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Reconstruct User Manual

1. Evaluating Alignments As discussed in Chapter 6 the alignment between two sections can be compared using either blending or flickering Blending overlays the two sections Fig 10 2 while flickering rapidly switches between the two sections Blend the sections just aligned using the spacebar To flicker hold the key down While either blending or flickering alone can be used to evaluate the alignment a combination of the two methods is advantageous With blending it is relatively easy to tell when there is good alignment Many of the cross sectioned features edges come into sharp focus while misaligned features are blurry However with blending it is often difficult to tell the direction of misalignment Flickering between sections generates apparent motion of misaligned features that quickly indicates the direction and magnitude of the offsets Some misalignment between features is to be expected even when the sections are correctly aligned since not 138 Reconstruct v1 0 6 1 Undoing and Adjusting Alignments every object in the series will be perfectly cross sectioned If the apparent motion generated during flickering is not uniform in direction and magnitude throughout the section but is instead locally random this indicates that the alignment is cor rect even though there is some blurriness with blending If an Align command produces a bizarre distortion of the section this probably means that there is a serious pr
2. unsimolified resect Area data column was included Note that open hidden 2 0 0202675 traces have zero enclosed area The in the G open 0 title bar is the default limit string which amp open unsimplified 0 effectively displays every trace in the section ZOOMING TO SELECTED TRACES The set of selected traces can be brought into maximum magnification by using the Zoom To item from the Trace menu This operation is accelerated by the Ctrl Home key sequence Trace List Traces can also be selected by name using the Trace List The Trace List window is opened by selecting List from the Trace menu Every trace in the section that matches the limit string specified in the Lists tab of the Series Options Fig 5 2 When new traces are drawn the list will be updated only when it is regenerated either by closing it and opening it again or by paging sections Each trace is listed by name in alphabetical order along with an icon that indicates some attributes of the trace Fig 8 1 A closed blue figure indicates that the trace is closed while an unclosed red line indicates that the trace is open Traces that are not simplified may contain loops see below so unsimplified traces are symbol ized by a figure eight shape Traces that are hidden from view are also listed with a special H icon In addition to the trace name and icon the list may contain several columns of additional data about each traces These Trace List
3. The Selection Tool signified by the arrow pointer can also be used to select traces in the section Place the tip of the arrow near a trace and press the left mouse button to select it Use the right mouse button to deselect it Pressing and holding the left mouse button allows a rectangular region to be delin eated When the button is released all traces that have a trace point inside the region will be selected When using any other tool depress the central mouse button or wheel to select the nearest trace This allows traces to be selected for deletion or moving without changing back to the selection tool For example when cutting traces with the Scalpel Tool one piece can be selected with the middle mouse button and deleted It is not possible to deselect individual traces without using the Selection Tool DESELECTING TRACES Traces can be deselected one at a time using the Selection Tool right mouse button The selected trace nearest the tip of the arrow cursor is deselected when the right mouse button is pressed To deselect all the selected traces simultaneously use the Deselect All item from the Trace menu Ctrl d Deselect All can be used with any tool Reconstruct v1 0 6 1 81 Traces x Figure 8 1 The Trace List window This Select Save Modify example list includes each of the possible list icons and trace names that correspond to the Trace Area attributes for these icons In this list only the
4. All traces can be hidden at once whenever a section is first displayed by using the checkbox in the General tab of the Series Options To prevent a permanent change to the section make sure the Autosave checkboxes for sections are unchecked as well Subsequently paging through the series will result in sections being displayed without any visible traces Go back to the General tab and uncheck the Hide all traces when loading section box Subsequent paging will display the traces again If the traces remain hidden then the sections were saved with Hidden attribute set on all traces To unhide them check the Unhide all traces when loading section box in the Series Options If both hide and unhide checkboxes are checked simul taneously all hidden traces will be revealed while all visible traces will be hidden This feature can be used to quickly switch between two sets of traces for the series SIMPLIFIED Trace simplification occurs automatically after traces are drawn with certain draw ing tools The trace is re pixelized to the resolution of the screen and then reduced to a set of line segments that match this path If the trace is closed all loops are 86 Reconstruct v1 0 6 1 Trace Attributes removed and the trace is reoriented to be clockwise Traces created as predefined shapes e g ellipses rectangles and stamp shapes do not undergo the simplifica tion process as they are assumed to be already simplified O
5. ified in the AutoTracing tab of the Series Options Reconstruct v1 0 6 1 105 Traces GENERATING WILDFIRES THROUGHOUT A REGION By pressing and holding the left mouse button the Wildfire Tool can be used to drag out a rectangular region Fig 8 11 When the mouse button is released all areas inside the stop criteria are automatically traced using the region growing algorithm When these regional wildfires are generated firebreaks are respected but existing traces are not Wildfires generated by the regional method cannot be automatically propagated to adjacent sections Regional wildfires can generate a very large number of traces and this can take a long time Make sure the stop criteria are set appropriately by testing them with a single mouse click before attempting to trace multiple wildfires using the mouse drag Use less than or greater than stop relations rather than differs by to generate fewer regions by tracing only white areas rather both white and black areas To further reduce the time required for regional wildfires work at lower magnification The large number of small areas sometimes generated by a regional wildfire can be controlled by setting the Ignore regional wildfires parameter in the AutoTrac ing tab to a larger value A value of zero the default allows all areas to be traced A value of 100 square pixels on the other hand will limit tracing to just the areas containing at l
6. in the AutoTracing tab of the Series Options Fig 8 10 If the resolution parameter is negative or zero then a resolution equal to 1 of the largest extent of the trace in the x or y direction is used Simplifying traces using a large positive resolution value results in traces with fewer trace points A smaller resolution value will produce many more points If the trace is large and the resolution is very small so many points may be generated that the response to a Simplify operation will become painfully slow To avoid slow simplifications make sure the resolution parameter is large enough If simpli fication is allowed to be performed automatically by checking this box in the Series Options the resolution will be chosen appropriately for the zoom factor allowing traces to be quickly simplified to a reasonable fidelity For simple shapes such as lines and rectangles the simplest possible representation is automatically generated For this reason these shapes are marked as Simplified as soon as they are created Unchecking the Simplified state of these traces and using the Simplify menu item will actually add trace points to these traces and may 102 Reconstruct v1 0 6 1 Wildfire Region Growing significantly change their shape For most other trace shapes some reduction in trace points without severe shape change may be possible provided the Simplify resolution is appropriately chosen Simplification will normally expand
