to hold all of the estimated locations for a given landmark point entering the points as Kronecker delta functions and then convolving the space with a Gaussian smoothing kernel The size of the smoothing kernel is set in the Fin sig field see Section 18 and is given in the coordinate system of the current sequence i e taking into account any down sampling applied during data loading The final refinement algorithm selects the location of highest point in the space after the convolution i e the most probable point location based on any sub set of the estimated locations from the database as the final estimated point location If no highest point exists since the estimated locations are all separated by more than three times the size of the Gaussian kernel used in the convolution then the point from the database with the lowest x is used instead and
20. allows the user to construct a text file listing TINA operations This system can be used to construct multiple macro files each of which performs the analysis listed above on a single image volume and then outputs the results in Raw file format An arbitrary number of samples can then be analysed e g overnight by running the macro files as a batch job the only user interaction required is to load each image volume and Raw format landmarks file once the automatic analysis is complete and check or correct any points identified as potential outliers See the TINA Users Manual 19 for more details on the construction of macro files 49 16 Approximate Projection It may be useful under some circumstances to have a set of very approximate point locations that whilst not accurate enough for shape analysis can instead be used to initialise a manual landmark procedure The software provides this option through the LM projection button This will take the set of landmark points in the database corresponding to each point in the landmark list project them into the current volume based on any registration results available i e point based global image based global and local whichever stages have been performed and find the mean of these projected positions writing the result to the current landmark list The Projection check list provides two options for this procedure All will replace all of the points in the landmark list eve
21. bone surface of a given sample testing on micro CT images of rodent skulls has indicated that the software is reliable if the database contains examples from the same genus as the current volume The list of landmark points must also be identical i e the must contain the same number of points marked up in the same locations and in the same order The software will not allow the addition of new entries to the database if the length of the list of landmark points differs from previous entries already stored in the database but it is up to the user to ensure that the lists of landmarks contain the same biological points marked in the same order Since the aim of the software is to facilitate the placement of landmark points for experiments in geometric morphometrics there will naturally be some difference in shape between the data sets being analysed Therefore the software uses a coarse to fine registration strategy in which all stages are based on the likelihood function given above in order to progressively align the two sets of image patches The first stage is a global registration in which the patches span the entire volume and intersect at its centre The shape difference between the structures in the images in the current volume and the database mean that the global patches from the former are a relatively poor model of the latter and so the result is an approximate alignment The registration then proceeds to align patches around individual la
22. eigenvector number to be displayed individually between one and the number of model components vector scale This is the scale value used to magnify the eigenvectors in order to improve visualisation on the display window as the absolute values of vectors may be too small to see display eigenvectors By pressing this button the eigenvectors are displayed taking into account the vector scale It is recommended that the source is selected to be the mean shape One may display eigenvectors corresponding to several model components superimposed by changing the eigenvector number and pressing display eigenvectors repeatedly error scale This is the scale value used to magnify the error bars in order to improve visualisation on the display window as the absolute values of errors may be too small to see display error bars By pressing this button the error bars are displayed taking into account the error scale It is recommended that the source is selected to be the mean shape One useful graph may also be obtained by displaying the aligned data superimposed In the case of Procrustes errors are only computed after the alignment and using the residuals left which can be displayed in a similar manner 20 4 Input Output Files It is strongly recommended that for every new experiment a new directory is created and the input data file is copied there This is to make sure all the input and output fil
23. frontal process of maxilla with frontal and lacrimal bones right side 5 Frontal squasmosal intersection at temporal crest left side 6 Frontal squasmosal intersection at temporal crest right side 7 Center of alveolar ridge over maxillary incisor left side 8 Center of alveolar ridge over maxillary incisor right side 9 Lateral intersection of maxilla and palatine bone posterior to the third molar left side 10 Lateral intersection of maxilla and palatine bone posterior to the third molar right side 11 Anterior notch on frontal process lateral to infraorbital fissure left side 12 Anterior notch on frontal process lateral to infraorbital fissure right side 13 Most posterior point of the anterior palatine foramen left side 14 Most posterior point of the anterior palatine foramen right side 15 Anterior most point at intersection of premaxillae and nasal bones left side 16 Anterior most point at intersection of premaxillae and nasal bones right side 17 Most infero lateral point on premaxilla maxilla suture left side 18 Most infero lateral point on premaxilla maxilla suture right side 19 Intersection of parietal temporal and interparietal bones left side 20 Intersection of parietal temporal and interparietal bones right side 21 Most inferior aspect of posterior tip of medial pterygoid process left side 22 Most inferior aspect of posterior tip of medial pterygoid process right side 23 Joining of squasmosal body
24. green lines linking equivalent landmarks on the left and right sides of the sample if the corresponding linking lines file has been prepared and loaded or the linking information has been manually entered into the Landmark Points dialog box see Section 7 Note that all points whether displayed as cross hairs or spheres will be shaded to indicate their interaction with the bone surface as specified in the Bone threshold field of the Manual Landmark tool Any part of a cross hair or sphere that lies above all bone surfaces from the current view direction will be displayed at 100 intensity Any part of a cross hair or sphere that lies below a bone surface from the current view direction will be displayed at 50 intensity This provides visual information on how the point interacts with the surface 19 6 The Volume Rendering Interface X VR Control SEE Render quality Fastest Fast High Highest Render type Opacity Greyscale Pseudo colour Full Colour Scalar Classification spline linear reset apply Gradient Classification spline linear reset apply Options DQ Front fac 1 0 DQ Density 1 0 Depth queing Off On Ambient R 01 G 01 B 01 Diffuse R 04 G 04 B 04 Specular R 05 G 05 B 05 Shinyness RGB 1 0 Lighting Control Foreground colour Background colour EE Scan Load Save Figure 6 The interface to the volume rendering engine The TINA Manual Landmark t
25. into the X Y and Z fields of the Manual Landmark tool e The arrow keys after each of the X Y and Z fields in the Manual Landmark tool allow the user to increment or decrement each coordinate of the 3D cursor the size of the increment can be chosen via the Resolution choice list in the Manual Landmark tool e When the 2D TV mouse interaction is in Pick mode the user can move the large red cross hair representing the 3D cursor by left clicking on a position in any 2D TV or by left clicking and dragging Alternatively the keyboard shortcuts can be used to shift the position of the 3D cursor see Section 5 2 1 e When the 3D TV mouse interaction is in Pick mode the user can left click on any position in the 3D TV the 3D cursor will then be moved to the upper most bone surface under the mouse cursor The bone surface is detected using a simple intensity threshold specified in the Bone Threshold field of the Manual Landmark tool see Section 5 2 2 Once the 3D cursor is in the desired position its coordinates can be stored by clicking the Mark Point button in the Manual Landmark tool The coordinates will be transferred to the point specified in the Markup choice list If Curr LM is selected then the coordinates will be transferred to the current landmark i e the landmark specified in the No and Name fields in the Manual Landmark tool and also displayed in the middle of the five rows of
26. list in the Manual Landmark tool provides the option to display the positions of the G reg points in the 3D TV where they are displayed as orange spheres or cross hairs The stored coordinates for G reg points can be viewed in the Landmark Points dialog box The A P G point choice list selects which point will be viewed and the Name x y and z fields in the same row of the tool display the name and coordinates of the selected point if stored In order to identify a G reg point e Move the 3D cursor to the desired point in the 3D image e Select the desired point in the Markup list of the Manual Landmark tool e g G1 is the first G reg point e Press the Mark Point button in the Manual Landmark tool this will save the current coordinates of the 3D cursor to the point specified in the Markup list of the Manual Landmark tool The choice of whether to use the landmark list or the G reg points for the initial global alignment is controlled by the Point based reg type choice list LM based specifies use of landmark list points and G reg based specifies the use of the G reg points Obviously if G reg points are used then they must have been marked for all database entries i e identified in each database volume prior to clicking the Landmarks gt database button and so the user must consider the global registration technique during database preparat
27. must be in the same directory e If the sequence is too large to load into memory the Stride and Downsample fields can be used to down sample the data as it is loaded The Stride field down samples data in the inter slice direction For example if the Stride field is set to 2 the sequence is down sampled by a factor of 2 in the inter slice direction The Stride average switch controls the method used to achieve this if it is set to Off then down sampling is achieved by skipping over slices during loading No use is made of the skipped slices they are never loaded If the Stride average switch is set to On then all slices are loaded and linear averaging is applied in the inter slice direction to down sample the sequence The Downsample field down samples data within each slice For example if the Downsample field is set to 2 then each image is down sampled by a factor of 2 along its x and y axes after it is loaded and the down sampled image is entered into the sequence Linear averaging is used during this process reducing image noise e Press the Load button Any previous image sequence will be deleted from memory and the file s specified in the Image file field will be loaded in order to produce a new sequence The Image type choice list will be updated to show the variable type of the images loaded into the sequence The Start and End fields will be u
28. of sets of manually or automatically identified landmark points together with a statistically valid alternative to Procrustes alignment based on likelihood and taking full account of the anisotropic errors on individual landmarks This user manual covers Section 2 installation of TINA the Volpack volume rendering library and the TINA Geometric Morpho metrics toolkit on Linux systems Section 3 the TINA graphical subsystem TVs and TV tools Section 4 the TINA Sequence tool used for loading 3D medical data sets Section 5 the Manual Landmark tool definition of coordinate systems and the TVs available in the tool Section 6 the TINA interface to the Volpack volume rendering library used to produce the volume rendered images displayed in the 3D TV of the Manual Landmark tool Section 7 the Landmark Points dialog box used to load save and manipulate lists of landmark points Section 8 the use of the Manual Landmark tool to mark up the positions of landmarks specified in the landmark point list Section 9 the Keyboard Controls dialog box used to assign keyboard shortcuts to the TVs of the Manual Landmark tool Section 10 the TINA View tool which provides functions to save the images displayed in TINA TV tools Section 11 an example landmarking session Sections 12 to 18 the Automatic Landmark Point Placement tool which can semi automatically identify landmarks given a database of manually identified examples Sectio
29. propagation to account for the effects of smoothing differentiation and multiplication by y The use of image derivatives rather than intensities makes the software robust to the presence of any overall offset between the intensities in the two image volumes The y factor provides further robustness against any linear scaling between the intensities in the two volumes Since y must be taken into account in the calculation of ce and the noise estimate cannot be allowed to vary during maximum likelihood optimisation y is estimated once at the end of the first global registration stage see below and then fixed during local registration The optimisation itself is performed using the simplex algorithm 14 Accurate knowledge of n and ce also allows the calculation of a goodness of fit score from the final value of the likelihood function as the likelihood can be translated directly into a x using 2lnL x 2 the x per degree of freedom can then be obtained by dividing by the number of independent terms in the likelihood sum minus the number of parameters optimised Since the automatic point location process is based on affine transformations i e assumes that an affine transfor mation of the image volume currently loaded into the Sequence tool provides a good model of the image volumes stored in the database it is important that the current and stored volumes have similar shapes e g for identifi cation of morphometric landmarks on the
30. rendering Mouse interaction with the 3D TV can be selected using the 3D Tv Mouse switch in the Manual Landmark tool In Zoom mode mouse interaction controls the 3D image Clicking and dragging with the left mouse button will rotate the image Clicking and dragging with the middle mouse button will zoom in or out moving the mouse left and right or rotate the image within the image plane moving the mouse up or down Clicking and dragging with the right mouse button will move the image around within the TV When Pick mode is selected mouse interaction within the 3D TV controls the 3D cursor Left clicking within the 3D TV will move the 3D cursor to the first bone surface under the mouse pointer i e the position of the mouse pointer specifies a vector through the data depending on the current rotation of the 3D image the software will run along this vector starting at the uppermost point from the current view direction until it reaches a bone surface The 3D cursor will then be moved to this point on the bone surface The bone surface is defined by a threshold which the user can set in the Bone threshold field of the Manual Landmark tool see Section 18 The software also provides a profiling tool 17 to aid the user in selecting a suitable threshold Right clicking with the mouse within the 3D TV will produce a grey level profile along the vector under the mouse pointer and display it in the Imcalc Graph TV
31. that user inspection has occurred and the point is in the correct location e Proceed through the landmark list by clicking the Next outlier button inspecting points correcting them if necessary and clicking the Mark Point button whether or not any correction has been applied until the end of the landmark list has been reached 47 14 Using the Automatic Landmarking Tool A complete set of steps required to automatically identify a set of landmarks is as follows e Prepare the database as described in 13 1 by manually identifying a specific set of landmarks in multiple specimens of similar shape to the image volume intended for automatic landmarking and if desired the G reg points Click the Landmarks gt database button once all points have been identified in each volume before proceeding to the next volume e Load the image volume intended for automatic landmarking e Load the landmark list names file used to identify landmarks for the volumes in the database e Manually identify at least four points in the landmark list or alternatively the four G reg points e Ensure that the Point based reg type choice list is set to the correct option depending on whether G reg or landmark points were identified in the previous step e Ensure that both Point based and Image based are selected in the Reg stages check list e Click the Global reg button to perform global registration
32. the Linklines option of the 3D Tv draw checklist is used Move downwards in the list of landmark points Move downwards by five points in the list of landmark points Move to the next point in the landmark list that is flagged as an outlier after automatic landmark point location i e has a point type code of 3 or greater Move to the end of the landmark point list Select an axis plane or G reg point to view Ax 1 the first axis point Ax 2 the second axis point Pl 1 the first plane point Pl 2 the second plane point Pl 3 the third plane point G1 the first G reg point G2 the second G reg point G3 the third G reg point G4 the fourth G reg point The name of the selected axis plane or G reg point Stored x coordinate for the selected axis plane or G reg point Stored y coordinate for the selected axis plane or G reg point Stored z coordinate for the selected axis plane or G reg point TPS NTSYS files Study name TPS NTSYS files TPS and NTSYS files can hold multiple lists of points this field selects which one to load if there is more than one list in the file Delete the current axis points Delete the current plane points Delete the current G reg points Delete the current landmark links information Delete the current landmark points list Choose the type of file to load 75 Input pathname Scan Load Output format output pathname Scan Save Names a lis
33. the third G reg point G4 display information on the fourth G reg point The point number of the selected G reg point from the current database entry The point name of the selected G reg point from the current database entry The x coordinate of the selected G reg point from the current database entry The y coordinate of the selected G reg point from the current database entry The z coordinate of the selected G reg point from the current database entry The point type code of the selected G reg point from the current database entry Scroll upwards through the list of landmark points in the current automatch database entry the next ten rows of fields show the information stored for the landmark points the middle pair of rows is the current landmark Landmark point number Landmark point name Stored x coordinate for the landmark point Stored y coordinate for the landmark point Stored z coordinate for the landmark point Type code for the landmark point Projected x coordinate for the landmark point based on registration results Projected y coordinate for the landmark point based on registration results Projected z coordinate for the landmark point based on registration results x for the patches used in the final local registration stage using the global registra 80 Chi2 global local Down Image patch Push tion result x for the patches used in the final local registration stage using the global and local
34. to construct the database Analyse the results comparing the automatically estimated landmark point locations for each volume to the manually identified landmark point locations in order to estimate the errors Repeat the process varying the free parameters in order to determine the optimal parameters for the data This procedure involves large numbers of experiments and so will be considerably faster if the TINA macro file system is used to run the experiments see Section 15 Furthermore the outlier detector operates by placing thresholds on the distances between the landmark point location determined by the final refinement stage of the algorithm and the locations of the three corresponding points in the automatch database with the lowest x per degree of freedom after global and local registration Since all of this information is contained in the point positions files see Section 17 1 the optimal error detection thresholds can be determined by outputting such files and using them to construct ROC curves 48 15 Double Pass Operation of the Software As described in Section 13 2 1 the global registration stage of the automatic landmark point identification software incorporates an initial stage based on user identified landmark points to deal with any gross rotation of the specimen relative to the previous specimens entered into the automatch database This introduces a requirement for user interaction at the start of the automatic lan
35. within each slice and consisting of 1000 to 1200 slices per volume These were down sampled by a factor of 2 along all axes of the volumes during loading i e the Stride and Downsample fields were both set to 2 This produced volumes of 329x329 voxels in each of 500 to 600 slices after loading These figures can be used as a guide when setting the values of the Stride and Downsample fields as the volumes are small enough to be loaded onto any machine with 2Gb or more system memory and to allow the volume renderer to run in interactive time on typical hardware at the time of writing i e Intel Core 2 Duo or equivalent or better Switching the Stride average option to On will increase the time required to load an image volume since rather than loading a sub set of the slices all slices will be loaded and a linear averaging process applied to produce the down sampled sequence However this also has the effect of reducing the image noise on the volume producing higher quality renderings in the 3D TV see Section 5 2 2 More importantly the Automatic Landmark Point Placement tool will produce more accurate results and the error analysis stage of that software will be more reliable Therefore it is recommended that the stride averaging be switched on when using this software if Stride is set to a value greater than 1 The sequence tool can also save data to file although not all file formats are supported th
36. INA User s Guide 19 for full instructions on how to use these tools Two functions of these tools are of particular interest when using the TINA Manual Landmark tool the creation of movies from 3D images and the saving of images to files The View tool can be used to prepare a movie from any TV displaying a 3D image e g the 3D TV of the Manual Landmark tool in which the image rotates in 3D around its central point Each frame of the movie is rendered in turn and stored as a 2D image within the TV tool during playback these 2D images are displayed in the TV as a slide show Therefore no rendering is performed during playback allowing the movie to play in a much shorter time than it took to render This can significantly increase the viewer s perception of 3D and so can be useful in seeing exactly where landmark points are located in the 3D image The user can specify the angles of rotation the number of frames in the movie the time delay between each frame during playback and the number of times the movie loops during playback Note that the rotation angles are specified in radians around the current x axis i e left right axis and y axis i e up down axis of the TV i e they do not refer to the coordinate system used in the Manual Landmark tool Once a movie has been created it is stored in the TV tool until the user deletes it To create a movie in a TV tool displaying a 3D image e Select the TV for which you wish to produce