7. 8 2 The mouse or keyboard can be used to access the fields of the dialog Use the Tab key to move through the fields For the Fill mode box use the arrows to highlight a mode and the space bar to select If any fields are grayed as shown for the Fill mode items in Fig 8 2 then the current state of these items has mixed values among the selected traces If grayed fields are not changed there will be no change to these attributes of the selected traces The Get Default and Get Clipboard buttons can be used to fill the attribute fields with the values of the default attributes or the attributes of traces placed on the trace clipboard Alternatively the individual fields can be filled as described in the following NAME The current trace name is shown in the Name box A new name for the trace may be typed in the box If more than one trace was selected then all the traces will be given the new name The name in the Trace Attributes dialog only modifies the selected traces When new traces are drawn they use the default trace attributes for the series which are set in the Tracing tab of the Series Options or with the Trace Palette see below If the selected traces had different names when the Attributes dialog was opened then an asterisk will be displayed in the Name box Only when the asterisk is replaced with a new string will the names of the traces be changed This allows the colors or other attributes of a set of traces to b
8. PCX PCC GEM Raster IMG Portable Greyscale map binary P5 type PGM and Portable Pixel map binary P6 type PPM For most formats it is required that the image filename extension reflect the file format used For example a portable pixel map file could be named myim age ppm but not myimage junk Exceptions to this rule are the common for mats Windows Bitmap JPEG TIFF and GIF The filenames for these formats is Reconstruct v1 0 6 0 17 Software Overview arbitrary because the program can identify the file format from header information in the file System Requirements Reconstruct was written and tested under Microsoft Windows XP on a Dell Opti plex GX260 Pentium 4 2 GHz computer with 512 Mb of RAM The Reconstruct binary executable is compiled for Pentium compatible processors and the applica tion relies entirely on the Win32 libraries for interacting with the computer s user interface Reconstruct will therefore run on any Pentium compatible computer that supports Win32 OPERATING SYSTEM Reconstruct will run on all Microsoft Windows operating systems Windows 95 Windows 98 Windows 2000 Windows NT and Windows XP Reconstruct will run under other operating systems provided a Win32 emulator translator is installed A Win32 translation layer for X Windows such as WINE www winehq com translates the Win32 API calls from Reconstruct into the equivalent X Windows operations WINE is a
9. to be between 0 and 1 inclusive If e is zero then the lines are parallel and the squared x y distance D is if a00 0 and a11 0 then D c c if b1 lt 0 else if a00 0 then D 4all 2 blt c if b1 2all ca b1 all otherwise c if b0 lt 0 else if a11 0 then D a00 2 b0 c if b0 a00 cH b0 a00 otherwise c if a01 lt 0 and b0 lt 0 a00 2 b0 c if a01 gt 0 and b0 gt a00 else D d all 2 bl c if a01 gt 0 and b0 gt a01 h if a01 lt 0 and a01 b0 gt a00 c b0 a00 otherwise Reconstruct v1 0 6 1 127 Calibration and Measurement If e is not zero then the lines are not parallel If fis less than or equal to zero then D is computed by c if b0 lt 0 if lt 0 and b1 lt 0 then D Ja00 2 b0 ce if b0 gt a00 c b0 a00 otherwise all 2 bl c if a01 lt b0 else if g gt e and b1 gt a11 then D h if a01 b0 gt a00 all 2 b1 c a01 b0 a00 otherwise c if b1 lt 0 ele D Jall 2 bl e if bl gt all e b1 all otherwise If fis greater than or equal to e D is instead computed by c if b0 lt 0 if g lt 0 and a01 b1 lt 0 then D 4a00 2 b0 c if b0 gt a00 b0 a00 otherwise all 2 bl c if a01 lt b0 else if g gt e and a01 61 gt a11 thenD h if a01 b0 gt a00 a00 2 b0 ce a0l b1 all otherwise c if b1 lt 0 else D Jall 2 bl c if bl gt al1l1 c b 1 a 11 otherwise 128 Reconstru
10. use Select All from the Trace menu to select all the traces then use Zoom To from the Traces menu Ctrl Home ZOOMING AND PANNING The magnification of the section display can be changed in several ways Using Center or Actual Pixels will change the magnification to either center the section image data or display the image data at the screen resolution respectively The Reconstruct v1 0 6 1 53 Sections previous magnification and position can be restored by using the Last Zoom item or equivalently hitting the End key from the tools window Select the tool that looks like a magnifying glass as shown to the left Depress and hold the left mouse button when the cur sor is at the corner of the desired region in the main window Drag the rectangle to cover the desired region and release the button The section will be re rendered to the largest magnification at which this region still fits entirely within the main win dow The previous magnification can be regained by clicking the right mouse but ton G The magnification can also be changed with the Select Zoom Region tool magnification and position When this tool has been selected the cursor will be crossed arrows Drag with the left mouse button depressed to pan the section After panning the hourglass may again appear while the sec tion is re rendered with the new settings Holding the right mouse button down changes the tool to the zoom cursor Dragging with the right mo
11. 0 000000 microns dendrite2 bmp BMP 2 0 000000 0 000000 microns dendrite3 bmp BMP 3 0 000000 0 000000 microns dendrite4 bmp BMP 4 0 000000 0 000000 microns dendriteS bmp BMP 5 0 000000 0 000000 microns dendrite6 bmp BMP 6 0 000000 0 000000 microns dendrite7 bmp BMP 7 0 000000 0 000000 microns dendrite8 bmp BMP 8 0 000000 0 000000 microns dendrite9 bmp BMP 9 0 000000 0 000000 microns Figure 1 1 The Import Images dialog box after images have been selected using the Select button Since the file names contain the section index automatic numbering is realized by having a 1 specified in the First Section box Figure 1 1 shows the Import Images dialog after some images have been selected The source directory is given at the top of the dialog The filenames of recognized image files and their formats are listed in the left most columns of the list The third column gives the section number to which each image will be imported Set the first section number of the sequence by typing the number typically 1 in the First Section box If the sections are not numbered correctly and the filenames con tain numbers indicating the correct order you can use the Sort Numerically check box to reorder the files in the list For finer control of section numbering you may have to import images in stages Create the section files by selecting the Import button in the dialog As the image files are added to the series they will be deleted from the list Af
12. Chapter 10 but it is usually much easier to align sections by specifying correspondence points and using the Align command from the Trace menu This option is outlined in the fol Reconstruct v1 0 6 1 5 Quick Start lowing steps It will only work if appropriate landmarks are visible in adjacent sec tions For best alignment results read Chapter 10 Step 1 Go to a section which will serve as the stationary reference Then page to the adjacent section that will be aligned to the stationary one Press the key to switch between the two sections Step 2 In the Trace menu select Palette to open the trace palette window Click on the first palette button and pick up the Stamp Tool from the tools window It is assumed that this is the default palette for a new series such that the first palette button is an orange circle with the default name a Step 3 Use the Stamp Tool to place traces at points to be aligned Use the key to alternate between sections as points are entered Enter the landmarks in the same order on both sections so that traces of corresponding points will be numbered the same To delete a point if you make a mistake use Ctrl D to deselect all the points pick up the Arrow Tool 1st tool button and select the point to be deleted then use the Delete key to delete it Use Ctrl S to reselect all the points to be aligned Step 4 After entering 3 or 4 correspondence points switch to the section
13. Last Section 9 r Save section images to Series Name mySeries_aligned cancel 62 Reconstruct v1 0 6 1 Exporting Section Images Figure 6 5 The effect of Fill with border colors Left Original section as it appeared in the main window Middle After Render Section with border color fill Right The effect is altered by a different order of the traces In this case the yellow region was on top so the interior regions were colored first and then yellow colored over everything so the interior appear only lightly darkened In the middle image the interior region were on top so they were colored last When the option to make images resemble current view is selected the pixel size to that of the main window the width and height is set to that of the main window and the offset is set to the current view offset Thus each section will be made to appear as it does in the main window For all other pixel sizes the width height and offset is set to fully contain all domains in all sections at the selected resolu tion The file format of the images can be selected to be either an uncompressed 24 bit Windows Bitmap or a compressed JPEG image file The Quality factor determines how much compression is applied to the JPEG output A value of 100 has the least compression and the best preservation of the image data while a value has max imum compression at the expense of image quality A value of 80 is usu
14. Series Options dialog Ctrl P Switch to from precision cursor when drawing Ctrl R Repeat last movement Ctrl S Select all traces on section Ctrl U Undo all current changes to the section Ctrl V Paste traces into section Ctrl X Cut selected traces from section for later pasting Ctrl Z Undo section or domain changes Home Center section image in view Ctrl Home Zoom and center selected traces in view End Undo redo last zoom Insert Paste traces into section Delete Delete selected traces or domain Reconstruct v1 0 6 1 29 User Interface Tools Window The Tools Window is a floating toolbar that allows the user to select different modes of mouse input in the main window The toolbar can be opened or closed by selecting the Tools Window item from the Program menu The Tools Window can also be closed by selecting the close box on the right hand side of the title bar A description of the tool appears in the title bar of the Tools Window whenever the a tool button is pressed Pressing the tool button also selects the tool for use in the main window The keyboard can be used to change tools without moving the mouse pointer To do this give the tools window the focus using Ctrl Tab Then use the Tab key to move through the tool buttons The highlighted button can be selected with the Enter key The Tools Window can be resized and placed anywhere on the screen The tool buttons will automatically reorganize to fit within the new window
15. The Contour element contains no other elements but has many attributes name the identifier of object to which the trace belongs hidden whether trace is displayed closed whether the trace closes on itself simplified whether trace points have been sim plified border the displayed color of the polygonal representation fill the color of the interior of the trace when filled mode the type of filling operation used comment a string of user information points the sequence of x y coordinates of of the trace Fig 3 2 shows a valid XML section data file based on the DTD Note that a similar DTD is defined for the series data file series dtd but it is not expounded here because the large number of series options makes the file very long To read the DTD format open the series dtd file using any text editor IMAGE FILE FORMATS Reconstruct currently supports several common single image file formats includ ing Windows Bitmaps JPEG TIFF and GIF The complete set of acceptable file formats is determined by the image input output library developed by Andy Key see Credits below These include Windows and OS 2 bitmaps BMP VGA BGA RLE DIB RL4 RL8 except for CCITT G3 MH Fax compressed and 24 bit RLE files CompuServe GIF files GIF JPEG File Interchange Format files JPG JPEG JPE TIFF files TIF TIFF with 1 4 8 and 24 bits per pixel True vision Targa Vista bitmaps TGA VST AFI ZSoft PC Paintbrush Image format
16. The effects of slant and deformation movements centered on a rectangle region Shown are the default movement directions When Deformations are selected F5 F8 produce the deformations shown in the top row In the default mode the slants skews shown in the bottom row are produced Each keystroke produces a fixed increment in the transformation that maps the data into the section The size of increments can be modified by holding the Ctrl or Shift key down while pressing one of the movement keys of Table 10 1 Typically Ctrl reduces the size of the movement while Shift makes the movement larger The size of the movement increments including the effect of Ctrl and Shift keys can be adjusted by using the Movement tab in the Series Options If the arrows keys do not produce a movement even when the section is unlocked try increasing the movement parameters in the Series Options The Movement Options tab includes a checkbox which changes the operation of the F5 F8 keys When the box is unchecked the F5 F8 keys produce skew Unchecking the box allows deformations in which one side of the object is scaled relative to the other side Figure 10 1 shows the difference between slants and deformations for the movement directions defined by the default movement parameters All keyboard movements are performed with respect to x and y axes aligned with the main window Movements are initially made relative to the origin of these axes a
17. abort without creating a trace use the Esc key The Line Tool is used to create a trace consisting of a single line segment This tool is useful for creating calibration lines as described in Chapter 9 Press and release the left mouse button to start the segment A candidate line will then follow the cursor until the left mouse button is clicked again to create the line trace To abort without creating a trace use the Esc key When a trace is created it is added to the set of selected traces on the current sec tion Selected traces are described next Selecting Traces When a section is first displayed none of the traces are selected When no traces are selected most of the operations in the Trace menu are disabled since the oper 80 Reconstruct v1 0 6 1 Selecting Traces ations are defined on selected traces only To modify or use traces it is necessary to first select them Traces can be selected in several ways One way is to list the traces by selecting List from the Trace menu Select one or more traces from the list and then click on the Select item at the top of the Trace List window When traces are selected they are drawn with a dashed border and possibly a colored interior Selecting other traces from the trace list will first deselect any existing selections To quickly select all the traces on the section use the Select All item from the Trace menu This operation is accelerated by the Ctrl s key sequence
18. and Mouse Actions in Trace List Window Command Action Up amp down arrows Highlight next previous item in list Page Up amp Down Move through the list by the number of visible traces Home Move to the first trace in the list End Move to the last trace in the list Delete Delete the highlighted traces Enter Select the trace in the main window Ctrl or Shift Highlight multiple traces Left Mouse Click Highlight the trace under the pointer Left Mouse Dbl Click Select the trace under the pointer Trace Palette Window The Trace Palette Window can be opened or closed using the Palette item from the Trace menu The window displays the set of predefined trace attributes as a set of buttons Each palette entry represents the trace name border color fill color and fill mode for drawing new traces This is displayed on the button by a trace drawn in the shape of the stamp trace also defined in the palette entry For more on palette entries see Chapter 8 The Trace Palette Window works much like the Tools Window It can be resized and placed arbitrarily on the screen The default trace attributes can be set by selecting the palette entry button with a left mouse click The Tab key can be used to move through the palette buttons The highlighted button can be selected with the Enter key When a palette button is pressed the default trace name of the palette entry is displayed on the title bar TABLE 4 9 Keyboard and Mouse Actions in Trac