37. Tina Memo No 2010 007 Technical Memo The TINA Geometric Morphometrics Toolkit P A Bromiley H Ragheb and N A Thacker Last updated 9 2 2012 TINA WWW TINA VISION NET Imaging Science and Biomedical Engineering Division Medical School University of Manchester Stopford Building Oxford Road Manchester M13 9PT 1 The TINA Geometric Morphometrics Toolkit P A Bromiley H Ragheb and N A Thacker Imaging Science and Biomedical Engineering Division Medical School University of Manchester Manchester M13 9PT UK paul bromiley manchester ac uk Introduction The TINA Geometric Morphometrics Toolkit comprises three main tools the Manual Landmark tool the Automatic Landmark Point Placement tool the Shape Analysis tool The TINA Manual Landmark tool is designed to support the identification and specification of morphological landmarks for subsequent processing using techniques such as Procrustes analysis However the time required to manually identify sufficient numbers of landmarks in sufficient numbers of specimens for landmark based geometric morphometric studies can be prohibitive Therefore the Automatic Landmark Point Placement tool can given multiple examples of manually identified landmarks semi automatically identify those landmarks in further spec imens greatly reducing the user input required and therefore increasing productivity The Shape Analysis tool provides standard Procrustes alignment
38. ULL X 1 0 y 1 0 Z 1 0 Type 1 Proj x 1 0 Proj y 1 0 Proj z 1 0 Chi2 global 0 0 Chi2 global local 0 0 No 1 Name NULL x 1 0 y 1 0 Z 1 0 Type 1 Proj x 1 0 Proj y 1 1 0 Proj z 1 0 Chi2 global 0 0 Chi2 global local 0 0 Down Image patch X y zZ DX DY DZ RX RY RZ Push Figure 18 The Automatch Database Inspector dialog box The Automatch Database Inspector dialog box allows inspection of the information stored in the automatch database It can be started by clicking on the Automatch DB Inspector button in the Automatic Landmark Point Placement tool and is shown in Fig 18 The lt and gt keys allow the user to scroll through the entries in the database the total number of entries and the ordinal number of the one currently being displayed are indicated in the Entry field The Name field gives the filename of the image volume used to create the entry The Noise field displays the estimated standard deviation of the image noise this is used in the calculation of the likelihood function for image based registration If global registration has been performed the Et Er and Scale fields display the parameters of the optimised transformation model the rotation is displayed as a rotation matrix although in effect only three parameters are optimised the optimisation itself uses a quaternion representation of the rotation parameters in order to avoid probl
39. a movie by clicking on its entry in the TV list of the tool it is associated with e g click on 3D in the TV list of the Manual Landmark tool e Start the View tool by clicking the View button in the tinaTool window The View tool will start and will display the name of the currently selected TV in the Current Tv field e Select the parameters of the movie Rot x the rotation to apply about the x axis of the image in radians Rot y the rotation to apply about the y axis of the image in radians Steps the number of frames in the movie Timer 0 01 sec the time delay between each frame during playback Count the number of times to loop through the movie e Click Make seq to prepare the movie and store it to the TV e Click Show to play the movie in the TV e Clicking Init will delete any previously stored movie allowing a new one to be prepared The Dump tool which is a sub tool of the View tool can save the image displayed in any TINA TV tool as a TIFF or Encapsulated Postscript file TIFF files can be used to transfer images to Windows machines e g for incorporation into MS Word documents whilst EPS files can be used to incorporate images into documents prepared under Linux e g using Latex 35 To save the image displayed in a TV tool to a file Select the TV for which you wish to produce a movie by clicking on its entry in the TV list of the tool it
40. a small subset of samples rather than the whole population Note that for the analysis to be statistically valid depending on the data a minimum number of samples is needed e g 30 samples outlier threshold This is the standard deviation value used as threshold in the outlier detection routine This is usually set to a value between 3 0 and 5 0 depending on the data under study left reference point Enter here the left landmark point of the base line Note that each landmark number is between one and the number of landmarks right reference point Enter here the right landmark point of the base line third reference point This landmark point is not needed for the case of 2D data but must be entered appropriately for the case of 3D data to form a planar reference together with the left and right points model components This is the number of eigenvectors values used when constructing the linear model Obviously one needs to have a good idea of the optimum number of model components for each data set as using too many components would result in over fitting and using insufficient number of components would result in the model failing to fit well to the data This number is usually smaller for the Likelihood method than that for the Procrustes We outline an experiment in Section 2 4 and Box C that helps users to find an optimum number for each data set as the number of model components number of iteration
41. a warning message is displayed in the top level tinaTool window This should be considered an error condition for the reasons outlined above The Fin sig field should be set to a value representing the expected accuracy of the registration result for points that successfully find the correct location i e ignoring any outliers One way to determine this value is to manually identify landmarks in a representative image volume twice the median absolute error in voxels between the manually identified positions for each landmark provides a guide to the achievable landmarking accuracy and can be used as the value for Fin sig However the user must remember that the landmark positions output 45 to file are in the original coordinate system of the data i e ignoring any down sampling applied during loading whilst the Fin sig parameter is in the coordinate system of the current sequence i e takes any down sampling applied during loading into account The final refinement has two additional options First the Chi sq weight option weights the Kronecker delta functions using the x at the end of the local registration i e down weights the points that may not have fitted correctly This option requires careful testing before use since the estimated x values themselves are not com pletely reliable and so it is recommended that Off should be selected Second the Snap points option will take the location predict
42. ace as specified by the Bone threshold field of the Manual Landmark tool then clicking on this button will move it to the nearest bone surface 8 1 Axis and Plane Points In addition to landmark points the TINA Manual Landmark tool supports three plane points and two axis points The plane points can be used for example to specify the plane of bilateral symmetry The axis points are used to specify an arbitrary axis within the 3D image Both sets of points are used to implement various utility functions that make the manual landmarking process easier Axis and plane points can be specified regardless of whether a landmark list is loaded or not 32 In order to mark up an axis or plane point e Move the 3D cursor to the desired point in the 3D image e Select the desired point in the Markup list of the Manual Landmark tool e g P1 1 is the first plane point e Press the Mark Point button in the Manual Landmark tool or use the keyboard shortcut assigned to this function in any of the Manual Landmark tool TVs see Sections 5 2 1 and 5 2 2 this will save the current coordinates of the 3D cursor to the point specified in the Markup list of the Manual Landmark tool In order to re use an existing landmark point as an axis or plane point e Scroll through the list of landmark points either in the Landmark Points dialog box or in the Manual Landmark tool e Press the Jump to button in the Ma
43. aded the names and numbers of the landmarks can be viewed in the Landmark Points dialog box The top half of the dialog box shows five landmarks at a time displaying their number name any stored x y and z coordinates a type field see below and a link no field which specifies which if any other point the point is linked to when the Linklines option is used in the 3D Tv draw checklist The user is free to edit any of these fields manually however the type code has specific meaning that affects the operation of the software particularly the Automatic Landmark Point Placement tool see Section 7 1 For example the Manual Landmark tool assigns a type of 1 to any point that has not yet been identified and a type of 0 to any point that has been manually identified Any point with a type of 1 will not be displayed in the 3D TV Therefore the point type code should not in general be altered by the user After loading a landmark names file one landmark in the list is always considered to be the current landmark During manual landmarking whenever a point is identified the coordinates of the 3D cursor will be transferred to the current landmark The current landmark is always the one displayed in the middle of the five rows of landmark information in the Landmark Points dialog box The user can specify the current landmark so that points can be marked up in any order by scrolling up and down through the landmark list using the
44. al alignment is also displayed 13 4 Checking the Global Registration Result The results from the global registration stage of the software form the initialisation for the coarse to fine local registration of patches of image data around each landmark point If this initialisation is not accurate enough the local registration will fail to find the correct locations for the landmark points and the automatic landmark point location software will perform poorly Failures in the global registration stage of the software can indicate several different problems e The points used in the initial point based stage of the global registration are not consistent i e different points have been selected for different entries in the database or for the current volume e The points used in the initial point based stage of the global registration are poorly located e g errors have occurred during manual landmarking or the points themselves vary significantly between different image volumes e The points used in the initial point based stage of the global registration are poorly selected e g they are close to coplanar or cover a small volume of the total sample e The shapes of the specimens in the automatch database vary too much from the shape of the specimen in the current volume to allow a good image based alignment Therefore the user should check the global registration result for a representative sample of the image volumes to which the automa
45. andmark tool Start four new TV tools and install them on the four TVs of the Manual Landmark tool Start the VR Control dialog box and the Lighting Control dialog box and manipulate the volume renderer options to produce a good volume rendering see Sections 6 and 6 1 for suggested settings Use the techniques described in Section 5 2 2 to specify a suitable bone threshold Load the landmark names file using the Landmark Points dialog box see Section 7 The current landmark will now be the first one in the list manipulate the rotation of the image in the 3D TV with the 3D TV mouse interaction in Zoom mode until you can see the corresponding point on the bone surface Switch the 3D TV mouse interaction to Pick mode and then left click on the position of the landmark point in the 3D TV the 3D cursor will be moved to that point Check the position of the 3D cursor in the 2D TVs if necessary refine it using the arrow keys in the Manual Landmark tool or by using mouse or keyboard interaction with the images in the 2D TVs see Section 5 2 1 When you are happy with the position of the 3D cursor click the Mark point button in the Manual Landmark tool or use the keyboard shortcut in the 2D or 3D TVs see Sections 5 2 1 and 5 2 2 The coordinates of the 3D cursor will then be transferred to the current landmark and the current landmark will then be incremented i e the next landmark in the list will become the current
46. ane This is mainly for the case of 3D data When working with 2D data selection of planes does not matter and the xy plane default is always active source Select which source of point data you want to be used in the display routines Select data for the input data aligned for the final aligned data mean for the final mean shape and model for the final linear model constructed sample number The user may enter the sample number to display individual samples between one and the number of samples display sample points By pressing this button the landmark points of the sample number chosen will be y y g displayed display all samples By pressing this button the landmark points of all the samples studied are displayed superimposed This is a useful button when used to display the input data before starting the process or to display aligned data and models once the process is over Looking at such data spreads gives an idea of the quality of alignment landmark number The user may enter a landmark number to be highlighted on the display window between one and the number of landmarks highlight landmark By pressing this button the landmark point of the sample number chosen will be high y 8 lighted next landmark By pressing this button the next landmark point of the sample number chosen will be high lighted eigenvector The user may enter the
47. ar curve linking the control points reset reset the curve to a linear ramp apply implement the changes made to the curve The thickness of the depth cueing fog at the front of the image gt 0 The rate at which the depth cueing fog becomes thicker with depth through the image gt 0 Switch depth cueing on off depth cueing acts like a fog in the rendered image so that more distant parts of the data are dimmer The reflection coefficients of surfaces for ambient light red green and blue 0 0 to 1 0 The reflection coefficients of surfaces for diffuse light red green and blue 0 0 to 1 0 The specular reflection coefficients of surfaces red green and blue 0 0 to 1 0 The shininess of surfaces 1 0 to 100 0 Start the Lighting Control dialog box Set the foreground colour for pseudo colour rendering Set the background colour for pseudo colour and full colour rendering Specify a file from to which all the options of the renderer can be loaded saved Start a file browser for the options file Load renderer options from the file specified in the Options field Save renderer options to the file specified in the Options field 73 21 4 Lighting Control Dialog Box 1 6 On off x X Z r 8 b Reset to defaults Apply Switch the light on or off a maximum of six lights are supported The x component of the direction vector of the light 1 0 to 1 0 The y component of the direction vector o
48. assing through the centre of the volume This functionality can be useful in viewing the extremal extents of some structures The viewpoint of the 3D TV can also be aligned using the Rotate to Axis and Rotate to Plane buttons in the Manual Landmarking tool Once three plane points have been selected clicking the Rotate to Plane button will rotate the volume such that the viewing direction after rotation is the normal to the plane using the smallest rotation angle possible Clicking the button a second time will flip the volume over to give the view from the other side of the plane Once all axis and plane points have been defined clicking the Rotate to Axis button will rotate the volume such that the projection of the axis onto the plane is aligned with the left right axis of the 3D TV so that using both buttons in combination will completely align the volume Again the rotation applied is through the smallest angle possible and clicking the button a second time will flip the volume over to give the view from the opposite side 33 9 Keyboard Controls X Tv keyl Select the text entry field for the key you wish to reassign then press the required key on the keyboard 2D Tvs Up Up Left Left Right Right Down Down 2D amp 3D Tvs Pick Shift R Zoom Control R Mark Return Prev landmark Page Up Next landmark Page Down Figure 15 The TV Keyboard Controls dialog box As described in Sections 5 2 1 and 5 2 2
49. ble estimate The function currently included in the software has been subjected to basic testing i e it runs and produces the correct single point estimates of the minimum variance bound However more rigorous stability testing and possibly modification in order to improve numerical stability is required prior to use The parameter covariances could be used in two ways First they could be included in the output files and thus made available for use in later analysis However they could also be used to set the size of the Gaussian kernel used in the final refinement stage of automatic landmark location this would provide a more optimal combination of the available data and thus a more accurate final hypothesised landmark location 18 3 Bone Threshold Estimation Several functions in the software depend on the value entered into the Bone threshold field in the Manual Landmark tool including mouse interaction with the volume rendering displayed in the 3D TV in Pick mode landmark point display in the 3D TV and the various Snap functions in the Manual Landmark tool and the Automatic Landmark Point Placement tool The procedure required to set this value is given in Section 5 2 2 However using a single value is sub optimal for several reasons For example the true threshold for points on bone surfaces in typical geometric morphometrics studies varies across the image and is different e g at the interface 54 between toot
50. c functionality TV tools can also be freely reassigned by selecting another TV and pressing the install button again Note that when a TV is installed onto a TV tool any other TV tool displaying that TV will be un installed the title of the TV tool will change to reflect this Each TV tool has the following menus and buttons e Size set the size of the TV tool to various default values 256x256 512x512 and 768x768 TV tools can also be freely resized by clicking and dragging the corner of the TV tool window with the mouse e Mouse sets the mouse interaction with the image in the TV tool e ROI select a region of interest within the image e Proj select the projection function for the TV tool e install install the currently selected TV onto the TV tool e clone install a duplicate of the currently selected TV onto the TV tool allows multiple TV tools to display the same TV e init re initialise the image display to the default viewpoint removing any translation rotation or zooming of the image e repaint re display the image with the current translation rotation or zooming used if the image display should become corrupted for any reason See the TINA User s Guide 19 for full details Each TV tool also displays two messages the name of the TV installed onto the TV tool displayed in the title bar and the current mouse functionality for the TV tool displayed just below the button row The mouse fun
51. cated landmark points that have failed an error check see Section 12 will be displayed in green and red chequers e Axis display yellow cross hairs for any specified axis points Plane display cyan cross hairs for any specified plane points e Global reg display orange cross hairs for any specified G reg points 18 e Text display text labels for any displayed landmark plane axis or G reg points The text label will appear next to the point and will be displayed in the same colour green for landmark points yellow for axis points cyan for plane points and orange for G reg points Landmark points are labelled with their point numbers axis points as Al and A2 plane points as P1 P2 and P3 and G reg points as G1 G2 G3 and G4 Since a single point might be used simultaneously as a landmark axis plane and G reg point the labels are arranged around the point such that they should not overlap However only the labels corresponding to the selected drawing options will be displayed e g if a single point is identified both as a landmark point and an axis point but the Landmarks option is selected and the Axis option is not selected then the landmark number label will be displayed but the axis point label will not e Poly display a yellow line representing the axis and a cyan grid representing the plane e Ball display 3D spheres instead of cross hairs for the landmark axis plane and G reg points e Linklines display
52. changed if Curr LM is selected in the markup choice list the 3D cursor will automatically by moved to the stored coordinates for that point if any Move the 3D cursor to the coordinates of the point specified in the markup choice list if those coordinates have already been marked up Lock the rotation of the image in the 3D TV so that rotation only occurs around an axis parallel to the axis defined by the two axis points and passing through the centre of the volume Rotate the image in the 3D TV such that the view direction is normal to the plane pressing the button a second time will rotate the view by 180 degrees around the y axis to show the opposite side of the volume Rotate the image in the 3D TV such that the projection of the axis defined by the two axis points onto the plane defined by the three plane points is aligned with the 71 Reflect cursor Snap Mark point left right axis of the TV pressing the button a second time will rotate the view by 180 degrees around the y axis of the TV to show the opposite side of the volume Project the cursor through the plane defined by the 3 plane points Move the 3D cursor to the nearest bone surface Save the coordinates of the current 3D cursor position to the point specified in the markup choice list i e the current landmark or one of the axis plane or G reg points A landmark list must be loaded before landmarks can be marked up 72 21 3 Volume Renderer Control D
53. city of each voxel i e with no surface shading so that the Gradient classification lighting and material parameters are all ignored 20 This provides a transparent view of the data as shown in Fig 8 The Greyscale choice will render an image with both opacity and surface shading so that the Gradient Classification lighting and shading parameters are all applied However only the overall intensity of light from each voxel is displayed and so the resulting image is greyscale The Pseudo colour choice produces a greyscale image but then colours the result using the colour chosen with the Foreground colour button and sets the background to a uniform colour as specified with the Background colour button The Full colour choice gives the renderer complete control of image colour so that coloured lights and material properties can be used see below the Background colour field can also be used to change the colour of the background in this mode Pressing the Lighting Control button spawns the Lighting Control dialog box which provides the user with full control over the lighting parameters used by the volume renderer See Section 6 1 for more details The volume renderer also supports depth cueing This function allows a user controlled fog to be applied to the volume rendering which grows thicker with depth through the image More distant objects therefore appear dimmer which helps to
54. contained in a separate file use the character as a wildcard For example if the directory contains 10 files named im 00 to im 10 enter the pathname of the first file into the Image file field either by typing or by using the file browser Then replace the part of the name that changes between files with the character so that it becomes im Enter the replaced part of the filename from the first file into the Start field including any leading zeros 00 in this example and the relevant part of the filename from the last file into the End field 10 in this case During image loading the wildcard characters in the pathname will be replaced with the contents of the Start field and the number contained there will be incremented until it is equal to or greater than the End field loading a file each time If the numbers in the filenames increase by more than 1 between each file then the Stride field can be used to specify the increment For example entering im into the Image file field 00 into the Start field 10 into the End field and 2 into the Stride field will load the images im 00 im 02 im 04 im 06 im 08 and im 10 in that order Note that there are some restrictions on loading multiple files in this way The substitution only works for numbers can only be applied to one contiguous segment of the pathname and only works in ascending or descending order In addition all files
55. create a more three dimensional effect Depth cueing can be switched off or on using the Depth cueing buttons and is controlled by the two parameters Front fac the thickness of the fog at the front of the image and Density the rate at which the fog grows thicker with depth through the image according to fog thickness front factor ie MMe dente Both Front fac and Density must be positive numbers Changes to the these parameters are not implemented in the rendered image until the next time it is regenerated the user can force the image to be rendered with the new parameters by clicking the Repaint button in the TV tool displaying the 3D TV When the rendering type is set to greyscale pseudo colour or full colour the intensity gradient of the original 3D image data is used to shade the surface of each voxel Finer control over the reflectivity of the surfaces can be achieved using the material property parameters contained in the Ambient Diffuse Specular and Shininess fields The ambient and diffuse fields control how the surface reflects ambient and diffuse light respectively the red green and blue components can be set independently The specular field controls the intensity of specular reflections i e highlights and again the red green and blue components can be controlled independently The shininess field controls the overall shininess of surfaces for which there is only a s
56. ction P1 thresh 35 0 P2thresh 35 0 P3 thresh 35 0 Error checks P1 P2 P3 Error analysis Output Scan Pos output Input database Output database Figure 17 The Automatic Landmark Point Placement tool In order to accelerate the process of landmark identification the TINA Geometric Morphometrics toolkit is capable of automatically estimating landmark positions based on a database of previous examples of manually identified landmarks This functionality is available via the Automatic Landmark Point Placement tool which can be launched by clicking on the Automatch button in the Manual Landmark tool The Automatic Landmark Point Placement tool is shown in Fig 17 The automatic landmark identification operates by storing information from manually identified sets of landmarks including both the coordinates of the landmark points and image data from around the points in a database the automatch database These image patches are then aligned with the data set currently loaded into the Sequence tool using a coarse to fine registration process producing multiple estimates of the position of each landmark one for each entry in the database The registration is based on a maximum likelihood estimation of a nine parameter affine transformation model i e a 3D translation rotation and scaling Rather than using 3D blocks of data around each landmark point the software extracts 2D images on the three orthogonal planes inte
57. ctionality lists the name of the function type followed by the functionality of the left middle and right mouse buttons e g zoom3D rot zoom shift indicates that the mouse has been set for 3D interaction with the image displayed in the TV tool clicking and dragging with the left mouse button rotates the image in 3D clicking and dragging with the middle mouse button zooms the image and clicking and dragging with the right mouse button moves the image around in the TV tool 4 Loading 3D Medical Image Data The Sequence Tool A o equence T DICOM re scale Help Tv sequence File AIFF _ANLZ RAD 7 NEMA PGM DICOM Image type bin chr int fit Start 0 Stride 1 Downsample 1 Cur frame 0 End o Image File Scan Scales x 1 0 y 1 0 Zz 1 0 jae 1 0 Load Save First lt gt End Jumpto Stride average Off On Del Seq Del Ins Rep Push Stack gt Seq Figure 2 The Sequence tool used for loading 3D medical image data The TINA Manual Landmark tool 18 allows manual identification of anatomical landmarks in 3D medical image data it was designed for use with micro CT images of rodent skulls but can interact with any other tomographic medical image data e g MR images Tomographic medical image data is loaded into TINA using the sequence tool shown in Fig 2 The current sequence can be viewed by installing a TV tool onto the Sequence TV This displays the slice specified in the Curr frame
58. d that the 3D cursor position will always be displayed in the coordinates of the down sampled volume but all landmark positions displayed in the Landmark Points dialog box and saved to file using that dialog will automatically be multiplied by the relevant down sampling factors so that they are in the coordinate system of the original data For example if the original image volume consists of 1000 slices each 1000x1000 voxels in size and this is down sampled by a factor of 2 on each axis when it is loaded i e the Downsample and Stride fields in the sequence tool are both set to 2 then the coordinates of the 3D cursor placed at the centre point of the volume will be 256 256 256 and this will be displayed in the X Y and Z fields of the Manual Landmark tool however if this point is marked up as a landmark point then the coordinates will automatically be multiplied by the down sampling factors to become 512 512 512 before they are displayed in the Landmark Points dialog box or saved to file This prevents the choice of 13 Figure 4 The coordinate system used to refer to the data top left the z direction is the inter slice direction the x direction is the left right axis of images seen in the sequence tool TV and the y direction is the top bottom direction of images seen in the Sequence tool TV The images shown in the three 2D TVs of the Manual Landmark tool are generated from orthogonal planes passing through the current
59. d with its major axes Simplex optimisation is used to optimise the parameters of a nine parameter affine transformation through a maximum likelihood method based on the likelihood function described above The y factor is set to 1 during global registration it is then calculated from the aligned full sized patches once the global registration is complete and used during local registration this procedure is based on the theory underlying the expectation maximisation algorithm The smoothing applied during the image based stage of global registration is controlled by the Glob sig field in the Automatic Landmark Point Placement tool this specifies the standard deviation of the smoothing kernel in voxels in the down sampled coordinate system i e the coordinate system of the data currently loaded into the database and the Sequence tool As mentioned above the Glob border field must have a value at least three times the size of the Glob sig field The software will display a progress bar during image based global alignment In addition the number of iterations of the simplex optimisation and the value of the cost function at the end of optimisation will be displayed in the top level tinaTool window Once transformation models have been calculated for all database entries they will be stored in the automatch database and can be inspected in the Automatch Database Inspector dialog box The x per degree of freedom at the end of glob
60. database with the third lowest y Run the outlier detection algorithm Select the sets of coordinates from the automatch database to save to the positions output file Proj glob the projected positions using the transformation from the global reg istration Proj 1 the projected positions using the transformation from the global registration and stage one of the coarse to fine local registration Proj 2 the projected positions using the transformation from the global registration and stages one and two of the coarse to fine local registration Proj the projected positions using the transformation from the global registration and stages one two and three of the coarse to fine local registration Pos the original positions of the points Specify a path and filename relative or absolute for database and positions file I O Launch a file browser for the Output field Output a point positions file containing the locations of all points in the automatch database and the current landmark list plus all stored error information and land mark point codes to the file specified in the Output field The set of coordinates from the automatch database points is selected using the Pos output field choice list Input the database file specified in the Output field Output the current database to the file specified in the Output field 79 21 8 Automatch Database Inspector Dialog Box lt Entry n
61. dmarking process However in large landmark based geometric morphometric studies involving hundreds of specimens it may be desirable to eliminate this stage of the analysis so that no user interaction with the software is required prior to checking and correcting any outliers Double pass operation of the software allows this but relies on careful positioning of the specimen in the CT scanner such that e All specimens involved in the analysis including those used to build the automatch database are in approx imately the same location and orientation in the image volumes e All specimens are of approximately the same size e Each image volume contains only one specimen Double pass operation of the software involves running the automatic point location procedure twice The first pass is performed with no initial point based registration The number of automatically located points that pass error checks in this process will be relatively low typically 60 of the points in tests with relatively well aligned micro CT images of mouse skulls However these points can be used to perform the point based stage of registration in a second pass through the automatic point location process resulting in a more accurate global alignment and thus a higher proportion of automatically located points that pass error checks typically 80 90 of the points in tests with relatively well aligned micro CT images of mouse skulls The double pass op
62. dmarks have been manually identified press the Landmarks gt database button in the Automatch Dialog box This will extract all of the information required for subsequent operation of the automatic landmark software and enter it into the database All image based registration stages allow the application of smoothing during the preparation of the image patches in order to reduce image noise and thus provide a smoother cost function for the optimisation reducing the probability that the optimisation will get trapped in a local minimum In order to allow accurate calculation of the smoothed images the software supports the use of a border around the patches i e it stores and smooths a patch of the requested patch size plus the border size and then ignores the data in the border region during calculation of the likelihood The border size should be set to at least three times the size of the smoothing kernel in order to ensure that no part of the smoothing kernel falls outside the patch when calculating any smoothed image intensity that will be used in the calculation of the likelihood since the smoothing function truncates the smoothing kernel at the 30 points Borders patch sizes are controlled by two parameters Storage patch size and Glob border The first controls the size of the images patches around each landmark point that are stored in the database The patches must be large enough to include the largest patch size used in the l
63. dy been processed The files corresponding to the model the mean and eigenvectors are automatically generated by the Likelihood analysis and made available in the data directory every time a data set is processed by estimating the covariances Hence for this Monte Carlo test the covariance file should also be provided The default selection is No rest rand seed This button only has an effect on the Monte Carlo data generation routine By pressing this button one can reset the seed value using the system time for the random number generator Once the seed value is changed the software can generate a new set of Monte Carlo data which is different from a previous set before resetting the seed settings Before pressing the process data button the user must set all processing choices Settings dialog box appropriately These are clearly explained below process data By pressing this button the process starts based on the choices made using other buttons of the tool display tool This tool box provides the means needed to display data and results graphically Detailed descriptions are given below 20 2 Settings Here we explain what is the purpose of different settings appearing on the Settings dialog box number of samples This can be set to a number smaller or equal to the number of samples which have been successfully read from the TPS file This is useful if user decides to experiment with
64. e Resolution choice list in the Manual Landmark tool Select a shortcut key to move the cursor left in the currently selected 2D TV by the number of voxels specified in the Resolution choice list in the Manual Landmark tool Select a shortcut key to move the cursor right in the currently selected 2D TV by the number of voxels specified in the Resolution choice list in the Manual Landmark tool Select a shortcut key to move the cursor downwards in the currently selected 2D TV by the number of voxels specified in the Resolution choice list in the Manual Landmark tool Select a shortcut key that switches the mouse functions to Pick mode for all TVs Select a shortcut key that switches the mouse functions to Zoom mode for all TVs Select a shortcut key for the Mark Point button i e markup the point currently selected in the Markup choice of the Manual Landmark tool with the current co ordinates of the 3D cursor and then if Curr LM is selected move the current landmark to the next one in the landmark list Select a shortcut key that scrolls up through the landmark points list Select a shortcut key that scrolls down through the landmark points list TT 21 7 Automatic Landmark Point Placement Tool Automatch DB Inspector Storage patch size Glob border Landmarks gt database Free database Free curr entry Reg stages Glob sig Point based reg type Greg 1
65. e init repaint zoom3D rot zoom shift imzoom shift scale roi X X Axis o x X Z Axis SE Size Mouse ROI Proj Size Mouse ROI Proj install clone init repaint install clone init repaint imzoom shift scale roi imzoom shift scale roi Figure 5 The four TVs of the Manual Landmark tool arranged so that the cross hairs for the three 2D TVs are consistent across all three windows which the left right axis is the z axis of the sequence and the top bottom axis is the y axis of the sequence The y axis TV displays an image in which the left right axis is the x axis of the sequence and the top bottom axis is the z axis of the sequence The z axis TV displays an image in which the left right axis is the x axis of the sequence and the top bottom axis is the y axis of the sequence Mouse interaction with the 2D TVs can be selected using the 2D Tv Mouse switch in the Manual Landmark tool The functions assigned to each mouse button are displayed in the TV tool just above the image When Zoom is selected the mouse can be used to manipulate the image Clicking and dragging with the left mouse button will move the image around in the TV Clicking and dragging with the middle mouse button will zoom in or out Clicking and dragging with the right mouse button will select a rectangular region of interest and then zoom into that region when the mouse button is released During the moving or zooming
66. e in the following instructions to configure CFLAGS O2 fopenmp the O2 flag switches on the optimiser which also speeds up the execution of the software In addition the main limitation on the execution speed of the automatic landmark point location software when built with parallelisa tion options switched on is not the number of available execution cores but the memory bandwidth Therefore the execution of the software will be slowed down if hyperthreading is used and it is recommended that if hyper threading is provided by the processor of the machine used to run the software it should be disabled this can be done through the bios setup programme although the exact procedure will vary between computers To build the TINA libraries open a shell tool and as root create a directory called Tina6 in usr local Copy both tarballs into the new directory Then unzip both tarballs using e g gunzip tina libs 6 0rcbuild008 tar gz gunzip tina tools 6 0rcbuild008 tar gz and then unpack both using tar xvf tina libs 6 0rcbuild008 tar tar xvf tina tools 6 0rcbuild008 tar Build the tina libs libraries first cd into the tina libs xxx where xxx is the build number directory created when the tina libs tarball was unpacked and type configure make make install Build the tina tools libraries after the tina libs libraries cd into the tina tools xxx where xxx is the build number directory created when the tina tools ta
67. e of the automatic point location can be completely accurate Therefore errors may exist on both the automatic point locations and the estimated errors on those locations provided by the x per degree of freedom The outlier detection therefore inspects consistency across both estimated location and estimated error so that an error on either quantity will result in a point being flagged as an outlier A leave one out experiment involving 14 micro CT datasets of mice wood mice voles hamsters and gerbils indicated that this provides a reliable outlier detection method with a false negative rate of 0 at the expense of a false positive rate of 20 on image volumes for which the majority of the database consists of examples from the same genus The outlier detection operates by sorting the predicted locations for a given landmark point from the database into order based on their xy per degree of freedom at the end of local registration and then flagging any point as an outlier if the estimated locations for the points with the lowest x differ from the location resulting from the final refinement stage by more than a given threshold The number of points included in the check is controlled by the Error checks check list P1 checks the point with the lowest x P2 the point with the second lowest x and P3 the point with the third lowest x The distance thresholds are set in the P1 thresh P2 thresh and P3 thre
68. e Tina dialog box Proceed if you are happy with them otherwise go back to step 1 If you wish to use existing covariance estimates press scan again locate the CovarianceFile then press input covariance button Check the messages warnings appearing on the Tina dialog box Proceed if you are happy with them otherwise go back to step 1 Ignore this step if you wish to estimate covariances or if you wish to work with Procrustes Select the analysis method by pressing either on Likelihood or on Procrustes If Likelihood method is selected decide whether you wish to estimate covariances or use the input co variance data that you have already provided by selecting Yes or No in front of estimate covariances Ignore this step if you have selected Procrustes To apply the Likelihood method to any manual mark up data make sure that No is selected in front of input eigenvectors Ignore this selection if you have selected Procrustes Press on the settings button to open the Setting dialog box Make sure you enter appropriate values for the three reference points left right and third based on your knowledge about the configuration of shape data under study You may need to use the Display Tool to view data samples and highlight landmarks in order to choose these reference points If you wish to experiment with a subset of data samples enter the number of samples as well I
69. ed by the final refinement and then move it to the closest surface as defined by the Bone threshold option in the Manual Landmark tool The final refinement can be run by clicking the Gauss conv button A progress bar will be displayed however for small Gaussian kernels the process will be completed relatively quickly The point locations predicted by the final refinement will be copied into the current landmark list replacing any coordinates already stored there A current landmark list must exist in the software i e at least a landmark point names file must have been loaded The type code of each point will be changed to 2 so that the user can easily see which points in the landmark list have been automatically estimated 13 7 Outlier Detection With careful database construction the automatic point location software is capable of achieving point location errors equivalent to those from manual point identification for 95 of the points in typical landmarking experiments However occasional outliers will remain and so the user is expected to manually inspect automatic point locations and refine them where necessary this should be much faster than manually identifying all points The outlier detection stage of the software provides a guide to this process attempting to identify these outliers and indicate them to the user Lacking a model of shape variation between the image volumes in the database and the current volume no stag
70. een that there are several landmarks for which the error estimates are much larger than expected We cannot argue for an increased model order as this then reduces other values to well below the observed repeatability over fitting As the additional variance seen is due to the inability of the model to predict correlations in the data our conclusion must be that either this data is not well described by a linear model or the repeatability estimate systematically underestimates the true accuracy with which points can be meaningfully located This can happen if local image features which are themselves not well biologically related to the main structures such as the brightest pixel are used to identify locations 63 O major eigen values 5 components O major eigen values 6 components gt major eigen values 7 components Figure 24 Mouse 2D mandible data major error eigenvalues estimated using our 5 6 and 7 component models based on MM1 against those computed using the corresponding repeatability test the two dashed lines show the 2 80 range 64 Box A In order to perform shape analysis on a mark up data set using Likelihood 1 2 9 Select data dimension by pressing 2D or 3D Press scan button to browse in your computer disk and locate the directory and then the TPS file you wish to use as input mark up data Press input samples button Check the messages warnings appearing on th
71. ems with gimbal lock The Centroid error field provides an estimate of the error on the point based stage of global registration using the distance in voxels in the coordinate system of the current volume i e including the effects of any down sampling applied during data loading between the centroids of the registration point sets in each volume The Chisq field gives the x per degree of freedom at the end of the image based stage of global registration which provides an estimate of how well the transformed full sized patches through the centre of the current volume model the equivalent patches stored in the database entry The G reg point choice list allows information about the G reg points to be displayed if any are stored The options G1 G2 G3 and G4 select which point will be displayed and the No Name x y z and Type fields in the next row of the tool display the point number name coordinates and type code The next section of the dialog displays information about the individual landmark points contained in the database entry The List Length field displays the number of points As in the Landmark Points dialog box information about five points is displayed simultaneously and the user can scroll through the list of points using the Up and Down buttons For each point the point number name and type code are displayed The x y a
72. en in the Landmark Points dialog box These are used by the software to represent information about the point and should not in general be changed by the user Point type codes 1 NULL point not yet allocated 0 Point loaded from TPS NTSYS names file 1 Point with manually specified coordinates 2 Point with automatically identified coordinates 3 9 Automatically identified point flagged as an outlier 101 First axis point 102 Second axis point 103 First plane point 104 Second plane point 105 Third plane point 106 First global registration point 107 Second global registration point 108 Third global registration point 109 Fourth global registration point 31 8 Manual Landmark Identification The aim of the Manual Landmark tool is to allow the user to manually specify morphological landmarks in any 3D medical image volume that has been loaded into TINA via the Sequence tool In order to do this the user must manipulate the position of the 3D cursor until it corresponds to the desired point and then store the coordinates of the 3D cursor When data is first loaded the 3D cursor if initialised at the centre of the volume Its position is displayed graphically in all TVs as the red cross hair Its coordinates are also displayed numerically in the X Y and Z fields of the Manual Landmark tool The position of the 3D cursor can be manipulated in a number of ways e Numerical values can be entered directly