19. any configuration However if opening the 3D Scene generates an error or crashes the program a likely culprit is the OpenGL hardware interface An updated video driver might be available to fix the problem The speed and quality of the 3D Scene rendering is a result of the OpenGL imple mentation of the graphics hardware a k a the video display adaptor Most Win dows computers are designed for 3D video games and have excellent OpenGL graphics displays appropriate for Reconstruct For example the 32Mb ATI Radeon VE graphics adaptor www ati com was used during code development and gave nice results To render 3D scenes with even better resolution and quality use third party software specifically designed for that purpose such as 3D Studio MAX www discreet com 0 00821684 0 052455 60 0241702 0 466433 6 0148161 Figure 12 1 A screen shot of Reconstruct with a 3D Scene window open The objects highlighted in the Object List with blue icons reside in the scene These objects include several dendrites various colors and axons orange reconstructed from a series of electron micrographs from the hippocampus The color of the axons was changed from their trace colors using the In Scene Attributes dialog 164 Reconstruct v1 0 6 1 Composing a Scene All OpenGL surfaces are generated as collections of triangular surface patches as described in Chapter 11 This allows the surfaces to be quickly rendered and ani mated us
20. area in domain list include midpoint in domain list include comments in trace list include lengths in trace list include areas in trace list include x centroids in trace list include y centroids in trace list include z distance in trace list include sect thickness in trace list include section range in object list include trace count in object list include surface area in object list include flat area in object list include volume in object list include z trace comment in list include section range in z trace list include length in z trace list custom colors for trace borders Reconstruct v1 0 6 1 45 Series TABLE 5 1 Series Options Stored in the Series File Option Type Default s Description fillColors floats 0 0 0 custom colors for trace fills offset3D floats 0 0 0 positional shift applied to object max3Dconnection float 1 Boissonnat surfacing parameter first3Dsection integer 1 first section for 3D generation last3 Dsection integer 9999 last section for 3D generation type3 Dobject integer 0 type of 3D representation to use upper3 Dfaces boolean true Boissonnat surfacing parameter lower3 Dfaces boolean true Boissonnat surfacing parameter faceNormals boolean false whether to create face normals vertexNormals boolean true whether to create vertex normals facets3D integer 8 density of faces in 3D output dim3D floats 1 1 1 dimensions of 3D object gridType integer 0 default type of grid trace to draw gridNumber intege
21. asterisk the question mark the pound sign the plus sign and the dollar sign The dollar and plus signs can be used any where in the name string to automatically number trace names The dollar sign in a trace name string is replaced with the current section number when a trace is created For example the name string s would result in the string s101 for all traces created on section 101 and s45 for all traces created on section 45 A plus sign in a trace name results in the immediately preceding integer being incremented The number is incremented every time a new trace is drawn For example the default name string vesicle would result in vesicle1 for the first trace vesicle2 for the second trace and so on Multiple plus signs can be used but plus signs occurring together will be reduced to a single one Initialize the increment at a particular value by preceding the plus sign with an integer For Reconstruct v1 0 6 1 83 Traces Name D1 Border E M Hidden f Simplified Closed Fill Ba Fill when not selected Mode J None V Darken f Solid Lighten Comment Get Default Get Clipboard Apply Cancel Figure 8 2 The Trace Attributes dialog The title bar indicates how many traces were selected when the dialog was invoked All of these traces will receive the changes made in the dialog Grayed checkboxes or buttons e g the Hidden checkbox indicate
22. be represented at a resolution of 0 001 series units A trace point of more than 6 significant digits would be rounded when it is stored e g 10000 059 to 10000 1 so the significant digits parameter must be large enough to capture the desired precision of the data over the whole range of the series volume The number of significant digits that are displayed and stored can be modified in the General tab of the Series Options Increasing the significant digits will allow a larger range and or increased resolution but will also increase the storage require ments for the section files The significant digits parameter also determines how many digits are displayed in lists and output to csv dxf and wrl files Decreasing the parameter is generally not a good idea as this can result in making traces inac curate but saving fewer significant digits has the benefit of reducing the size of the files The actual position of a trace on the section is additionally determined by the asso ciated transformation also stored in series units Because the transformation con sists of just a few parameters it is always stored at the full resolution Since all real values are represented by 64 bit floating point numbers the full resolution is 15 significant digits plus an exponent 110 Reconstruct v1 0 6 1 Units of Measurement IMAGE PIXEL SIZE Images are stored as raster bitmaps in the original image files When this data is imported into
23. black region will be ignored i e it won t be part of the final boundary Wildfires will not spread beyond the display of the window except for the part under the status bar and will not spread into a region in which there is no domain image If an attempt is made to ignite a wildfire trace on a black region no trace will be created The stop criteria for the Wildfire tool can be changed in the AutoTracing tab of the Series Options Fig 8 10 Existing traces can be used as stop criteria by checking Reconstruct v1 0 6 1 103 Traces the appropriate box in the AutoTracing tab but the main criteria that stop region growing are the relations set on the hue saturation and brightness values The default stop value of Brightness is less than 100 means that Wildfires will not pass through pixels that have an intensity less than 100 where 0 is black and 252 is white Changing this option to Brightness differs by 50 will allow either white or black regions to be automatically traced with the Wildfire tool Saturation refers to the amount of color A saturation value of 0 has no color i e it is gray scale Hue is actual color red green blue etc To get a visual picture of the col ors defined by different hue saturation and brightness values open a color dialog by clicking on the Fill color button in Names Colors tab Note that brightness is sometimes referred to as luminance A stop criterion can be disabled by sel
24. chosen a subsequent dialog right allows for applying the pixel size adjustment to all domains in the series inaccurate point placement at the ends Draw more lines to also reduce inaccura cies of a single trace When possible draw lines in both directions on the calibra tion grid CALIBRATE SELECTED TRACES Once calibration trace are drawn select the traces on the section With only the calibration traces selected select the Calibrate item from the Traces menu A dia log box will appear for each selected trace displaying the current length of the trace Enter the actual length of the trace in place of the current length For an open trace the length is the sum of the distances between trace points The length of closed traces is the entire perimeter including the closing segment RESIZE TRACES SET PIXEL SIZE OR SCALE SECTION The end result of calibration can be one of three things resizing the selected traces to the length specified changing the pixel size of the domain in which the traces were drawn or scaling all the transformations in the section domains and traces Which of these three operations will be performed is determined in the Calibrate Traces dialog Fig 9 2 presented after the lengths of all selected traces have been entered Complete the calibration operation by choosing one of the options and selecting OK No changes will be made if the Cancel button is selected 114 Reconstruct v1 0 6 1 Calibra