73. ence tool into alignment with the entries in the automatch database 13 2 1 Initial Point based Global Registration Image based registration algorithms cannot reliably handle gross misalignments for example if the current vol ume is rotated by approximately 180 degrees compared to those stored in the database Therefore an initial approximate stage of alignment is required prior to image based registration In the current version of the software see Section 18 this requires the user to mark four landmark points in each volume the first stage of the global registration is based on aligning these points by minimising the sum squared distances between corresponding points using the simplex optimisation algorithm These points must span all three dimensions of the space and be well separated in order to provide an accurate alignment i e they must not be co planar The points can be landmark points i e points in the current landmark list simply mark up any four or more of these and the software will calculate the initial global registration by aligning them to the corresponding points in the database Alternatively the user may wish to use points not included in the landmark point list for the global alignment The four G reg points provide this functionality they can be manually identified in the same way as any other landmark point using the G1 G2 G3 and G4 selections in the markup choice list The 3D Tv draw check
74. eration of the automatic point location software e Load the data set in which landmarks are to be automatically located the automatch database to be used and the landmark points names file e Deselect the Point based option in the Reg stages check list of the Automatic Landmark Point Placement tool and select the Image based option e Run the automatic landmark location software by clicking on Global Reg Automatch Gauss conv and Error analysis in turn in the Automatic Landmark Point Placement tool e Select both the Point based and Image based options in the Reg stages check list of the Automatic Landmark Point Placement tool e Select LM based in the Point based reg type choice list of the Automatic Landmark Point Placement tool e Run the automatic landmark location software a second time by clicking on Global Reg Automatch Gauss conv and Error analysis in turn in the Automatic Landmark Point Placement tool e Check and correct any remaining outliers using the procedure given in Section 13 8 Prior to using the software in this way users should check that the specimens in their scans are well enough aligned by constructing an automatch database then loading a few example scans and running the global registration stage of the software checking the results as described in Section 13 4 TINA incorporates a macro system that
75. ering Computer Graphics 22 4 65 74 1988 R L Gregory Perceptual illusions and brain models Proc Roy Soc Lond B 171 1024 279 296 1968 P Lacroute and M Levoy The Volpack volume rendering library http graphics stanford edu software volpack P Lacroute and M Levoy Fast volume rendering using a shear warp factorization of the viewing transform In Proc SIGGRAPH 794 July 24 29 Orlando Florida pages 451 458 1994 S I Olsen Estimation of noise in images An evaluation CVGIP Graphical Models and Image Processing 59 319 323 1993 W H Press B P Flannery S A Teukolsky and W T Vetterling Numerical Recipes in C Cambridge University Press New York 2nd edition 1992 H Ragheb and N A Thacker Estimating anisotropic measurement errors on landmarks http www tina vision net docs memos 2012 001 pdf H Ragheb and N A Thacker Estimating anisotropic measurement errors on landmarks Extension from 2D to 3D http www tina vision net docs memos 2011 009 pdf H Ragheb and N A Thacker Morphometric shape analysis with measurement covariance estimates http wiw tina vision net docs memos 2011 006 pdf A C Schunke P A Bromiley D Tautz and N A Thacker TINA manual landmarking tool software for the precise digitization of 3D landmarks Frontiers in Zoology 9 6 2012 N A Thacker and P A Bromiley TINA 5 0 User s Guide http www tina vision net docs memos 2005 002 pdf 82
76. es are the correct ones for the specific settings provided for the specific experiment The files CovarianceFile WhiteData MonteCarlo MeanShape EigenVectors Eigen Values ChisqAdjustFile and ChisqTest only correspond to the Likelihood analysis and are not generated used when working with Procrustes TPS This is a common file format in geometric morphometrics for reading and writing mark up data and our software expects this format when reading samples from the input file while the file name is arbitrary The file extension must be in capital letters TPS SampleLabels This file contains the labels IDs corresponding to the input samples received by the software This file is generated when new mark up data are fed to the software by pressing the input samples button These labels will be used later when generating the Monte Carlo and the white data files Outliers The list of potential outliers is printed on the Tinatool dialog box where each sample number is between one and the number of samples and each landmark number is between one and the number of landmarks The 60 threshold that effects the extent of this list is outlier threshold in the Settings dialog box There will be no list of potential outliers when working with Procrustes CovarianceFile The covariance estimates are written to a text file called CovarianceFile cvr where the number of lines is equal to the number of landmarks and the number of col
77. f for example a simple threshold is used then this is equivalent to the assumption that all bone surfaces in the volume have a well defined grey level At any point where this assumption fails e g where the bone is thin and so obscured by partial voluming is of low density or is corrupted by image noise errors will be generated in the rendering e g pseudofenestrations holes in the rendered bone surface that do not exist in the original data The TINA Manual Landmark tool uses a volume rendering algorithm in order to avoid such problems Volume rendering produces an image using the entire 3D volume each pixel in the rendered image represents a vector through the 3D volume and the renderer runs along these vectors converting the intensity and intensity gradient of every voxel in the 3D volume into opacity and reflectivity and combining these together to produce the intensity of the final pixel in the rendered image The final rendered image thus provides a more complete view of the data than a surface rendering at the expense of significantly more processing In order to produce volume rendered images in interactive time the TINA Manual Landmark tool uses a fast volume rendering algorithm known as the shear warp algorithm The operation of this algorithm is illustrated in Fig 13 Projecting along vectors from some arbitrary viewpoint would involve complex geometrical calculations in order to find which voxels were intersected by which vector
78. f the 3D cursor by the number of pixels spec ified in the resolution choice The number of voxels by which to move the 3D cursor when the arrow keys are pressed Specify an intensity value that corresponds to bone surface Display of the current landmark in the landmark list Move to the start of the landmark list Move to the previous point in the landmark list flagged as an outlier after automatic landmark point location Move up by five points in the landmark list Move to the previous landmark point in the landmark list Move to the next landmark point in the landmark list Move down by five points in the landmark list Move to the next point in the landmark list flagged as an outlier after automatic landmark point location Move to the end of the landmark list The number of the current landmark point A text description of the current landmark point The type code of the current landmark point Choose which point to mark up when the Mark point button is pressed Curr LM the current landmark from the landmark list Ax 1 the first axis point Ax 2 the second axis point Pl 1 the first plane point Pl 2 the second plane point Pl 3 the third plane point G1 the first G reg point G2 the second G reg point G3 the third G reg point G4 the fourth G reg point If this switch is on then when the selection in the markup choice list is changed or when the choice of current landmark is
79. f the light 1 0 to 1 0 The z component of the direction vector of the light 1 0 to 1 0 The red component of the colour of the light 0 0 to 1 0 The green component of the colour of the light 0 0 to 1 0 The blue component of the colour of the light 0 0 to 1 0 Reset all options to the default of a single white light shining down the z axis Implement any changes to the lighting options 74 21 5 Landmark Points Dialog Box Up 5 Last outlier Start No Name X y z Type Link no Down 5 Next outlier End A P G point Name X y Z TPS NTSYS ID Field no Clear axis Clear plane Clear G reg Clear links Clear LM Input format Move upwards in the list of landmark points the next five rows of fields show the information stored for the landmark points the middle row of the five is the current landmark Move upwards by five points in the list of landmark points Move to the previous point in the landmark list that is flagged as an outlier after automatic landmark point location i e has a point type code of 3 or greater Move to the start of the landmark point list Landmark point number Landmark point name Stored x coordinate for the landmark point Stored y coordinate for the landmark point Stored z coordinate for the landmark point An integer used to to store additional information for the landmark point The point that this point is linked to when
80. f this image displayed in green is the full sized y z slice through the centre of the image volume stored in the current automatch database entry i e the entry currently displayed in the dialog box The imaginary part displayed in red is the corresponding slice through the current volume obtained using the transformation model optimised during the global registration The two images should be approximately aligned they will not align exactly due to shape differences between the specimens but should be in the same approximate orientation and of the same overall size Any gross misalignment indicates that the shape differences between the specimens are too great to allow a good alignment i e the specimen stored in the database cannot be used to automatically identify landmark points in the current volume e Repeat the previous step for the RY and RZ image patches e Repeat the process for each entry in the database if there are not at least 6 7 specimens in the database with a good alignment to the current image volume then the automatic landmark point location software will perform poorly If persistent problems occur with global registration consider constructing a database from specimens more similar in shape to those that will be automatically analysed or using a different selection of points in the initial point based stage of the global registration 13 5 Coarse to fine Local Registration The global registratio
81. fied by the mouse cursor However this is not done using the 3D renderer instead it is done by searching through the original 3D image data as contained in the Sequence tool until a voxel with an intensity equal to or greater than the intensity specified in the Bone threshold field of the Manual Landmark tool is found None of the options contained in the VR Control dialog box are used in this process and so the user is free to reset them in order to produce the best possible rendering of the data without this process having any effect on subsequent analysis of the landmark points generated using the software Significant user experimentation with the options displayed in the VR Control dialog box may be required in order to produce the best possible 3D display of the data Therefore the software provides the ability to save load all of the currently selected volume rendering and lighting options to from a file The pathname of the file can be entered into the Options field at the bottom of the VR Control dialog box either by typing the pathname directly or by using the file browser started with the Scan button The Load and Save buttons then load or save the options to or from the file Several example volume renderer options files are available in the extra_files sub directory of the max planck toolkit directory that is created when the Geometric Morphometrics Toolkit is extracted e tmlt_surface_style txt the optio
82. field When a sequence is loaded the current slice is set to be the first slice in the sequence Pressing the gt button increments the current slice number i e moves forwards through the sequence Pressing the lt button decrements the current slice number i e moves backwards through the sequence The First and Last buttons set the current slice to be the first and last slice of the sequence respectively To view a specific slice enter the slice number into the Curr frame field and press the Jumpto button Individual image slices can be manipulated using the buttons at the bottom of the Sequence tool The Push button copies the current slice to the top of the Imcalc stack see the TINA User s Guide 19 where it can be processed or passed to other tools The Ins button takes the image from the top of the Imcalc stack and places it into the sequence after the current slice if no sequence is loaded then a new sequence will be created with the image as the first slice The Rep button takes the image from the top of the Imcalc stack and replaces the current slice of the sequence with it The Del seq button deletes the entire current sequence allowing it to be replaced with other images from the Imcalc stack The Stack gt Seq button will take all of the images currently held in the Imcalc stack and use them to create a new sequence any current sequence is deleted in this process the images wil
83. h enamel and air space compared to the interface between soft tissue and bone The software has been designed to avoid consequent problems e g by encouraging the user to refine manual landmark points using the 2D TVs in order to ensure that the use of a simple threshold does not result in any bias on the landmark locations TINA includes an advanced medical image segmentation algorithm 6 7 8 1 that could be used in the first instance to set the bone threshold automatically However it could be used in a more advanced fashion to analyse the intensity profile along any vector through the image data during landmark point selection using the 3D TV in Pick mode and automatically locate interfaces between any pair of tissues removing the need for a simple intensity threshold and avoiding any consequent bias on landmark point locations Finally the optimised tissue model produced by the TINA medical image segmentation algorithm could be used to enable the multi material rendering options in the Volpack library allowing for example soft tissues to be displayed as a transparent surface in a different colour above the bone surface in the 3D rendering This would allow the user to consider biomechanical aspects of the specimen such as the size of particular muscle groups 55 Part 3 The Shape Analysis Tool H Ragheb and N A Thacker Imaging Science and Biomedical Engineering Division Medical School University of Manchester Stopford B
84. he Output AM Database button Note that database files are in a binary format and cannot be manually edited In order to load a previously prepared database enter the path and filename into the Output field and click the Input AM Database button any database currently loaded into the software will be replaced The database currently loaded into the software can be freed by clicking on the Free Database button The contents of the database can be examined using the Automatch Database Inspector dialog box see Section 17 Additionally the current entry in the automatch database i e the one displayed in the Automatch Database Inspector dialog box can be deleted by clicking the Free curr entry button in the Automatic Landmark Point Placement tool this allows correction of errors during database construction 41 13 2 Global Registration The global registration consists of two stages an initial landmark point based stage and a final image based stage These are described in detail in the following sections The Reg stages check list determines which stages will be run in general both Point based and Image based should be selected however see Section 15 for an alternative way to operate the software Global registration is performed by clicking on the Global Reg button the software will then proceed to optimise transformation models that bring the image volume currently loaded into the sequ
85. he list of automatically located landmark points is as follows e Switch the Jump lock option in the Manual Landmark tool to On e Scroll to the start of the landmark list by clicking the Start button in the Manual Landmark tool or the Landmark Points dialog box e Click the Next outlier button The current landmark will be advanced to the first landmark in the list that has been identified as a potential outlier indicated by a type code of 3 or greater Since the Jump lock option is on the 3D cursor will be moved to the automatically identified position for this landmark and all TVs will be updated accordingly e Inspect the indicated position of the landmark If an error has occurred move the 3D cursor to the correct position using the arrow keys in the Manual Landmark tool or by using mouse or keyboard interaction with the images in the TVs e Click the Mark Point button in the Manual Landmark tool or use the keyboard shortcut assigned to this button in any TV The corrected coordinates of the 3D cursor will be transferred to the landmark point and the point type code will be changed to 1 to indicate that the point has been manually identified Note that even if the point was in the correct position and has not been adjusted clicking on the Mark Point button in the Manual Landmark tool or using the keyboard shortcut assigned to this button in any TV will update the type code to 1 to indicate
86. he point has fitted correctly The registration will abort if the global registration has not been performed i e there are no stored global transformation models in the database All stored transformation models will be deleted if a new image volume is loaded into the Sequence tool since they will no longer be valid However if the automatch database is stored to file then the results of all stages of registration are stored in that file This allows all registrations to be optimised and stored for a specific current volume facilitating rapid testing of the final refinement and error detection stages of the software However it also permits the situation where the user saves a database containing global and local registration results for a specific current image volume loads a different volume into the sequence tool and then loads the database for the original current volume ending up with a set of global and local registrations for the wrong current volume The software has no way to detect this error and so the user must be careful to avoid it 13 6 Final Refinement of Landmark Estimates Once the global and local registration stages have been completed the database will contain multiple estimates of the position of each point in the landmark list one for each entry in the database The purpose of the final refinement stage is to combine these estimates into a single hypothesised location for the landmark the coordinates of which can then be e
87. he two dashed lines show the 2 80 statistical allowable range 1 st error eigen values O 2 nd error eigen values D gt 3 rd error eigen values Figure 21 Original Apodemus mandible 3D data 87 samples 20 landmarks error eigenvalues estimated using the Monte Carlo data against the expected ones which were used when generating the simulated data independent models using 5 model components the two dashed lines show the 2 80 statistical allowable range The outputs written in the result file could be used to generate another useful plot We have demonstrated in a previous report 15 how linear model order selection can be performed by comparing baseline reproducibility errors with those estimated from the model In Fig 24 we plot the eigenvalues corresponding to the errors estimated 62 1 st error eigen values O 2 nd error eigen values D gt 3 rd error eigen values Figure 22 Apodemus mandible 3D data after outlier removal 85 samples 20 landmarks error eigenvalues estimated using the Monte Carlo data against the expected ones which were used when generating the simulated data independent models using 5 model components the two dashed lines show the 2 80 statistical allowable range Fs ge ao Figure 23 Mouse 2D mandible data 337 samples 14 landmarks error bars x20 computed from repeat data against those computed from the repeat data Fig 23 It can be s
88. ialog Box Render quality Render type Scalar classification Gradient classification DQ Front Fac DQ Density Depth cueing Off On Ambient R G B Diffuse R G B Specular R G B Shininess RGB Lighting Control Foreground colour Background colour Options Scan Load Save Set the balance of speed vs rendering quality by ignoring low opacity voxels the recommended option is Fast Choose the type of rendering Opacity show an image of opacity transformed intensity only Greyscale show both opacity and surface shading transformed gradient Pseudo colour show a greyscale image in which the object is tinted with the Fore ground colour and the background is filled with the Background colour Full Colour give the renderer full control of image colour this allows the use of coloured lights and material properties A user defined curve controlling the transformation from intensity in the original data to opacity in the rendered image spline produce a spline curve linking the control points linear produce a piecewise linear curve linking the control points reset reset the curve to a linear ramp apply implement the changes made to the curve A user defined curve controlling the transformation from intensity gradient in the original data to surface shading in the rendered image spline produce a spline curve linking the control points linear produce a piecewise line
89. id false negatives For example if the Loc patch size 3 parameter is set to 10 but the sequence was loaded with Stride and Downsample set to 2 then the error detection thresholds should be set to around 20 The outlier detection requires that all previous stages of the automatic point location have been applied and will not operate if any manual alteration of the automatically located points has been performed This is enforced by 46 checking the type code of all points in the current landmark list they must all be of type 2 The user can set these type codes through the Landmark Points dialog box to work around this check but does so at their own risk 13 8 Correction of Outliers The parameters of the outlier detector should be tuned to provide a minimum number of false positives i e points that whilst in the wrong location are not flagged as outliers This reduces the probability that erroneous points will be passed to subsequent Procrustes analysis but inevitably means that a large number of false negatives will be generated i e points that whilst in the correct location are flagged as outliers However such points require only brief inspection by the user and an alteration of their type code to indicate that they are in the correct position This can be done efficiently by making use of the Jump lock option in the Manual Landmark tool A complete set of steps required to efficiently correct outliers in t
90. identified in the software Note that neither the white data nor the Monte Carlo data are in the original coordinate system Hence these files may contain negative coordinates MeanShape The file MeanShape mrk contains the coordinates of all landmark points in the mean shape written in one single row Hence there are two components x and y for each 2D landmark and three components x y and z for each 3D landmark Here the order of landmarks is identical to the original input data EigenVectors The file EigenVectors egv contains the coordinates of all the eigenvectors used to construct the linear model The number of rows is equal to the number of model components set in the Setting dialog box Each row contains normalised values of the corresponding eigenvector Note that these vectors are in the whitened space and not in the original coordinate system EigenValues The file EigenValues egv contains single eigenvalues corresponding to the eigenvectors used to construct the model The number of eigenvalues is equal to the number of model components set in the Setting dialog box These values are all written in one single row ChisqAdjustFile The software stores one adjustment factor per landmark per line to be taken into account when if a x test is performed later on The file containing these adjustment factors is called ChisqAdjustFile adj ChisqTest A x test is performed when the user uses the covariances estima