25. clockwise loop It is possible to change sections while drawing with the pencil The trace will be created only on the section in which the trace is terminated even if points were added while other sections were displayed This is similar to the behavior of other tools that involve mouse dragging such as the Ellipse and Rectangle Tools 98 Reconstruct v1 0 6 1 Editing Traces Like the stamp and grid tools the section can be panned by dragging the right mouse button near the edge of the main window when the Pencil Tool is used Editing Traces Existing traces can be edited using the tool that looks like scissors The tool is used to break a trace at the cursor point and start redrawing it using the point by point method Backspace can be used to undo a portion of the trace after breaking it with the scissors If the trace is clockwise as most are then be sure to select a point further clockwise than the part of the trace to be redrawn This will ensure that backspace which is usually counterclockwise gets rid of the unwanted part of the trace When the left mouse button is pressed the segment of the trace nearest the cursor is removed and the tool is immediately switch to a point by point drawing tool If an open trace is edited the portion of the trace after the break point is removed before redrawing For a closed trace only one trace point is removed by the Scissors Tool The subsequent drawing operation can be aborted b
26. column and once with the object in the right column Use more specific limit strings to speed up the list cal culations For example to generate only distances from numbered vesicles vesiclel vesicle2 to a synapse named mySynapse enter vesicle for the first limit string and mySynapse for the second limit string 3D Object Representations An object is added to the 3D Scene Chapter 12 using one of several possible 3D representations Which representation is generated is determined by the selection in the Generate box of the 3D tab of the Series Options dialog Fig 11 2 The 3D representation is generated only for the traces of the object in the section range indicated WARNING Closing the series during 3D generation will crash Reconstruct 152 Reconstruct v1 0 6 2 3D Object Representations Figure 11 2 The 3D tab of the Series aixi Options determines how an object will be aa Proses Thumbrois General Tracing Grids 3D Lists represented in the 3D Scene Specify how 3D scene objects will be generated from traces m Generate Traces C Traceareas Trace midpoints C Box Boissonnatsurface Cylinder C Ellipsoid Sphere using traces in sections 1 through 48 m Shift object by X 0 Y Oa 0 microns Surface Normals C None Face Vertex Faceting Density C4 8 C CO2 Size A 1 ee 1 1 microns Es Surfa
27. determined by the limit string in the Limit To box The limit strings are reset to the default value whenever a series is opened The rows of this list will be all elements that match the limit string A limit string can be a specific value such as mito which would display only rows named mito The wildcard characters asterisk question mark and pound sign allow a range of values to be selected An asterisk matches any string of characters A question mark matches any single character And a pound sign matches any single digit For example D will limit the list rows to those that begin with a capital D The limit string 42 Reconstruct v1 0 6 1 Series Options IX Fi l e 2x Figure 5 2 The Lists tab of the Series Movements Proxies Thumbnails AutoTracing H H H i Se a Options Dialog The settings in the Lists tab determine what rows and columns will Use wildcard string to limit rows Check columns to display be displayed in the list windows Any p Section List p Trace List p Object List changes will be accepted with the OK eee ee am button and rejected with the Cancel i button Shown are the default settings for v Thickness JV Comment Range a new series fv Length I Count r Domain List v Area I Surface area Limit To V Centroid Flatarea Foo Exen 7 Volume JV Z distance ae Iv Source ASM Limit To Iv Pixel size p Distances List m I Length Lim
28. frame TABLE 4 4 Keyboard and Mouse Actions in Tools Window Command Action Tab Move to next tool button Enter Select highlighted tool Left mouse click Select the tool under the pointer Status Bar The Status Bar can be opened or closed by selecting the Status Bar item from the Windows submenu in the Program Menu The Status Bar displays information about the data under the pointer when a series is open The left most element on the Status Bar is a lock unlock icon that indi cates whether the current section can be moved or aligned Chapter 6 The Status Bar also contains the number of the section being displayed the name of the trace nearest to the mouse pointer and the position of the pointer on the section in series units On a domain the Status Bar displays the position of the pointer in image pixels When drawing the Status Bar displays the name of trace being drawing and either position length or area information for the drawing increment 30 Reconstruct v1 0 6 1 Section List Window Section List Window The Section List Window is opened by selecting the List item from the Section menu The section list displays the sections of the series in order of ascending sec tion number The two attributes of each section can be displayed the lock unlock status and the section thickness in series units The columns of the list can be resized by dragging the column borders with the left mouse When the section
29. i Area The area is the size in square units of the region enclosed by a trace A nega tive area indicates that the trace has a counterclockwise orientation A positive area indicates a clockwise orientation The area of an open trace is zero Area and centroids are calculated from a formula based on Green s Theorem 1 area 5 is Xi 1Yi f for each segment from x y to X 1 yj in the trace The accuracy of this com putation is dependent on the trace being free of internal loops and the values x y not being too large Since 64 bit floating point values are used for all such calcula tions the second factor should not be an issue for most series Centroid X This column reports the x position of the centroid of the trace in series units The calculation is 122 Reconstruct v1 0 6 1 Object List Measurements 20 TX 4 MOV 41 Xi 4 x SV Odin X Vi Centroid Y This column reports the y position of the trace centroid in series units calculated by Vo FY DOVi41 X14 Wd L y SV Odie Xj4 i Min X This column reports the minimum x position of the trace in series units Min Y This column reports the minimum y position of the trace in series units Max X This column reports the maximum x position of the trace in series units Max Y This column reports the maximum y position of the trace in series units Z Position The Z distance of the section computed as described in the previous sectio
30. image data that is stored in separate image files To delete these image files along with the section files set the checkbox in the Delete Sec tions dialog The UNDO Stack Each section has an undo stack associated with it The undo stack keeps copies of the section data as changes are made allowing the user to return to previous ver sions of the section Changes saved in the undo stack include movements of sec tions domains and traces adding or deleting traces and domains etc The undo stack can hold up to 16 changes The undo stack for a section is cleared each time 58 Reconstruct v1 0 6 1 Thumbnails the section is loaded into memory A section is loaded into memory whenever it is displayed in the main window as the current section The exception is when it was the previous section displayed in which case the undo stack is retained in memory Only when two new sections have been displayed is the undo stack for a section finally deleted from memory To restore the section to the state prior to the last change select the Undo item from the Section menu This can be accomplished more quickly by using Ctrl z on the keyboard If the Undo item is disabled then the undo stack is empty To return to the state prior to the first change in the undo stack select the Reset item from the Section menu Immediately after an undo is made the state of the section prior to the undo can be restored using the Redo command from the
31. image in the 3D Scene window in a JPEG file Since JPEG is a compressed image format the user is prompted for the amount of compression to use before the file is written 172 Reconstruct v1 0 6 1 Scene Information x Figure 12 3 The Scene Into dialog box reports the total number of scene elements including those not visible Scene contains by transparency occlusion or view point 1 Objects 264 Vertices 532 Normal vectors 0 Line segments 532 Triangles Scene Information The number of data elements that compose a scene determines the speed of render ing the size of VRML files and the complexity of future scene processing There fore it is often desirable to reduce the total number of elements in a 3D Scene The total number of elements including vertices line segments triangles and normal vectors is tallied in the Scene Info dialog box Fig 12 3 To reduce the number of vertices and triangles it is necessary to reduce the number of trace segments prior to generating the 3D representation This can be done using the Simplify command Chapter 8 but note that such changes are permanent so you may want to make copy of any objects before you Simplify them Reconstruct v1 0 6 1 173 The 3D Scene 174 Reconstruct v1 0 6 1 Bibliography Boissonnat J D 1988 Shape reconstruction from planar cross sections Computer Vision Graphics and Image Processing 44 1 29 Fiala JC 2005 Reconstruct A fre
32. is ineffective in terminating these opera tions Thumbnails The Thumbnail options apply to the thumbnail window described in Chapter 6 The thumbnail window displays a view of the sections in pre rendered form These section thumbnails can be quickly browsed and animated When viewing sections in the main window each section is redrawn when it is accessed This process is slow because each image is transformed into position using com plex calculations Checking the box next to Overlay thumbnails to make a flip book causes the thumbnail window to display sections on top of each other creating a fast version of the normally slow Page Up and Page Down operation Autotracing The final tab contains the options related to automatic simplification and region growing of traces Initially automatic simplification is always turned on and traces are simplified after drawing Simplification eliminates loops and other artifacts that can reduce the accuracy of measurements and three dimen sional representations It is advisable to keep the option on at all times For more details on the other simplification options see Chapter 8 Wildfires are a form of autotracing based on a region growing algorithm The Stop parameters control when region growing will terminate These parameters may need to be set care fully to get the Wildfire tool to work at all For more details see Chapter 8 TABLE 5 1 Series Options Stored in the Series Fi
33. is sized to the maximum dimension of the object When the object is a single trace on a single section this representation gives a reasonable point representation of the trace Alternatively a Sphere object can be given an explicit diameter by entering a positive value in the Size A box of the Options tab 3D PARAMETERS In addition to the type of representation and the section range several other param eters can be used to adjust the object s 3D representation Not all parameters are applicable to every type of reconstruction Shift Object By These parameters define an offset in the x y and z directions to be applied to the 3D representation These parameters let you position any type of Reconstruct v1 0 6 2 159 Objects 3D representation anywhere in the scene but they should be used carefully In par ticular set the parameters to zero when done shifting objects so that future recon structions from the series will not be mistakenly shifted Surface Normals All representations except Traces define surfaces To properly reflect light and produce a shaded 3D shape each part of the surface should have a surface normal vector For Trace Areas and Trace Midpoints no normals are gener ated because these representations use emissive color rather than diffuse color see Chapter 12 For all other representations the Surface Normals option allows nor mals to be generated for faces vertices or eliminated altogether Eliminat
34. keyboard movements as well Undoing Movements Movements of a selected trace or domain or of an unlocked section can be undone by using the Undo item from the Section menu or equivalently Ctrl z as described in Chapter 6 Each incremental movement is pushed onto the undo stack so that multiple undos will back up through a movement sequence The state of the section prior to any changes can be recovered by the Reset command To move a domain it must first be selected from the section by using the domain list or the domain selection tool Chapter 7 An Undo stack is created for a selected domain so that keyboard movements can be undone Although the Undo stack has a limited depth the original position of the domain is retained at the bot tom of the stack Backing up through the entire stack will eventually lead to the original position the one prior to selection of the domain This starting position 134 Reconstruct v1 0 6 1 Repeating Movements can also be restored by using the Reset item from the Section menu Once the domain is deselected the movements are no longer undoable However the entire domain change can be undone by using Undo on the section In rare circumstances it may be necessary to completely reinitialize the domain transformation returning the domain to the origin just as though it is newly imported This can be accomplished by using the Reinitialize gt Transform item from the Domain menu This options is on
35. listZTraceLength borderColors Type boolean boolean boolean integer integer boolean boolean boolean integer integer integer integer boolean integer integer float floats floats integer string string boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean boolean floats Default s false false false 128 96 true false false 1 9999 1 5 true 2048 1536 0 25 1 0 1 1 0 1 9 domain true true true false false false true false true false false false false true true false false false true true true 0 0 0 Description hide all domains when paging unhide all domains when paging absolute vs relative path to image width of thumbnail buttons height of thumbnail buttons display traces on thumbnail images thumbnail overlay flag center sections on thumbnails first section for thumbnails last section for thumbnails distance between thumbnails frequency of shuttle play in Hz proxy images will be used threshold width for creating proxy threshold height for creating proxy scale factor for proxy dimensions default trace border color default trace fill color default trace fill mode default trace name default trace comment string include thickness in section list include source path in domain list include pixel size in domain list include length in domain list include
36. must be fully visi ble in both sections Another way to limit the alignment region is to edit the domain boundary Only the part of the image within the new domain boundary will be displayed and therefore used By Correlation Tips for Aligning Sections Alignment and reconstruction of serial sections is an art Obtaining accurate results depends on many factors including the experience of the user and her familiarity with the material to be reconstructed A number of good practices that can improve the outcome are presented here For a simple protocol see Chapter 1 Section the Material Appropriately Experience has shown that sectioning is a very important consideration Sections should be as thin as is reasonable and at least thin enough that lots of common features are available in adjacent section Too much disparity between sections and alignment is very difficult if not impos sible In addition sectioning should be done such that as many objects as possible are cross sectioned with the long axes perpendicular to the sections This ensures that cross sectioned features rather than oblique features will be available for alignment Beware of Ambiguous Alignments Ambiguous alignments arise when multiple interpretations are possible for the profiles of an object Figure 10 4 Aligning whole fields of objects with a single smooth transformation can minimize the dis torting consequences of these local ambiguities since the ambiguities o