91. ignment is a placeholder used during software development since it allows rapid testing of the software when automatically identifying landmarks for comparison to manually identified gold standard landmark locations However it is arguably not the ideal method for user interaction with the software Fortunately it would be possible to implement a much more convenient method The user could be prompted to rotate the view in the 3D TV to a fixed orientation it does not matter what this orientation is as long as it is repeatable across different specimens to reasonable accuracy The viewing transform could then be obtained call get viewing transform in tlmedMousetool volpack interface c and stored in the automatch database The combination of the viewing transform from the database with one from the current volume would provide the rotation parameters for an affine registration of the two it would then be trivial to obtain the translation and scaling in an image based registration limited to only those parameters Alternatively the bone threshold in the Manual Landmark tool could be used to identify a set of surface points the centroid and standard deviation of which would provide the translation and scaling parameters A second image based stage of global registration could then be performed to refine the transformation model This procedure would avoid the need to mark up any landmark points prior to automatic point location 18 2 Covariance Esti
92. increment the current image Set the last image in the sequence as the current image Set the image specified in the Cur frame field to be the current image Select the method used to down sample in the inter slice direction if Stride is set to a value greater than 1 Off down sample by skipping over slices On down sample by loading all slices and applying linear averaging Delete the current sequence from memory Delete the current frame from the sequence Take the image from the top of the Imcalc stack and insert it into the sequence after the current image 68 Rep Take the image from the top of the Imcalc stack and insert it into the sequence replacing the current image Push Copy the current image to the top of the Imcalc stack Stack gt Seq Take the entire Imcalc stack and produce a new sequence with it in reverse order 69 21 2 Manual Landmark Tool VR Control Landmark List Keyboard Controls Automatch Shape Analysis Tv 3D Tv 3D Tv Mouse 2D Tv Mouse 3D Tv draw 2D Tv draw 3D Cursor Position Starts the Volume Renderer Control dialog box which allows the user to set the various options used to produce the image in the 3D TV Starts the Landmark Points dialog box which allows the user to load view and save lists of landmark points Starts the Keyboard Controls dialog box which allows the keyboard controls for interaction with the TVs to be re defined Star
93. indow Behaviour tab from the menu on the left Select the Policy drop down menu in the Focus box Select Focus follows mouse from the drop down menu e Click the Apply button at the bottom of the window In order to switch to focus follows pointer mode in Suse 11 3 e Click on the start button in the bottom left corner of the screen select System Settings Select the General tab at the top of the System Settings dialog Select the Window Behaviour tab from the Look and Feel section of the dialog Select the Window Behaviour tab from the menu on the left hand side of the dialog Select Focus follows mouse from the Policy drop down menu Click the Apply button at the bottom of the window The window manager focus will now follow the mouse pointer These instructions are valid for openSuse using the KDE desktop Other window managers may vary see the instructions for the window manager you are using if necessary The keyboard shortcuts can be reassigned to any set of keys with the exception of a few reserved by the window manager e g the print screen key using the TV Keyboard Controls dialog box see Section 9 Additional display functionality can be accessed via the 2D Draw check list in the Manual Landmark tool If Axis is selected then any currently specified axis points see Section 8 1 will be displayed as small yellow cross ha
94. ing the covariance estimates with the ones from reproducibility studies based on repeat manual mark ups 5 Using the anisotropic measurement covariance output data as input data to process independent repeat data where x ratios are used to assess statistical equivalence 6 Using the list of potential outliers detected in the data to identify problematic data samples for subsequent elimination or manual correction 7 Processing Monte Carlo data using the model the mean and eigenvectors together with the covariance estimates used when generating them to generate plots of expected versus obtained measurement errors This is the less challenging Monte Carlo test 8 The Monte Carlo data can also be processed by constructing an independent model and estimating covariances accordingly This is the more challenging Monte Carlo test 20 Software Guide In what follows we outline how to use different dialog boxes and buttons to perform shape analysis experiments 20 1 Shape Analysis Tool In this section we describe how to work with the buttons appearing on the Shape Analysis Tool dialog box data dimensions One must select the dimensionality of the input data before pressing any other buttons This tells the software whether each landmark is 2D x and y components only or 3D x y and z components The default dimension is 3D If 3D is selected but a 2D data file is provided data will not be accepted However if 2D is
95. ingle parameter Valid parameters for the ambient diffuse and specular RGB components are in the range 0 0 to 1 0 the shininess parameter should be in the range 1 0 to 100 0 The colouration of surfaces will only be visible when the renderer is in full colour mode in greyscale or pseudo colour modes the rendered image is produced using only the overall intensity of the reflected light i e the intensity of each of the red green and blue components combined Although the Volpack library provides the ability to assign different material properties to different tissues using the techniques described in 9 the current version of the Manual Landmarking tool assumes a single material type Changes to the material properties are not implemented in the rendered image until the next time it is regenerated the user can force the image to be rendered with the new parameters by clicking the Repaint button in the TV tool displaying the 3D TV It is important to note that none of the options in the VR Control or Lighting Control dialog boxes will affect the accuracy of landmark point placement except to the extent that they affect the user s ability to see where the landmark point should be placed When the user selects a point in the 3D image by placing the mouse cursor over that point and then left clicking with the Pick mode of the 83D Tv Mouse function selected the 3D cursor is moved to the first bone surface lying under the point speci
96. ion The software implements a number of tests on the point based stage of global registration These will ensure that at least four points are available and that they are sufficiently non coplanar In addition the optimal alignment of any sets of points should bring the centroids of the point sets into alignment This could be used to calculate the translation parameters of the transformation model directly without optimisation However the software actually optimises all nine parameters of the transformation model This allows the distance between the point set centroids after alignment to be used as an error estimate it is displayed in the Automatch Database Inspector dialog box after global registration has been performed and should in general be less than one voxel 13 2 2 The G reg Point Filler The G reg point filler provides functionality to copy locations from four points in the current landmark list to the four G reg points Enter the numbers of the landmark points to be copied into the Greg 1 Greg 2 Greg 3 and Greg 4 fields in the Automatic Landmark Point Placement tool and then click the Fill G reg points button If any of the specified points cannot be copied e g a non existent landmark point number is entered then a warning will be issued The G reg point filler can be used to accelerate the automatic landmark point placement process in two ways First 42 the user is required to manually identif
97. ion and error messages 3 TVs and TV Tools X Tv Tool1 yax Size Mouse ROI Proj install clone init repaint null Figure 1 A TV Tool The TINA image display system is designed to provide the maximum range of configuration options to the user It consists of two components TVs and TV tools The majority of the TINA sub tools will have a choice list at the top of the sub tool window labelled Tv Each button in the list represents an available graphics channel however TVs are not displayed by default in order to save space on the screen In order to display a TV graphics channel it must be associated with a TV tool e Start a new TV tool by pressing the New Tvtool button in the top level TINA tool A new TV tool window will appear on the screen as shown in Fig 1 e Select the TV you wish to display by left clicking on its field in a Tv list Note that since multiple lists are available in various TINA sub tools this may be required even if it appears that the desired field is already selected e Click the install button in the new TV tool The title of the TV tool will change to the name of the TV it is displaying Once a TV has been installed on a TV tool the TV tool will display the graphics produced by that TV auto matically updating whenever the graphics change without further user interaction TV tools can be minimised or closed at any time without affecting algorithmi
98. ior nasal spine 45 Intersection of the right occipital condyle and the foramen magnum taken at the lateral most curvature right side 46 Intersection of the left occipital condyle and the foramen magnum taken at the lateral most curvature left side 28 An example landmark point links file for mouse skulls corresponding to the names file above 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 38 41 39 42 40 43 45 46 Names files and other files containing landmark points are loaded and saved using the Landmark Points dialog box which also displays the currently loaded list of landmark points and provides some basic tools to manipulate it This dialog box is started by clicking on the Landmark List button at the top of the Manual Landmark tool and is shown in Fig 14 In order to load a landmark names file e Prepare the names file in advance using any text editor Optionally prepare the corresponding linklines file e In the Landmark Points Dialog box enter the absolute or relative pathname of the landmark names file into the Input Pathname field either directly or by using the file browser started by clicking on the Scan button e Select the format of the file in this case Names from the Input format choice list e Click the Load button in the Landmark Points dialog box Once a names file has been lo
99. irs in the 2D TVs Similarly Plane will display any currently specified plane points see Section 8 1 as small cyan cross hairs Note that these are projected onto the TVs so that they are displayed even if they do not lie in the plane of the image 16 The 2D Draw check list in the Manual Landmark tool also provides two options linked to the display in the 3D TV see 5 2 2 If Rotate is selected then any rotation that the user applies to the image displayed in the 3D TV will also be applied to the images displayed in the 2D TVs This allows the user to align the 3D image in a specific way for example looking along the anterior posterior axis of the specimen with the plane of bilateral symmetry aligned with the vertical axis of the image and then apply this rotation to the images shown in the 3D TVs to provide views of the data that are easier to interpret However any subsequent rotation of the image in the 3D TV will also be applied to the images in the 2D TVs The Lock rotation option modifies this behaviour so that any rotation applied to the image displayed in the 3D TV at the time the lock rotation option is selected is stored and used for all subsequent drawing operations on the 2D TVs until the lock rotation option is deselected The image in the 3D TV can then be manipulated without disrupting any useful alignment that has been achieved in the 2D TVs The Lock Rotation option over rides the Rotate option
100. is is to prevent the user from saving sequences where essential header data is not available or is no longer valid due to processing that has been applied to the images In order to save a tomographic image data set e Enter the pathname of the image file files into the Image file field of the tool e Select the format of the image file from the File choice list AIFF RAD ANLZ and PGM are supported NEMA and DICOM are not supported For file types e g AIFF where each slice is contained in a separate file use the character as a wildcard For example if the sequence contains 10 slices to be saved to files named im 00 to im 10 enter the pathname of the first file into the Image file field Then replace the part of the name that changes between files with the character so that it becomes im During image saving the wildcard characters in the pathname will be replaced with the slice numbers of the images in the sequence If the numbers in the filenames increase by more than 1 between each file then the Stride field can be used to specify the increment For example entering im into the Image file field and 2 into the Stride field will save the images im 00 im 02 im 04 etc Note that there are some restrictions on saving multiple files in this way The substitution only works for numbers can only be applied to one contiguous segment of the pathname and only works in ascending or de
101. isplays lines linking these pairs of points This provides a quick visual check at the end of the manual landmarking process the lines should approximately be parallel and if this is not the case it may indicate that the landmarks have not been identified in the correct order e g a pair of points has been transposed In order to use this option a file listing the links must be prepared Each line of the file contains two landmark point numbers corresponding to the landmark point numbers in the names file separated by whitespace spaces or tab characters The file must have the same filename as the landmark names file and must have the extension llf e g if the names file is called points txt then the linking lines file must be called points llf It must also be placed in the same directory The linking lines file will then be automatically loaded or saved whenever the names file is loaded or saved Lines will then be drawn in the 3D TV between the specified points whenever the Linklines option is selected in the 3D Tv draw check list and coordinates have been specified for both points An example linklines file is shown below 27 An example landmark point names file for mouse skulls 1 Most anterior point of the anterior palatine foramen left side 2 Most anterior point of the anterior palatine foramen right side 3 Intersection of frontal process of maxilla with frontal and lacrimal bones left side 4 Intersection of
102. l be placed into the sequence in reverse order i e the image on the top of the stack will become the last image in the new sequence In order to load a tomographic image data set e Enter the pathname of the image file files into the Image file field of the tool either directly or using the file browser started by pressing the Scan button For AIFF RAD NEMA and PGM file types the sequence tool will automatically add the correct file name extension aiff rad ani and pgm respectively and so the filename should be entered without the extension However for Analyze or DICOM files the sequence tool will not automatically add an extension and so the correct extension must be entered into the filename field The file browser started using the Scan button will automatically strip the extension from any selected file and so when loading DICOM files the extension must be manually re entered into the filename field e Select the format of the image file from the File choice list For file types e g Analyze where multiple slices of data are contained within a single file enter the number of slices in the file into the End field If you are unsure of the number of slices then enter a number that is too high the Sequence tool will load data until it reaches the end of the file and then reset the End field to show the number of slices loaded For file types e g AIFF where each slice is
103. landmark Proceed through the landmark list marking up each point in turn until the coordinates of all points have been specified Save the landmark list to file using the Landmark Points dialog box see Section 7 At this point the user can load another 3D image volume and repeat the process Note that the previous landmark list will still be in memory and so can be used as starting points if the data sets are similar enough or can be deleted using the Clear Landmarks button in the Landmark Points dialog box allowing the names list to be re loaded 37 Part 2 The Automatic Landmark Point Placement Tool P A Bromiley Imaging Science and Biomedical Engineering Division Medical School University of Manchester Stopford Building Oxford Road Manchester M13 9PT 38 12 Introduction A utomatic Landmark Point Placement Automatch DB Inspector Storage patch size 100 Glob border 15 Landmarks gt database Free database Free curr entry Global Registration Reg stages Point based Image based Glob sig 5 0 Point based reg type LM based G_reg based Greg1 2 Greg2 14 Greg3 27 Greg4 39 Fill G_reg points Global Reg Projection AIII Snap LM projection Local Registration Loc patch size 1 40 Loc sig 1 20 0 Loc patch size2 20 Loc sig 2 10 0 Loc patch size3 10 Loc sig3 5 0 Automatch Refinement Snap points Off On Chi sq weight Off On Fin sig 5 0 Gauss conv Outlier Reje
104. landmark information in the Landmark Points dialog box A list of landmark names must already have been loaded in order to do this otherwise the software will issue a warning that no landmark list has been loaded After transferring the coordinates the software will also increment the current landmark i e move to the next one in the landmark list so that the user does not need to scroll through the landmark list during landmarking The other choices in the Markup list allow the user to specify axis plane and G reg points see Sections 8 1 and 13 2 1 The Jump lock switch and the Jump stored button allow the user to re use coordinates of landmarks that have previously been identified If the user scrolls through the landmark list using e g the Up and Down buttons in the Manual Landmark tool or in the Landmark Points dialog box to change the current landmark to a landmark for which coordinates have already been stored and then clicks the Jump stored button the 3D cursor will be moved to the stored coordinates for the current landmark If the Jump lock switch is set to On then this becomes the default behaviour when scrolling through the landmark list the 3D cursor will automatically be moved to the stored coordinates of the current landmark whenever such stored coordinates exist The Snap button provides a basic snap to grid style functionality If the 3D cursor is not located on a bone surf
105. lighting control dialog box Viewing Rays Shear A A A Volume Slices project t warp 4 Image Plane a b Figure 13 Volume rendering a 3D data block from an arbitrary viewing direction results in arbitrary angles of intersection between the viewing rays and the slices of the 3D data a Shear warp volume rendering pre calculates an overall shear of the volume that makes the viewing rays parallel to one axis of the sheared volume b The sheared data is then projected along the parallel rays into a buffer which is finally warped onto the image plane to produce the rendered image This results in a much more favourable alignment of the data in memory vastly increasing rendering speed at the expense of a small reduction in the quality of the rendered image the direction of the light values between 1 0 and 1 0 are valid The final three components r g and b specify the colour of the light as normalised RGB components values between 0 0 and 1 0 are valid The default is to have a single white light shining down the z axis the Reset to defaults button resets all parameters to these default values Changes to the lighting parameters are not implemented until the Apply button at the bottom of the dialog box is pressed and even then have no effect unless the volume renderer is running If the Apply button is pressed with invalid parameters in the dialog box error mes
106. lled The header file should be picked up automatically by the compiler and the library location should be automatically identified if usr local lib is listed in etc ld so conf and if ldconfig has been run or the machine rebooted since Volpack was installed If these files cannot be located the build will fail with error messages about functions with names starting with vp e g vpRenderRawVolume these are the functions provided by the Volpack library In that case the locations of the Volpack library and header file can be specified manually to the configure command using the flags with volpack include PATH with volpack library PATH The executable file for the toolkit is located in the src subdirectory alternatively typing make install will copy it to the bin subdirectory In either case cd to that directory and type tinaTool to run the toolkit Upon typing tinaTool the top level TINA tool will appear on the screen This provides access to various sub tools that in turn provide access to the algorithmic functionality The sub tools of most interest for geometric morphometrics are the Manual Landmark tool itself and the Automatic Landmark Point Placement and Shape Analysis tools which can be accessed through the Manual Landmark tool the sequence tool used for loading medical image data and the TV tools used to display graphics The text window at the bottom of the top level TINA tool is used to display informat
107. mark tool is based around the concept of a 3D cursor a single point within the volume whose coordinates can be manipulated by the user in a number of ways The aim is to move the 3D cursor to the point the user wishes to mark and then to store the coordinates of the cursor in a list of landmark points When all landmark points have been specified in this way the list of points can be saved to a file for further processing e g Procrustes analysis and or added to the database used by the Automatic Landmark Point Placement tool 5 1 Definition of Coordinates The coordinate system used in the TINA Manual Landmark tool is derived from the original data and relates to the images displayed by the sequence tool as follows the z dimension is the inter slice dimension such that Z Zmin S the first slice and z Zmaz is the last slice Within each image the x dimension runs from left to right such that min is the left hand edge of the image and X naz is the right hand edge and the y dimension runs from top to bottom such that y ymin is the top edge of the image and y Ymax is the bottom This definition is visible to the user in several places such as in the definitions of the 2D TVs and in the landmark coordinates and is shown in Fig 4 When loading large medical image volumes it may be necessary to use the Downsample and Stride fields of the sequence tool depending on available memory In this case it should be note