37. names such that traces can be paired by name This can often be achieved by using the naming convention when creating traces Chapter 8 To align traces between sections select in one section the traces that will be the stationary reference Page to the other section and select the traces which will be moved With the section locked open the Align traces submenu and select the desired alignment operation e g Rigid The selected traces will be immediately repositioned ALIGNING ENTIRE SECTIONS When the section is unlocked the Align submenu is changed from Align traces to Align section This indicates that the entire section will be moved by the alignment command rather than just the traces The same procedure is used for aligning sections as with aligning traces Select in one section the traces that will be the stationary reference Page to the other section and select the traces which will be aligned to this reference Unlock the section Chapter 6 From the Align section submenu select the desired alignment oper Reconstruct v1 0 6 1 137 Movements and Alignments Figure 10 2 Blending of unaligned left and aligned right sections Misalignments produce a blurry image on the left while on the right the blending of aligned images results in sharp clear features where they are in cross section ation The selected traces and the rest of the section will be immediately reposi tioned
38. order that they are painted onto the section Fig 7 4 The domain name is the name of the domain boundary trace so it will be created from the default trace attributes at the time the domain is added to the section In addition to the domain name the list can contain columns denoting the path to the source image file the pixel size of the image the domain boundary length the domain boundary area and the midpoint of the domain all in series units The columns displayed in the Domain List are determined by the check Reconstruct v1 0 6 1 69 Domain Images boxes in the Lists tab of the Series Options The entire list can be saved to a file using the Save command at the top of the list window Only a single domain can be highlighted in the list Pressing Enter or clicking the Select menu item at the top of the list will select the domain from the section and display it in isolation Whenever a domain is selected the Domain List will be automatically closed The Modify menu at the top of the Domain List window allows the highlighted domain to be modified in several ways The domain can be deleted hidden from view or unhidden The Attributes menu item allows the attributes including the domain name the source path and the pixel size of the highlighted domain to be edited without selecting the domain from the section Selecting a Domain A domain in a section can be selected using the Select Domain Image is Tool f
39. pairs of sections in a series by blending Blending can be turned off by selecting the Blend menu item again or by hitting the key Reconstruct v1 0 6 1 55 Sections Listing all Sections All the sections in a series can be listed using the Section List window described in Chapter 4 The section list window is opened by selecting List from the Section menu The columns and rows displayed in the Section List are determined by the settings in the Lists tab of the Series Options The entire list can be saved to file using the Save command at the top of the list window To speed up the display of the section list it is created from the section files in the series folder when the series is opened The list is updated as changes are made within Reconstruct but changes made outside the program such as deleting sec tion files will not be incorporated in the section list until the series is reopened from the Series menu Editing Section Attributes Each section has three modifiable attributes the section thickness the movement lock flag and the section index Changes to these attributes can be made from the Section menu or from the Modify menu at the top of the list MOVEMENT LOCK The lock unlock attribute can be toggled by selecting the Lock or Unlock item from the Movement submenu of the Section Menu When a section is unlocked movements of the entire section are permitted Accidentally hitting an arrow or function k
40. section By creating multiple domains within a section a large montage can be formed from smaller images Fig 7 1 A section can contain no more than 254 image domains iojxj Figure 7 1 A section composed of two m domains Each domain is a separate image imported into the section Domains are easily positioned so that they overlap where they contain the same data The inset window is the Domain List for the domain2 section domain1 Copyright 2006 John C Fiala 65 Domain Images Source C fiala RECONSTRUCT test Import Pixel Size 0 002540 OfsetXx 0 0 y o0 v Copy files to series folder Quit Randomize J Sort numerically Start numbering at oa and increment by HE Place Image of Type into Section at Position J100CX 1885 JPEG 1885 0 000000 0 000000 microns 100CX 1886 JPEG 1886 0 000000 0 000000 microns 100CX 1887 JPEG 1887 0 000000 0 000000 microns 100CX 1888 JPEG 1888 0 000000 0 000000 microns 100CX 1889 JPEG 1889 0 000000 0 000000 microns 100CX 1890 JPEG 1890 0 000000 0 000000 microns J100CX 1891 JPEG 1891 0 000000 0 000000 microns 100CX 1892 JPEG 1892 0 000000 0 000000 microns 100CX 1893 JPEG 1893 0 000000 0 000000 microns J100CX 1894 JPEG 1894 0 000000 0 000000 microns KA _100cx 1895 JPEG 1895 0 000000 0 000000 microns Figure 7 2 The Import Images dialog The list shows the selected image files in the same order as they were listed in the folder Since the F
41. section These sections are stored along with the x and y posi tions of the trace points so that 3D measurements and shapes can be generated from the Z trace data Pressing the left mouse button starts the trace A drag line follows the cursor to the next point Page up or down to change sections and add trace points with left mouse clicks on the desired sections Clicking the right mouse button ends the z trace at the last point entered with the left mouse button The creation of a z trace can be aborted by using the Esc key Z traces cannot be edited or undone but they can be reviewed and deleted from the Z Trace List window Chapter 10 Z traces are not scaled during recalibration so they should be created only after the calibration is determined Reconstruct v1 0 6 1 107 Traces 108 Reconstruct v1 0 6 1 CHAPTER 9 Calibr ation and Measurement This chapter deals with the quantitative aspects of Reconstruct such as calibrating the dimensions of the series and getting accurate measurement data from the traces Units of Measurement Reconstruct works in an arbitrary system of units The user can specify the name of the units in the General tab of Series Options Each series has its own units Note that Reconstruct does not interpret or understand the units string It is only a comment entered by the user to signify what units are used when data is entered None the less the choice of units should be