108. mation The covariances of the parameters optimised in any maximum likelihood technique can be estimated using the minimum variance bound as long as the likelihood is properly normalised Properly normalised can be defined using 2inL x 3 i e normalised such that the distribution of 2 nL is given by the x distribution 4 2 3 5 Therefore the maximum likelihood optimisation can simultaneously provide optimised model parameters an estimate of the goodness of fit of the model in the form of the x per degree of freedom at the end of optimisation and an estimate of the errors on the optimised parameters in the form of the minimum variance bound Of course the errors given by this technique represent the extent to which the estimated parameters are destabilised by the noise on the data and will only correspond to the total error on the parameters if the model is a good fit to the data i e the residuals consist only of noise and the optimisation procedure has successfully located the global minimum of the cost function A parameter covariance calculation function based on the minimum variance bound is included in the software compute covar in thnedMousetool patchmatch c However calculation of the minimum variance bound can be problematic in practice for example in 2 3 5 it was found necessary to calculate the derivatives of the likelihood function over a range of values and then take the median in order to get a sta
109. n Box C we describe how to find an appropriate number for the model components In short the idea is to start from a relatively small number perform a repeatability test using the results increase the number while monitoring the repeatability plot until you reach the optimum number of model components Finally increase decrease the outlier threshold between 3 0 and 5 0 to get a shorter longer list of potential outliers Press the process data button in order to start the optimisation process Once the process is finished one can display the results see Section 2 3 and also use the output files to generate possible plots Whatever graphics which are displayed on the mono display may be stored as an image either using the Print Screen key usually as a png file or using the dump button on the view tool where the image name type may also be chosen The image from the former case would include everything displayed on the computer screen while in the latter case the image only contain the mono display In order to be able to plot the results later versus those from a Monte Carlo test or versus those from a repeatability test one must rename the output file called results txt to show that the error eigenvalues which are listed in the file correspond to the expected errors e g to results EXP txt Hence later when the second set of error eigenvalues are in hand the corresponding sets of numbers ma
110. n has already been loaded In such situations the lengths of the two lists will be compared If they are the same then the software will assume that the points are in the same order and will load all of the information contained in the new file into the relevant fields of the current landmark list If the lengths are different then the software will either refuse to load the new file or will discard the current landmark list and replace it with the information from the file always choosing the option that discards the least data i e if the user attempts to load a names file after a TPS or Raw file has been loaded the software will refuse to load the file if the user attempts to load a TPS or Raw file after loading a names file then the names will be replaced with those from the file However the Landmark Points dialog box provides a Clear Landmarks button that allows the current landmark list to be cleared and also provides Clear Axis Clear Plane Clear links and Clear G reg buttons to allow the user to discard any currently stored axis point plane points see Section 8 1 linklines information or G reg points see Section 13 2 1 30 In order to load a landmark list e In the Landmark Points Dialog box enter the absolute or relative pathname of the landmark file into the Input pathname field either directly or by using the file browser started by clicking on the Scan button e Select the fo
111. n if some of them have been manually identified un select this option to avoid overwriting landmark points that were manually located for the point based stage of the global registration Snap will take the mean projected location and then move it to the closest surface defined by the Bone threshold field in the Manual Landmark tool before entering it into the current landmark list The landmark projection can operate using only global registration results making it much faster than the full automatic point location procedure However it is not considered to be automatic point location since all results will have to be manually refined and so the point type code is not changed by this procedure 50 17 Automatch Database Inspector X Automatch database lt Entry 0 of 0 Name NULL Noise 0 0 List Length 0 gt Scale 1 0 1 0 1 0 Centroid error 0 0 Chisq 0 0 Ers 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 Et 0 0 0 0 0 0 G_reg point Gl G2 G3 G4 No 1 Name NULL Xi 1 0 y 1 0 n 1 0 Type 1 Up No 1 Name NULL Xi 1 0 yw 1 0 z 1 0 Type 1 Proj x 1 0 Proj y 1 0 Proj z 1 0 Chi2 global 0 0 Chi2 global local 0 0 No 1 Name NULL X 1 0 y 1 0 2 1 0 Type 1 Proj x 1 0 Proj y 1 0 Proj z 1 0 Chi2 global 0 0 Chi2 global local 0 0 No 1 Name NULL Xi 1 0 y 1 0 Z 1 0 Type 1 Proj x 1 0 Proj y 1 1 0 Proj z 1 0 Chi2 global 0 0 Chi2 global local 0 0 No 1 Name N
112. n provides an approximate alignment of the current image volume to those volumes repre sented in the automatch database This acts as an initialisation for a local registration of smaller image patches around each landmark point The local registration must both search the cost function for the global minimum and then refine the estimate of the global minimum for maximum accuracy These two objectives are in some ways diametrically opposed since efficient searching requires large patches and significant smoothing whereas maximum accuracy requires small patches in order to improve the fit of the model to the data and low smooth ing Therefore the local registration uses a coarse to fine strategy involving three stages with decreasing patch and smoothing kernel sizes The stopping criterion for the first two stages is less strict than for the third stage since only an approximate solution is required to initialise the following stage The local registration optimises only the translation and scaling parameters of the nine parameter transformation model i e does not optimise the rotation This avoids degeneracy when aligning landmarks located on surfaces of constant curvature for example a sector taken from a circle can be aligned to any other sector of that circle if rotation is allowed but will only match its original position if the transformation is limited to translation and scaling only The patch and smoothing kernel sizes can be controlled
113. nd z fields display the coordinates of the point in its original image volume these are in the original coordinate system of the data i e ignore any down sampling that was applied during data loading If any registration has been performed then the Proj x Proj y and Proj z fields display the coordinates of the point projected ol through the optimised transformation model i e the estimated coordinates of the corresponding point in the current volume The Chi2 global local field displays x per degree of freedom at the end of local registration the Chi2 global field displays the equivalent i e calculated from a patch of the same size as the one used in the last stage of global registration but using only the global registration parameters Any error score that has not yet been calculated has a default value of zero The last section of the dialog allows the user to inspect any image patches stored in the database The Push button will push the selected image onto the Imcalc stack allowing it to be viewed in the Imcalc TVs X Y and Z select the local patches for the current point of the current database entry the current database entry is the one being displayed the current point is the one displayed on the middle two rows of the ten rows of landmark point information in the form in which they are stored in the database DX DY and DZ select the local patche
114. ndmark points Three stages of this local registration occur with progressively smaller patches each time This is based on the concept that as the patch size is decreased the effect of shape differences between the database and current volumes is reduced i e the latter becomes a progressively better model of the former The global and initial local stages of registration therefore search the image volume to find approximate positions which are refined in the later stages of local registration to improve accuracy 40 13 Use of the Software Automatic landmark identification involves the following stages e Database construction Initial point based global alignment Image based refinement of global alignment Coarse to fine local alignment e Final refinement of landmark estimates Outlier detection These are described in detail in the following sections 13 1 Database construction Automatic landmark identification is based on a database of examples of manually identified landmarks which must be constructed by the user The database should contain typically around eight to twelve sets of manually identified landmarks In order to construct the database load an image volume and manually identify the list of landmarks or load them from file if available The user must also consider the initial point based stage of registration see Section 13 2 1 and so may wish to mark the four G reg points Once the coordinates of all lan
115. ne points Global reg display orange cross hairs for the G reg points Text display the number of each marked up landmark axis plane and G reg point Poly display a line representing the axis and a grid representing the plane Ball display 3D spheres instead of cross hairs for the landmark axis plane and G reg points Linklines display green lines linking symmetrical pairs of points Select how the images in the 2D TVs will be displayed Rotate display the 2D TVs using the rotation from the 3D TV Lock rotation display the 2D TVs using the rotation from the 3D TV at the time this option is switched on Subsequent rotation of the 3D TV will have no effect on the rotation of the 2D TVs until this option is switched off This option overrides the Rotate option Axis display yellow cross hairs for the axis points Plane display cyan cross hairs for the plane points Displays the coordinates of the 3D cursor 70 Resolution Bone threshold Current landmark Start Last outlier 5 Up Down 5 Next outlier End No Name Type Markup Jump lock Jump stored Axis lock Rotate to plane Rotate to axis X the x coordinate of the 3D cursor Y the y coordinate of the 3D cursor Z the z coordinate of the 3D cursor lt decrement the relevant coordinate of the 3D cursor by the number of pixels spec ified in the resolution choice gt increment the relevant coordinate o
116. ns 19 and 20 the Shape Analysis tool used to analyse morphometric landmark points Section 21 quick reference guides explaining the functions of each button data entry field etc in the Sequence tool Geometric Morphometrics toolkit and Geometric Morphometrics toolkit dialog boxes This manual covers the Geometric Morphometrics Toolkit v 2 0 which requires TINA 6 0 build number 008 or higher Some familiarity with the Linux OS is assumed e g how to start and use a shell tool Throughout this guide boxed sections contain detailed instructions on how to perform specific tasks with the TINA Geometric Morphometrics toolkit 1 1 Citation The authors would appreciate it if the following reference would be included in any published work that uses the TINA Geometric Morphometrics toolkit A C Schunke P A Bromiley D Tautz and N A Thacker TINA manual landmarking tool software for the precise digitization of 3D landmarks Frontiers in Zoology 9 1 6 2012 2 Installation Installation of the TINA Geometric Morphometrics toolkit is performed in three stages installation of the TINA libraries which provide the TINA machine vision functionality installation of the Volpack library which is used by the TINA Manual Landmark tool to produce 3D volume rendered images of tomographic image data and installation of the TINA Geometric Morphometrics toolkit which provides the Manual Landmark Automatic Landmark Point Placement and Shape analy
117. ns used in Fig 9 but with multiple coloured lights enabled similar to the options used to produce the image on the front page of this document e tmlt_printing txt similar to the options used in Fig 9 but with pseudo colour rendering enabled with a white background and with all landmark points displayed as spheres rather than cross hairs these options are useful for producing images to be incorporated into publications 6 1 The Lighting Control Interface The volume rendering engine supports up to six fully configurable light sources Fig 12 shows the lighting control dialog box which allows the parameters of the lights to be specified by the user The dialog box is created by pressing the Lighting Control button in the VR Control dialog box Each line of the lighting control dialog box controls one of the lights The buttons on the far left specify whether the light is switched on or off The next three fields x y and z specify the normalised x y and z components of 24 X Lighting Control 1 Off On x 0 0 y 0 0 N e g 2 Off On x 0 0 y 0 0 z 10 r 1 0 g 1 0 b 1 0 3 0fflOn x 0 0 y 10 0 z 1 0 a1 0 g 1 0 b 1 0 4 Off On x 0 0 y 10 0 z 1 0 r 1 0 g 1 0 b 1 0 5 Off On x 0 0 y 10 0 z 10 r 10 g 1 0 b 11 0 6 Off On x 0 0 y 10 0 z 1 0 r 1 0 g 1 0 b 11 0 Reset to defaults Apply Figure 12 The
118. ns used in Fig 9 these provide a surface rendering style 3D image for many micro CT scans of rodent skulls 21 yo VR Control O x Render quality Fastest Fast High Highest Render type Opacity Greyscale Pseudo colour Full Colour Scalar Classification spline linear reset apply X Gradient Classification Size Mouse ROI Proj spline install clone init repaint linear zoom3D rot zoom shift reset apply DQ Front fac 1 0 DQ Density 11 0 Depth queing Off On Ambient R 01 G 01 B 01 Diffuse R 10 44 G 0 4 B 0 4 Specular R 05 G 05 B 05 Shinyness RGB 1 0 Lighting Control Foreground colour Background colour E Options Scan Load Save Figure 7 The volume renderer using the default options XX VR Control ox Render quality Fastest Fast High Highest Render type Opacity Greyscale Pseudo colour Full Colour Scalar Classification spline linear reset apply X m Gradient Classification Size Mouse ROI Proj spline install clone init repaint linear zoom3D rot zoom shift reset apply DQ Front fac 1 0 DQ Density 11 0 Depth queing Off On Ambient R 01 G 01 B 01 Diffuse R 10 44 G 0 4 B 0 44 Specular R 0 5 G 0 5 B 05 Shinyness RGB 1 0 Lighting Control Foreground colour Background colour Options Scan Load Save Figure 8 Opacity only volume rendering 22 Figure 10 X VR Control a x Fas
119. ntered into the current landmark list However as with all optimisation based machine vision systems there is a chance that some of the estimated locations contained in the database will be fit failures i e will be in the wrong location This can be caused by several different effects shape differences can result in the structures around the landmark point in the current volume being a poor model for the structures in the database entry similar structures in different locations within the images can generate local minima in the likelihood cost function that trap the optimisation and local minima can also be generated by image noise Therefore the final refinement must take account of the fact that some of the estimated locations for each landmark may differ significantly from the correct location i e may be outliers The potential presence of outliers in the database militates against using any procedure based on a logical AND process e g taking the mean or median of the estimated locations to produce the final hypothesised location for each landmark Furthermore taking the single estimated location with the lowest estimated error would be undesirable for two reasons First lacking a model of shape variation between patches in the database and the current volume any error estimate such as the x per degree of freedom at the end of the local registration is itself unreliable which could lead to errors in point selection More importantly picking
120. nual Landmark tool this will move the 3D cursor to the coordinates of the current landmark point as specified in the Landmark Points dialog box and in the Manual Landmark tool e Select the desired point in the Markup list of the Manual Landmark tool e g P1 1 is the first plane point e Press the Mark Point button in the Manual Landmark tool this will save the current coordinates of the 3D cursor to the point specified in the Markup list of the Manual Landmark tool In order to display axis or plane points within the 3D rendering select the desired options in the 3D Tv Draw check list in the Manual Landmark tool Plane points are displayed in cyan and axis points in yellow As with landmark points and the 3D cursor the points are displayed as 3D cross hairs by default but can be displayed as spheres if the Ball option is selected in the 3D Tv Draw check list Any parts of a cross hair or sphere that lie behind a bone surface as specified by the Bone threshold field in the Manual Landmark tool from the current viewpoint are displayed in a lower intensity whilst points that are not behind a bone surface are displayed in a higher intensity this provides 3D cues to the user making it easier to see how each point interacts with the bone surface However this functionality is disabled during rotation of the 3D rendered image in order to prevent slowdown during rotation all points are di
121. ocal registration plus a border of three times the size of the smoothing kernel For example if the coarsest stage of local registration uses a patch size of 40 and a smoothing kernel size of 20 then the stored patch size must be at least 40 3 x 20 100 The software will abort the local registration if the stored patch size is too small Similarly Glob border controls the size of the border around the full sized patches through the centre of the volume that are used in the image based stage of global registration This should be at least three times the size of the smoothing kernel that will be used in the global registration The software will abort the global registration if the border is too small The user must take this into consideration if they wish to experiment with different settings of the smoothing kernel and local registration patch size parameters The patch border and smoothing kernel sizes are all measured in the coordinate system of the current volume i e taking into account any down sampling applied during data loading The patch size parameters refer to the distance from the centre of the patch to the edge i e if a patch size parameter is set to 10 the patch used measures 20 x 20 voxels in the down sampled coordinate system The database can be stored to disk by entering the desired path and filename pathnames can be either relative or absolute into the Output field of the Automatch Dialog box and then clicking on t
122. of N Name Noise List Length gt Scale Centroid error Chisq Er Et G reg point Type Type Proj x Proj y Proj z Chi2 global Scroll backwards through the entries in the automatch database The number of entries in the automatch database and the ordinal number of the entry currently displayed in the dialog box The path and filename of the image volume used to generate the current database entry Estimated image noise on the volume used to generate the current database entry The number of points in the landmark point list contained in the current database entry Scroll forwards through the entries in the automatch database The scale parameters of the global registration transformation model for the current database entry The distance between the centroids of the sets of points after the point based stage of global registration for the current database entry The x per degree of freedom at the end of the image based stage of global registra tion The rotation parameters of the global registration transformation model for the cur rent database entry displayed as a rotation matrix The translation parameters of the global registration transformation model for the current database entry Select a G reg point from the current database entry to display G1 display information on the first G reg point G2 display information on the second G reg point G3 display information on
123. om shift X VR Control ox Render quality Fastest Fast High Highest Render type Opacity Greyscale Pseudo colour Full Colour Scalar Classification i spline a 2 linear reset Hea apply X m SA Gradient Classification Size Mouse ROI Proj 4 goin instali clone int repaint j linear zoom3D rot zoom shift reset Gi apply DQ Front fac 1 0 DQ Density 1 0 Depth queing Off On Ambient R 0 1 G 01 B 01 Diffuse R 0 4 G 0 4 B 04 Specular R 05 G 05 B 05 Shinyness RGB 1 0 Lighting Control Foreground colour Background colour i Options Scan Load Save X Lighting Control O xX 1 Off On x 0 0 y 0 0 z 1 0 r 0 6 g 0 3 b 103 2 Off On x 0 0 y 11 0 z 00 r 0 3 g 10 6 b 10 3 3 0ffl On x 1 0 y 10 0 z 0 0 r 0 3 g 0 3 b 10 6 4 Off On x 10 0 y 10 0 z 1 0 r 1 0 g 1 0 b 1 0 5 Off On x 0 0 y 10 0 z 1 0 r 1 0 g 1 0 b 1 0 6 Off On x 0 0 y 10 0 z 1 0 r 11 0 g 11 0 b 1 0 Reset to defaults Apply Figure 11 Full colour volume rendering with options chosen to give a surface rendering style image showing the effects of coloured lighting e tmlt_pseudo_colour txt similar to the options used in Fig 9 but with pseudo colour rendering enabled and a light blue background allowing the user to see the opacity of the rendered image more easily e tmlt_full_colour txt similar to the optio
124. on using these geometrical constructs Note that the Rotate and Lock rotation options modify the description of the coordinate system given in Section 5 1 the images displayed in the 2D TVs are no longer produced from planes orthogonal to the major axes of the image volume when either of these options are selected 5 2 2 3D Image Display The 3D TV displays a volume rendering of the data loaded into the Sequence tool Since the preparation of this image involves a significant amount of processor time and memory usage the volume renderer is not activated by default and must be explicitly switched on by the user This is done using the 3D Tv choice list in the Manual Landmark tool if NULL is selected then the volume renderer is switched off Selecting VR will switch on the volume renderer and prepare the sequence for rendering this may take several tens of seconds to complete In order to use the volume renderer e Select VR in the 3D Tv choice list in the Manual Landmark tool e Select 3D in the Tv choice list of the Manual Landmark tool start a new TV tool and click Install in the TV tool the title bar of the TV tool will change to indicate that it is now displaying the 3D image stream e Click the VR Control button in the Manual Landmark tool to start the volume rendering controls dialog box e Use the volume rendering controls see below to produce a satisfactory
125. ool uses the Volpack volume rendering library 11 12 to produce 3D views of the data loaded into the Sequence tool In general terms it is easier to see the overall position of a morphological landmark when the data is displayed in 3D although for maximum accuracy it is important to refine the landmark position by checking the 2D views since these provide a more direct view of the data Pressing the VR Control button in the Manual Landmark tool spawns a dialog box that provides access to various user controlled parameters of the volume rendering engine as shown in Fig 6 The most important of these are the graphs contained in the Scalar Classification and Gradient Classification fields Each of these takes the form of a small user controlled graph with four associated buttons The curve shown in the Scalar Classification graph controls the way in which intensity grey level in the original 3D image data loaded into the Sequence tool and displayed on the x axis of the graph is converted into the opacity of the corresponding pixel in the volume rendered image the y axis of the graph The shape of the curve is controlled by a set of points placed within the graph by left clicking with the mouse and moved by left clicking and dragging with the mouse cursor placed over an existing point The spline and linear buttons set the shape of the curve to be a spline or piecewise linear curve through the control points The
126. operations the image will be replaced with a wire frame in order to increase speed When Pick is selected the mouse can be used to manipulate the 3D cursor via the 2D TVs Only the left mouse button has functionality assigned to it in this mode Clicking on any point in the image will move the 3D cursor to that point clicking and dragging will move the 3D cursor interactively Only the coordinates displayed in the TV will be altered e g the x axis TV can be used to manipulate the y and z coordinates of the 3D cursor When marking up large numbers of points in multiple images it is desirable to keep the mouse pointer within the TVs i e not to have to move it repeatedly back to the Manual Landmark tool window in order to click buttons there Therefore the TVs also provide a set of keyboard shortcuts for commonly used functions e Up arrow key move the 3D cursor position upwards by one increment e Down arrow key move the 3D cursor position downwards by one increment 15 e Left arrow key move the 3D cursor position left by one increment e Right arrow key move the 3D cursor position right by one increment e Right ctrl select Zoom mode for mouse interactions in all TVs e Right shift select Pick mode for mouse interactions in all TVs e Enter equivalent to clicking on the Mark point button in the Manual Landmark tool i e store the current 3D cursor coordinates as the currently selected landmark e Page Up
127. pdated to show the slice numbers found during loading The Scales fields will also be updated to show the size of each voxel if this information is contained in the image file headers Note that when the value entered into the Stride field is greater than 1 and the Stride average switch is on the software will load slices in multiples of Stride prior to averaging them together If the number of slices in the image volume is not an exact multiple of Stride then a warning will be issued during the final loading operation indicating that there are not enough slices to perform the final averaging operation and the last few slices will be discarded The End field will be updated to indicate how many slices have been loaded The size of the original image volume together with the options used during loading i e the values entered into the Stride and Downsample fields and the choice of the Stride average method will affect the memory requirements and execution speed of the Manual Landmark tool and the accuracy of the Automatic Landmark Point Placement tool In particular larger volumes will slow down user interaction with the 3D TV of the Manual 10 Landmark tool see Sections 5 2 2 and 6 as the volume renderer will require more processor time to render an image The software was tested during development using micro CT images of rodent skulls with original resolutions of 658x658 voxels
128. rball was unpacked and type configure make make install Note that the configure script in the tina tools directory provides some information about the required libraries in particular if the necessary GTK 2 libraries have not been installed see Section 2 1 or cannot be found a warning will be issued 2 3 Install Volpack The TINA Geometric Morphometrics toolkit requires the Volpack volume rendering library A version of this library can be found at http www tina vision net tarballs manual_landmark_toolkit Unzip and unpack the tarball this can be done in any directory although it is standard practice to put user installed software in usr local cd into the new Volpack directory created when the tarball was unpacked and use configure make make install as root to build the library On standard Linux systems the Volpack library and header file will be copied into standard locations usr local lib and usr local include respectively Then when the TINA Geometric Morphometrics Toolkit is built the compiler should automatically pick up the location of the header file and the location of the library will be automatically picked up if usr local lib is specified in etc ld so conf the system wide list of library locations If it is not then add usr local lib to the file Run ldconfig to refresh the list of libraries after the installation of Volpack or restart the machine 2 4 Install the Geometric Morphometrics Toolkit
129. reset button returns the shape of the curve to the default linear ramp After setting the curve to the desired shape the apply button must be pressed to pass the new curve to the volume renderer The effects of these parameters can be seen in Figs 7 8 9 10 and 11 In general the curves should both be set to sigmoid functions using steep sigmoids as shown in Fig 9 will produce a surface rendering style image The balance between rendering speed and image quality is controlled by the Render quality choice list These choices control an upper and lower threshold voxels with intensities below the lower threshold are ignored during rendering and voxels with intensities higher than the upper threshold are assigned 100 opacity so that no voxels behind them need to be rendered The Fastest choice sets these thresholds to their most extreme values so that many voxels are ignored the rendering is then faster at the expense of lower image quality The Highest choice disables the thresholds completely so that all voxels are rendered the rendering is then slow but produces the best image quality In general the Fast choice is the most suitable during mouse interaction with the image displayed in the 3D TV The volume renderer is also capable of producing several different types of image depending on the selection in the Render type choice list The Opacity choice will render an image showing only the opa
130. rmat of the file from the Input format choice list e If loading an NTSYS or TPS file containing multiple records enter the ordinal position of the record in the file into the Field no field e Click the Load button in the Landmark Points dialog box Ifa record from a TPS file is loaded the ID of the record will be copied to the TPS NTSYS ID field If a record from an NTSYS file is loaded any comments in that record will be displayed in the text window at the bottom of the top level tinaTool window Note that Raw NTSYS and TPS files do not contain the information from the line linking lines file However the user can load this information by loading a names file with a corresponding linking lines file after loading a Raw NTSYS or TPS file as long as all files contain corresponding lists of landmark points Note also that the Raw format contains more information that NTSYS or TPS In particular it records the landmark type code see Sec 7 1 which is used by the Automatic Landmark Point Placement tool see Sec 12 to record information about errors in the automatic point locations Therefore Raw is the preferred format for saving results from automatic landmark point location if the user intends to check and correct outliers at a later time 7 1 Landmark Type Codes Each landmark axis plane or global registration G reg point stored in the software is assigned a type code which can be se
131. rsecting at the position of the landmark point and aligned with the major axes of the image volume this provides enough data to estimate the parameters accurately and reduces the processor time required compared to the use of 3D data blocks Smoothing is applied to the patches in order to reduce the effects of noise and spatial derivatives are taken in the x and y directions of each patch producing a total of six image patches for each landmark point The likelihood function itself is the sum squared difference between the data in these patches and the data in the image volume currently loaded into the Sequence tool i e int gt HT 1 i 0 e 39 where L is the likelihood J represents smoothed image derivatives of the patches stored in the database J represents smoothed image derivatives of patches from the volume currently loaded into the sequence tool T represents the nine parameter affine transformation model being optimised in order to align the two sets of patches n represents iteration over corresponding voxels in the image patches y is a relative scaling between the I and J patches and Ge is the standard deviation of the noise on the numerator of the likelihood function The noise is calculated by measuring the noise on the original I and J image volumes using the standard TINA noise measurement function which is based on measuring the width of zero crossings in vertical and horizontal gradient histograms 13 and then applying error
132. s From our experience 10 iterations default is sufficient to reach convergence However this number may be set based on an idea of how light heavy the data set can be Note that a minimum of 2 iterations is needed for the methods to converge 20 3 Display Tool In this section we explain how to use different buttons appearing on the Display Tool dialog box rescale mono tv To use the display tool a display window should be prepared first by pressing the Mono button and then the New Tvtool button on the Tina dialog box followed by pressing the Install button on the New Tvtool Finally press the rescale mono tv button on the display tool to rescale the data in order to improve visualisation and its display coverage Also it is recommended to select the largest size available 768 on the display mono window Note that when there are negative coordinates in the 3D mark up data e g Monte Carlo data or data 59 generated by Morphoj software one may experience difficulties to rescale the mono tv properly and be able to view all parts of the projections on the 2D planes In such cases one should press Mouse button on the mono tv and select zoom then adjust the position and scale of the tv display in the usual way projections Selecting each of the projection planes xy xz and zy determines which plane is currently active so that the display buttons would only work on the selected pl
133. s Therefore the shear warp algorithm calculates an overall shearing of the volume that makes all of the vectors parallel to the nearest major axis of the volume The data is then projected into an intermediate image which is parallel to one face of the volume before being warped to produce the final rendered image This results in a much more favourable alignment of the data in memory vastly increasing rendering speed at the expense of a small reduction in image quality introduced by the warping Several additional limitations have been implemented in order to maintain rendering speed The renderer uses a parallel projection rather than a perspective projection i e assumes that all rays from the viewpoint through the data are parallel rather than diverging The drawback of this approach is that it can introduce an optical illusion known as the Necker Cube 10 in which particularly if the image has high transparency it can be difficult to tell which side of the volume is closest to the viewer Using renderer settings that minimise the translucency of bone surfaces will minimise this effect Furthermore the lighting model used by the renderer does not take account of radiosity i e diffuse lighting reflected from multiple surfaces within the volume Instead it only takes account of direct rays of light from the light sources reflected off of one surface to the viewer This can cause a characteristic artefact in which rough bone surfaces appear
134. s dialog box can also load and save a variety of other file formats as specified in the Input format and Output format choice lists Raw is a TINA specific ASCII text format that contains all of the information from the landmark list TPS and NTSYS are standard formats for morphological data All of these formats are ASCII text files and so can be viewed or edited in any text editor In order to save a landmark list e In the Landmark Points dialog box enter the absolute or relative pathname of the landmark file into the Output pathname field either directly or by using the file browser started by clicking on the Scan button e Select the format of the file from the Output format choice list Raw files can only hold one list of landmark points If a Raw file is overwritten then the previous version of the file will be replaced TPS files can hold multiple lists of landmark points If landmark points are saved to a TPS file that already exists then the new set of landmark points will be appended to the existing file The TPS format assumes that each set of landmarks in the file has a unique identifier the contents of the TPS NTSYS ID field will be used as the identifier The software does not check whether this is unique in the file i e it is up to the user to do this However the name of the directory containing the image files is copied to the TPS NTSYS ID field whenever
135. s for the current point of the current database entry in the form in which they are used in the image based registration i e with smoothing and differentiation applied This generates two images the derivatives with respect to the x and y directions of the image patch the y derivative image is pushed onto the stack first and so the derivative image will be on the top of the stack and the y derivative image will be second on stack once the process has been completed RX RY and RZ select the full sized patches used in global registration If an image volume is loaded into the sequence tool then the corresponding patch from the current volume will be extracted and combined with the patch to form a complex image in which the real part is the patch stored in the automatch database and the imaginary part is the corresponding patch extracted from the current volume If global registration has been performed then the result will be used when extracting the corresponding patches from the current volume The complex image is then pushed to the Imcalc stack and so can be viewed as an anaglyph image in the Imcalc TV The real part of this image displayed in green is the full sized y z slice through the centre of the image volume stored in the current automatch database entry i e the entry currently displayed in the dialog box The imaginary part displayed in red is the corresponding slice through the current volume obtained using the tran
136. s two three coordinates for each landmark point LM LM3 However one can provide the software with 3D data and force it to treat the 3D data as 2D by selecting 2D as data dimensions In this case the third coordinate values are ignored When generating outputs of the TPS file format the software writes two or three coordinates according to the current number of data dimensions 19 2 Methods The methodology behind the software functionalities have been described in detail in 17 and 15 Although in our technical report 17 it is assumed that data has two dimensions x and y the methods are applicable to 3D data with minor changes The extension of our methods from 2D to 3D has been explained in 16 19 3 General Use The shape analysis tool can be used to obtain a number of outputs results and conclusions as follows These have been explained in details with discussions on the results obtained from example data sets in our reports 15 and 16 We shall provide the list of steps needed to perform a number of experiments Boxes A B and C later on 57 1 Shape alignment by constructing a linear model and fitting the data based on an iterative optimisation approach 2 Estimating covariances on each landmark based on computing sample covariances and applying covariance corrections 3 Generating isotropic whitened output data for use later as input to popular packages which support a Procrustes analysis 4 Compar
137. sages will be printed to the text window of the top level tinaTool and any lights with invalid parameters will be disabled Finally the lighting control dialog features an emergency mode to ensure that even if all lights are disabled due to invalid parameters the default lighting will be applied so that a rendering is still produced Note that if coloured lights are specified with the renderer in greyscale mode then the rendering will show the intensity of the lighting but not the colour Switch to full colour mode to display the coloured lighting 25 6 2 Limitations of the Volume Renderer In order for the Manual Landmark tool to be usable mouse interaction with the 3D image must be fast enough that the image can be manipulated in interactive time i e the renderer must be able to produce several frames per second This places severe constraints on the amount of processing that can be performed during rendering Tradi tionally landmark identification software has used surface rendering algorithms in order to meet this requirement Such algorithms rely on identifying a set of points on the bone surface within the volume tesselating these points to produce a representation of the surface and then only rendering this surface i e only rendering a small fraction of the data However this approach has a drawback in that it relies on a specific interpretation of the data i e some technique must be used to identify the surface prior to rendering I
138. scending order e Press the Save button 11 Part 1 The Manual Landmark Tool P A Bromiley Imaging Science and Biomedical Engineering Division Medical School University of Manchester Stopford Building Oxford Road Manchester M13 9PT 12 5 The Manual Landmark Tool A VR Control Landmark List Keyboard Controls Automatch Shape Analysis Tv x axis y axis z axis 3D 3D Tv NULL VR 3D Tv Mouse Zoom Pick 2D Tv Mouse Zoom Pick 3D Tv draw v Cursor ___ Landmarks _ Axis __ Plane __ Global reg 3D Tv draw cont Text Poly Ball Linklines 2D Tv draw Rotate Lock rotation Axis Plane 3D cursor position X 128 0 lt gt Y 128 0 lt gt Z 128 0 lt gt Resolution 0 1 0 5 1 0 20 50 Bone threshold 1500 0 Current landmark Start Last outlier 5 Up Down 5 Next outlier End No 1 Name NULL Type 1 Markup Curr LM Ax 1 Ax 2 PI 1 PI 2 PI 3 G1 G2 G3 G4 Jump lock Off On Jump stored Axis lock Off On Rotate to plane Rotate to axis Reflect cursor Snap Mark point Figure 3 The Manual Landmark Tool The Manual Landmark tool Fig 3 18 allows morphological landmarks to be identified within any 3D data set loaded into the Sequence tool It provides multiple views of the data three 2D TVs showing orthogonal slices through the sequence and a 3D TV showing a volume rendering of the whole sequence The functionality of the Manual Land
139. selected while a 3D data file is provided the first and second coordinates of each point will be used and the third coordinate will be ignored input samples A directory needs to be created for every specific experiment on existing or new data The corresponding TPS file should be copied into the directory This leads the software to define this as its working directory read write The file and its directory full path is located using the scan button which provides a browsing facility Appropriate messages will appear on the Tina tool window informing user about the success or any possible problems should they occur For instance if there are missing coordinates in the data the corresponding samples will be removed from the data and a warning message confirms the action input covariances If there is a file containing covariance data in the format expected and consistent with the data dimensions and number of landmarks it is possible to locate this file via the browser and input these covariance data and use them rather than estimating new ones Although it is possible to generate these files manually it is strongly recommended that no other covariance file is used other than those generated by the software for similar shape data identical number of landmarks and dimensions Likelihood Procrustes The default method is Likelihood which is actually the method for which we have developed the software This generates
140. sformation model optimised during the global registration In all cases X Y and Z refer to the axis of the original volume normal to the plane of the image patch e g the X image is generated on a y z plane in the image volume 17 1 The Database Positions Output File During automatic landmark point location projected coordinates for all landmark points in the automatch database are stored after each stage of registration The Automatic Landmark Point Placement tool supports the output of files containing any of the sets of coordinates currently stored in the automatch database in ASCII text format Such files can be useful in analysing the operation of the software and in setting some parameters such as the error thresholds for the outlier detection stage The set of coordinates that will be output to file is controlled by the Pos output field choice list in the Automatic Landmark Point Placement tool e Proj glob the coordinates of the point after transformation using the result of the global registration e Proj 1 the coordinates of the point after transformation using the result of the global registration and the first stage of the local registration e Proj 2 the coordinates of the point after transformation using the result of the global registration and the first and second stages of the local registration e Proj the coordinates of the point after transformation using the result of the global registration and all
141. sh fields Click the Error analysis button to run the outlier detection If a point fails outlier detection an error code will be added to its type code based on translating a binary mask of failure flags from each stage into an integer e g failure of stages 1 and 2 results in the binary mask 011 which has integer value 3 resulting in a final type code of 5 since the type code will be 2 after automatic point location Points with type codes indicating an failed check will be displayed in the 3D TV as green and red chequered cross hairs or spheres The user should check the location of each of these points Note that unlike the other parameters in the Automatic Landmark Point Placement tool the error thresholds are given in the original coordinate system of the data not the down sampled coordinate system of the current sequence There is a reasonable direct proportionality between the error thresholds and the maximum error on landmark points that will pass error checks at those thresholds Therefore giving the error thresholds in the original coordinate system of the data allows the user to set them to the maximum acceptable error on landmark point locations in the same coordinate system used in the output files As a rough guide the error thresholds should be set to approximately the same value as the patch size parameter used in the last stage of local registration taking this coordinate system transformation into account in order to avo
142. sis tools and the user interface 2 1 Install GTK 2 The TINA libraries depend on the GTK 2 libraries which must be installed prior to installing TINA Both the gtk2 and gtk2 devel packages are required see the documentation for the Linux distribution you are using for instructions on how to install them Installation of these packages should also result in installation of various other packages atk pango fontconfig pkgconfig required by both GTK 2 and TINA 2 2 Install the TINA libraries Download the latest tina libs and tina tools tarballs from http www tina vision net tarballs The TINA Geometric Morphometrics toolkit version 2 0 requires rcbuild008 or greater Note that some sections of the Geometric Morphometrics toolkit particularly the volume renderer and the Au tomatic Landmark Point Placement tool are extremely processor intensive In order to mitigate the resultant time requirements the software makes extensive use of parallel processing through OpenMP However this is not enabled by default since it is not available in all compilers any version of GCC from 4 2 onwards released May 13th 2007 includes OpenMP the user must specifically select it In order to do so specific compiler options must be used when the TINA libraries and the Geometric Morphometrics toolkit are built If OpenMP is available in the compiler being used enable parallelisation using the fopenmp option by replacing all occurrences of configur
143. splayed in a medium intensity Additionally selecting the Poly option in the 3D Tv Draw check list will display a yellow line representing the axis if both axis points have been defined and the Axis option is on and a cyan grid representing the plane if all three plane points have been defined and the Plane option is on The stored coordinates for axis and plane points can be viewed in the Landmark Points dialog box The A P G point choice list selects which point will be viewed and the Name x y and z in the same row of the tool display the name and coordinates of the selected point if stored Any current axis and plane points can be deleted using the Clear Axis and Clear Plane buttons in the Landmark Points dialog box Plane points are used by the Reflect cursor button in the Manual Landmark tool Once three plane points have been specified pressing this button will project the current 3D cursor position through the plane to an equidistant point on the other side and reset the 3D cursor to that point This can be useful when marking up symmetrical structures Axis points are used to limit the rotation of the volume rendered image in the 3D TV Once both axis points have been specified press the Lock Rotation button in the Manual Landmark tool The volume rendered image will then only rotate around an axis parallel to that defined by the two axis points and p
144. ss is over rename the output file called results txt to show that the error eigenvalues listed in the file correspond to the estimated errors using Monte Carlo data e g to results MC A txt for Exp A or to the errors computed from the residuals left after alignment e g results MC B txt for Exp B As mentioned in Section 2 4 the number of vectors listed in the file results is equal to the number of model components while the number of elements in each vector is equal to the number of landmarks To plot only the major eigenvalues one should only plot the corresponding vectors on the first row of numbers in the result files results MC A txt versus results EXP txt Figs 21 22 or results MC B txt versus results EXP txt The rest of the vectors may be plotted similarly The 2 80 statistical allowable range should then be used to assess the statistical validity of the model Box C Here we outline a method for model selection i e selecting the optimum number of model compo nents It is based on comparing the model accuracy against the measurement accuracy If the measurement has been manual the measurement accuracy would be the manual repeatability which is straightforward to compute However if the measurement has been automatic the measurement accuracy would be the automatic repeatability obtained from two different landmark databases 1 Using two repeat data sets compute the
145. statistically valid estimates of parameters in a way which applies the Like lihood approach in a self consistent manner see technical reports Alternatively one can select the traditional Procrustes method for which no covariance data will be estimated used this has been included largely for com patibility with established methods within the morphometrics literature Output files from the Likelihood analysis are written see below which transform the data suitable for analysis in conventional Procrustes packages by enforcing the requirement of homogenous measurement noise This data set is already aligned so that subsequent Procrustes alignment should not modify relative sample positions but the new data now have homogenous error characteristics suitable for use in PCA 58 estimate covariances No matter whether the software has received covariance data or not the user can tick Yes to estimate covariances default or No to use the covariance data provided already as input In the latter case if no covariance file has been provided the identity matrix is used as the default covariance matrix In the case of using Procrustes even if a covariance file is provided it will not be used input eigenvectors Here Yes selection is specifically designed for one form of Monte Carlo test where the Monte Carlo data are fitted to the existing model So this experiment can only be done if the original data have alrea
146. t of landmark point numbers and names and corresponding linking lines file if one exists Raw a TINA specific ASCII text format that contains all of the information stored in the landmark list TPS a standard format for morphological data NTSYS a standard format for morphological data Absolute or relative pathname of the file from which to load landmark data Start a file browser to select the file to load Load data from the file specified in the input pathname field using the format specified in the Input format field and NTSYS TPS the field number Choose the type of file to save Names a list of landmark point numbers and names and corresponding linking lines file if link numbers exist Raw a TINA specific ASCII text format that contains all of the information stored in the landmark list TPS a standard format for morphological data NTSYS a standard format for morphological data Absolute or relative pathname of the file to which to save landmark data Start a file browser to select the file to save Save the current landmark point list to the file specified in the Output pathname field using the format specified in the Output format field 76 21 6 Keyboard Controls Dialog Box Up Left Right Down Pick Zoom Mark Prev landmark Next landmark Select a shortcut key to move the cursor upwards in the currently selected 2D TV by the number of voxels specified in th
147. ted on one data set as input covariance to process another similar e g repeat data set The results contain one x ratio per landmark per line which are written in a file called ChisqTest txt These ratios should be distributed around unity and may be plotted to investigate the statistical stability repeatability of the method Fig 20 shows an example plot of a typical x test on 2D fly wing repeat data see 15 for more details When the number of samples K is large enough e g K gt 30 the resulting statistic when applied to each 2D 3D landmark DoF 2K 3K is expected to be approximately Gaussian with mean u DoF standard deviation ce V2 DoF So when expecting 99 0 of data equivalent to 2 80 to fall inside the allowable range the range the dashed lines in Fig 20 is given by u 2 80 DoF results The file results txt firstly contains the eigenvalues extracted from the 2x2 or 3x3 covariance matri ces and secondly the Fisher information value computed based on variances estimated for all landmarks Note that although no covariances are estimated when working with Procrustes the Fisher information and the error eigenvalues are still computed and written out using the residuals left after the alignment We have shown in 15 and 16 how to use these error eigenvalues to produce useful plots For instance if we plot the eigenvalues corresponding to Monte Carlo data against those corresponding to the original data
148. test Highest Render quality Fast High Pseudo colour Full Colour Render type Opacity Greyscale Scalar Classification n I spline linear reset apply Gradient Classification ae spline linear reset Pi apply DQ Front fac 1 0 DQ Density 1 0 Depth queing Off On Ambient R 01 G 0 1 B 0 1 Diffuse R 04 G 04 B 04 Specular R 05 G 05 B 05 Shinyness RGB 1 0 Lighting Control Foreground colour Background colour EE Options Scan Load Save X VR Control ox Render quality Fastest Fast High Highest Render type Scalar Classification oce a OS Opacity Greyscale Pseudo colour Full Colour 3 spline a linear h reset Fi apply Gradient Classification ee spline linear 3 reset a apply DQ Front fac 1 0 DQ Density 1 0 Depth queing Off On Ambient R 01 G 01 B 01 Diffuse R 10 44 G 0 4 B 0 44 Specular R 0 5 G 0 5 B 05 Shinyness RGB 1 0 Lighting Control Foreground colour Background colour Options Scan Load Save Pseudo colour volume rendering with options chosen to give a surface rendering style image 23 X 3 Size Mouse ROI Proj install clone init repaint zoom3D rot zoom shift Figure 9 Greyscale volume rendering with options chosen to give a surface rendering style image X Size Mouse ROI Proj install clone init repaint zoom3D rot zo
149. the current volume using the full transformation model including both the global and local registration results The sixth number is the point type code The final line of the file contains equivalent information from the current landmark point list For example if the final refinement stage of the automatic point location algorithm has been performed then the automatically estimated point locations will be written to the file if the outlier detection stage of the automatic point location algorithm has been performed then the point type codes on the last line of the file will indicate any failed error checks In order to simplify the operation of loading the point positions files into analysis packages such as Mathematica all lines in the file are of the same length and any missing values are replaced with zeros For example there is no centroid error or x for the current volume therefore the first two numbers on the last line of the file are always zero Therefore the number of rows in the file is given by rows N 1 where N is the number of entries in the database and the number of columns is given by columns n 2 where n is the number of points in the landmark list 53 18 Notes on Future Updates This section lists some ideas for future updates to the software that could improve usability with little effort but which have not been implemented due to time constraints 18 1 Initial Global Alignment The point based global al
150. the point locations resulting from the final refinement stage of the automatic point location algorithm to the closest surface as defined by the 78 Chi sq weight Fin sig Gauss conv P1 thresh P2 thresh P3 thresh Error checks Error analysis Pos output field Output Scan Pos output Input database Output database Bone threshold field of the Manual Landmark tool prior to entering their coordi nates into the current landmark list Off do not move the points to the closest surface On move the points to the closest surface Specify whether to weight the points in the automatch database with their estimated x during final refinement Off do not weight On weight The standard deviation of the Gaussian convolution kernel applied during final re finement Apply the final refinement algorithm to calculate hypothesised point locations from the entries in the automatch database entering the results into the current landmark list Threshold for stage 1 of the outlier detection Threshold for stage 2 of the outlier detection Threshold for stage 3 of the outlier detection Specify which points to compare to the hypothesised location resulting from the final refinement algorithm during outlier detection P1 compare the point from the automatch database with the lowest 7 P2 compare the point from the automatch database with the second lowest x7 P3 compare the point from the automatch
151. the same comments about window manager focus apply to the 3D TV several keyboard shortcuts are available in the 3D TV e Right ctrl select Zoom mode for mouse interactions in all TVs e Right shift select Pick mode for mouse interactions in all TVs e Enter equivalent to clicking on the Mark point button in the Manual Landmark tool i e store the current 3D cursor coordinates as the currently selected landmark e Page Up scroll up to the previous landmark in the landmark list see Section 7 e Page Down scroll down to the next landmark in the landmark list see Section 7 These can be reassigned to any desired keys using the TV Keyboard Controls dialog box see Section 9 As well as displaying a volume rendered image of the data currently loaded into the sequence tool the 3D TV can display any stored landmark axis plane or G reg points see Sections 7 8 1 and 13 2 1 The user can choose which points to display using the 3D Tv draw check list in the Manual Landmark tool The available choices are e Cursor display a red cross hair representing the 3D cursor switched on by default e Landmarks display green cross hairs for all marked up landmark points Any landmark point outside the boundaries of the currently loaded image volume will be displayed in purple instead of green this is most likely to occur if landmarks corresponding to a different volume have been loaded from a file Any automatically lo
152. three stages of the local registration e Pos the coordinates of the point without any transformation i e the manually identified location of the point in its original image volume Clicking on the Pos output button will write these locations to the file specified in the Output field of the Automatic Landmark Point Placement tool Each line of the point positions file corresponds to one entry in the automatch database The first two numbers on the line are the centroid error and x per degree of freedom for the global registration The remainder of the line contains sets of six numbers corresponding to each landmark point in the database entry The first three of these are the x y and z coordinates of the point the set of coordinates that will be used is controlled by the Pos output 52 field choice list as described above The fourth number is the x per degree of freedom of the match between the stored local image patches around the point using the patch size specified in the Loc patch size 3 field and the corresponding patches around the transformed point in the current volume using the transformation model from the global registration i e prior to local registration The fifth number is the x per degree of freedom of the match between the stored local image patches around the point using the patch size specified in the Loc patch size 3 field and the corresponding patches around the transformed point in
153. tic landmark location software will be applied 43 In order to check the global registration result assuming that an image volume and automatch database have been loaded and global registration has been performed e Start the TINA Imcalc tool by clicking on the Imcalc button in the top level tinaTool window Start a new TV tool and install it on the Imcalc TV e Open the Automatch Database Inspector dialog box see Section 17 e Check the value in the Centroid error field it should be less than 1 If the value is greater than 1 then the point based stage of global registration has failed this usually indicates that the G reg points defined for the currently loaded image volume do not correspond to those defined for the specimen in the database or that errors have occurred during manual landmarking of these points e Check the value in the Chisq field this is a rough indication of the quality of the image based stage of global registration since the specimens in the current volume and the database will have different shapes and so no accurate guide can be given However the value should be less than around 30 and a value of less than around 15 indicates a high quality alignment e Select the RX option in the Image patch choice list of the dialog and then click Push A complex image will be pushed onto the Imcalc stack and displayed in the Imcalc TV tool as an anaglyph image The real part o
154. to be speckled with small dark patches Correcting this with a more realistic lighting model would increase the processor time requirements to the point where the renderer could not run in interactive time However the effect can be minimised with careful choice of lighting parameters e g using a single light shining onto the front of a rodent skull seems to give a good result Finally the TINA Manual Landmark tool was designed primarily for use on very large micro CT image volumes of rodent skulls typically several GB in size In order to make proper use of the accelerated 3D graphics hardware found in most PCs the data to be rendered must be copied to the video card memory in order to avoid continual paging of data from system memory to video card memory across the system bus However the majority of current video cards lack sufficient memory to hold an entire micro CT rodent skull volume Therefore the volume renderer used in the Manual Landmark tool does not use graphics hardware it runs on the CPU and from system memory 26 7 The Landmark Points Dialog Box X Landmark Point Up 5 Last outlier Start No 4 Name NULL x 1 0 y 1 0 z 1 0 Type 1 Link no 1 No 1 Name NULL x 1 0 y 1 0 z 1 0 Type 1 Link no 1 No 1 Name NULL xi 1 0 y 1 0 z 1 0 Type 1 Link no 1 No 1 Name NULL x 1 0 y 1 0 z 1 0 Type 1 Link no 1 No 1 Name NULL X 1 0 y 1 0 z 1 0
155. to zygomatic process of squasmosal left side 24 Joining of squasmosal body to zygomatic process of squasmosal right side 25 Intersection of zygomatic process of maxilla with zygoma jugal superior surface left side 26 Intersection of zygomatic process of maxilla with zygoma jugal superior surface right side 27 Intersection of zygoma jugal with zygomatic process of temporal superior aspect left side 28 Intersection of zygoma jugal with zygomatic process of temporal superior aspect right side 29 Basion 30 Bregma intersection of frontal bones and parietal bones at midline 31 Intersection of parietal bones with anterior aspect of interparietal bone at midline 32 Intersection of interparietal bones with squamous portion of occipital bone at midline 33 Nasion Intersection of nasal bones coudal point 34 Nasale Intersection of nasal bones rostral point 35 Opisthion 36 Intradentale superior 37 Most posterior projection of the posterior nasal spine 38 Most superior point on the external auditory meatus right side 39 Anterior edge of the alveolar process where right first molar hits alveolus right side 40 Most anterior medial point on the right carotid canal right side 41 Most superior point on the external auditory meatus left side 42 Anterior edge of the alveolar process where right first molar hits the alveolus left side 43 Most anterior medial point on the left carotid canal left side 44 Anter
156. ts the Automatic Landmark Point Placement tool Starts the Shape Analysis tool The list of available TVs in the Manual Landmark tool selecting an item in this list will make it available for installation on a TV tool x axis 2D TV showing images of the z y plane through the 3D cursor y axis 2D TV showing images of the x z plane through the 3D cursor z axis 2D TV showing images of the x y plane through the 3D cursor 3D 3D TV showing a volume rendering of the data loaded into the sequence tool Switch the volume renderer off on NULL switch the volume renderer off VR switch the volume renderer on Switch to control mouse interaction with the 3D TV Zoom mouse interaction moves rotates and zooms the image Pick mouse interaction picks points on bone surface or displays intensity profiles Switch to control mouse interaction with the 2D TVs Zoom mouse interaction moves or zooms the image Pick mouse interaction moves the 3D cursor cross hairs Select how landmark axis and plane points will be displayed in the 3D TV Cursor display a red cross hair representing the 3D cursor Landmarks display green cross hairs for all marked up landmark points Any points outside the volume will be displayed in purple Any automatically located points that have failed outlier detection tests will be displayed in green and red chequers Axis display yellow cross hairs for the axis points Plane display cyan cross hairs for the pla
157. uilding Oxford Road Manchester M13 9PT 56 19 Introduction A Shape Ana data dimensions 2D 3D display tool settings Directory Name scan A Settings Base Name S input samples input covariance Reset rand seed number ofsamples 42 outlier threshold 3 5 analysis method Likelihood Procrustes 3 left reference point 1 estimate covariances No Yes tight reference point 3 third reference point 9 input eigen vectors No Yes model components 8 process data number of iterations 10 a b X Display Toc y rescale mono tv projections xy XZ zy source data aligned mean model sample number 1 display sample points display all samples landmark number 1 highlight landmark next landmark eigen vector 1 vector scale 1 display eigen vectors error scale 1 display error bars c Figure 19 The Shape Analysis tool a the Settings dialog box b and the display dialog box c The purpose of the Shape Analysis tool shown in Fig 19 is to utilise the analysis of shape data with anisotropic measurement covariances as a tool to monitor manual and automatic placement of landmarks These methods are available via the automatic 3D landmarking tool as a system for quality assessment and validation of output data 19 1 Data The standard input data file format for this software is TPS Note that the extension must only consist of capital letters For the case of 2D 3D data the standard TPS file provide
158. umns is 4 2x2 for the case of 2D and 9 3x3 for the case of 3D data From left to right the values in each row of this file correspond to the first row elements of the 2x2 or 3x3 for 3D data covariance matrix for that landmark then its second row and then its third row only for 3D data Standard deviations for each landmark in different directions are then the square root of the elements on the diagonal line of the corresponding 2x2 or 3x3 matrix These elements are hence the values on the 1 st and 4 th columns for the case of 2D and 1 st 5 th and 9 th columns for the case of 3D data WhiteData The white data are written in a file with TPS format The file name is composed by concatenating the input TPS file name and the string WhiteData These data are then suitable to be fed to Procrustes if needed as these landmark points are guaranteed to have isotropic noise distribution around them which is consistent with the assumptions made in Procrustes PCA Note however that it may not be straightforward to make any obvious conclusions just by visualising the white data MonteCarlo The Monte Carlo data are also written in a file with TPS format The file name is composed by concatenating the input TPS file name and the string MonteCarlo Both in the MonteCarlo and the WhiteData files a sample number is added to each sample label ID in order to make a correspondence between each sample ID and the number with which it is
159. using the Loc patch size 1 Loc sig 1 Loc patch size 2 Loc sig 2 Loc patch size 3 and Loc sig 3 fields the number refers to the registration stage Local registration is performed by clicking on the Automatch button The software will automatically add a border around the specified patch size equal to three times the size of the smoothing kernel the local registration will be aborted if the stored patch size in the database is too small to allow this These images patches will then be aligned for all points in all records in the database this should take approximately one second per point 44 but will take considerably longer if the TINA libraries and the Geometric Morphometrics toolkit have not been compiled with parallelisation enabled see Section 2 A progress bar will be displayed informing the user of the current database record and point being aligned Once local registration has finished the projected location of each point in the database i e the estimated position of that point in the current volume based on the global and local registration results will be displayed in the Automatch Database Inspector dialog box together with the x per degree of freedom calculated from the third stage patch using both the global registration result only and the combined global and local registration results The x per degree of freedom for the global plus local registration should be close to 1 if t
160. y all landmark points in all image volumes that will be entered into the automatch database It may be more convenient to perform this process first outputting the results to file for later use in database construction The G reg point filler allows the locations of any four of these points to be rapidly copied into the G reg points during database construction Second once a database has been constructed it can be used to analyse an arbitrary number of further image volumes However the point based stage of registration requires four landmark points to be identified in each of these volumes It may prove more efficient to prepare a landmark point names file containing only four points and to manually identify these points in each volume to be automatically analysed saving the results to file These files can then be loaded prior to automatic analysis of the corresponding image volume and the four point locations copied to the four G reg points using the G reg point filer The landmark point names file for the full set of landmark points to be automatically located can then be loaded and the automatic landmark point location process applied 13 3 Image based Refinement of Global Registration The point based stage of global alignment provides the required initialisation for a second image based stage This uses full sized i e spanning the full size of the volume from edge to edge image patches intersecting at the centre point of the volume and aligne
161. y be fed as two vectors y versus x to any suitable software such as Matlab in order to generate the plot required see Fig 22 and Fig 24 65 Box B A Monte Carlo test is performed in order to assess the statistical validity of the model constructed based on the covariances estimated This experiment assumes that you have already obtained the outputs generated by the Likelihood method for the original data set see Section 2 4 and Box A These include the Monte Carlo data the covariance estimates the mean shape and the eigenvectors values All the numbers in the Settings dialog box must remain identical to the experiment which generated the Monte Carlo data 1 Repeat the steps listed in Box A while locating the Monte Carlo data file in step 2 as the TPS file for input samples Moreover use step 2 below in place of step 7 in Box A 2 Experiment A Select Yes in front of input eigenvectors if you wish to perform a Monte Carlo test with a given model This means that the model ingredients the mean and the eigenvectors values and the covariance estimates which are already available in the current directory should be used in the process rather than constructing a new model or estimating new covariances Experiment B Select No in front of input eigenvectors if you wish to perform a Monte Carlo test where an independent model will be constructed and covariances will be estimated accordingly Once the proce

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