42. selected in the section To also remove the selected traces from the section use Cut Ctrl x Copying or Cutting to the trace clipboard deletes any traces currently in the clipboard before storing the new ones Traces deleted with the Delete key or menu item are not put on the clipboard When the trace clipboard has data that can be pasted into the section the Paste item in the Trace menu is enabled Select Paste from the menu or use Ctrl v to add the traces to the section Any traces that are selected on the section are deselected and the pasted traces are selected Pasting data does not empty the trace clipboard and the same traces can be repeatedly pasted into sections Use Paste Attributes Ctrl b to copy the attributes of the first clipboard trace onto the selected traces Get Clipboard buttons in trace attribute dialogs also allow the attributes of a clipboard trace to copied to other traces or palette entries To see what is on the trace clipboard open the Trace Attributes dialog and use the Get Clipboard This is 90 Reconstruct v1 0 6 1 Setting Trace Attributes for Drawing ix reece 2x Figure 8 5 The Tracing tab of the Series Movements Proxies Thumbnails AutoTracing H i i i EE Tam pior doo Te seirene Trace names can be 63 characters long is replaced with the the name color and comment that will be section number A plus increments each new trace increments within a section Illegal char
43. series but all the sections above the offset were aligned and all the section below the off Reconstruct v1 0 6 1 135 Movements and Alignments set were also aligned Fixing the offset by moving the upper section would now cause this section to be misaligned with the next higher section By propagating the same movement to all higher sections the entire series could be put into align ment NOTE There is no undo for Propagate The Repeat menu item is only available when an unlocked section or a selected item is available for movement whereas the Propagate operation can be applied anytime after a movement has been made This is because the Propagate operation applies to entire sections and the sections do not have to be unlocked first REPEATING MOVEMENT SEQUENCES When the keyboard is used to move a trace or domain only the last keyboard increment is saved for repetition with Repeat or Propagate but it is sometimes desired to repeat a sequence of keyboard movements A sequence can be captured using the Record submenu of the Movement menu Selecting Start from this submenu clears the recording buffer and prepares for accumulating movements Any subsequent keyboard Type In or mouse drag movements are added to the recording until the Stop item is selected from the Record submenu Note that alignments or repeated movements are not added to the recording After Stop the recorded sequence is moved to the last movement buffer where
44. sets the default trace attributes to the corresponding user defined palette entry The Ctrl key in combina tion with the number key sets the current default attributes onto the corresponding palette entry When the palette name contains automatic numbering it does not get updated like the default trace name does during tracing Use the Ctrl number key command to update the palette after tracing For example if the Oth palette entry name is set to d00 then selected and used for drawing 4 traces the default trace name would be incremented d01 d02 d03 d04 but the palette entry would remain d00 Pressing Ctrl 0 would update the palette entry to d04 The palette is mainly for quickly changing the default attributes when drawing new traces However it can be used to modify existing traces as well To give the attributes of a palette entry to an existing trace select the trace and the palette entry from the Trace Palette Then open the Trace Attributes dialog Ctrl a and use the Get Defaults button to fill the dialog with the attributes from the palette entry Reconstruct v1 0 6 1 93 Traces Stamping Predefined Shapes rendered quickly with a single mouse click The shape is centered at the position of the stamp tool cursor The shape is drawn at a fixed pixel off set from the cursor position such that the shape is a constant size with respect to the screen This means that tracing at high magnification will produce a
45. shift it until the edges of the two pieces match Merge the second domain into the sec tion to complete the process Domain Boundary The domain boundary is a trace that defines what part of the image will be dis played The initial default boundary is a rectangle surrounding the entire image To change the domain boundary first select the domain from the section Then select a drawing tool that creates a closed boundary such as the Freehand Pencil or the Point by Point Tool Define the default trace attributes to use for the boundary trace including the name that will be given to the domain Draw the new boundary on the domain image When the boundary is completed the domain image will be repainted with only the portion interior to the boundary displayed To return to the original domain boundary select Boundary from the Domain Reinitialize menu 72 Reconstruct v1 0 6 1 Domain Attributes A domain boundary can also be created from an existing trace by pasting the trace from the trace clipboard onto the selected domain Place the trace on the trace clip board before selecting the domain Do this by selecting the trace and copying it Chapter 8 Domain Attributes When a domain is selected edit its attributes by selecting Attributes from the Domain menu at the top of the main window Fig 7 5 These attributes can also be changed without selecting the domain by using the Attributes item from the Modify menu at th
46. that will be moved Verify in the title bar that the stationary section is not displayed Step 5 Choose Linear from the Align section submenu of the Traces menu When the section is not unlocked this submenu will be titled Align traces instead of Align section Enter Ctrl L to unlock section movement The status bar will display an open lock image If traces are accidentally aligned instead of the images use Ctrl Z to undo the trace movement If the message Alignment needs more traces is displayed make sure you have entered at least 3 points on each section and that all points are selected Ctrl S on both sections If this message still appears make sure the corresponding traces in each section have the same name In other words a1 in section 1 must be matched to a trace a1 in section 2 a2 must match a2 and so on Step 6 After Align Section Linear has moved the section use or blend space bar to confirm that all parts of the section are aligned Chapter 10 If the align ment is acceptable lock Ctrl L the section to prevent further movements 6 Reconstruct v1 0 6 1 Getting Started Step 7 If the alignment is not acceptable use Ctrl Z to undo the movement Check the names and positions of the traces entered and modify or add traces as needed Go to Step 5 As a last resort delete all traces by entering Ctrl S followed by the Delete key Do this for each of the two se
47. the Dis tances List the Distances calculation can usually be aborted without adverse con sequences by closing the window However if a list view attempts to display too many items it may make the entire program unresponsive so that the window can not be closed When this happens you may need to abort the entire program to stop the operation Reconstruct can usually be terminated by closing the Main Window If a series is open it will be closed prior to exiting the program One exception is when adding objects to the 3D Scene from the Objects List When 3D representations are being generated the current series should not be closed nor should the program be termi nated This will cause a fatal error that may result in the loss of unsaved changes to the series During 3D object generation use the Abort button provided and wait for the operation to terminate on its own before closing the series or exiting the pro gram In the event that Reconstruct becomes unresponsive such as when trying to fill a Distances List with 65 000 measurements you may need to abort the program from the outside To do this open the task manager by using Ctrl Alt Delete or by selecting Run from the Start menu and entering Taskmgr In the Task Man ager select the Applications tab and find the Reconstruct icon beside the name of the series If the Status is listed as Not Responding highlight this entry with the mouse and then click on the End Task b
48. the screen resolution not at the resolution of the underlying image data Drawing when zoomed at high magnification allows subpixel traces with respect to the image data The resolution and density of trace segments is gen erally greater when drawing at higher magnification Copyright 2006 John C Fiala 79 Traces Several drawing tools are provided in the Tools window Tools window buttons indicate the type of trace to be drawn When the trace will be closed with the end connected back to the beginning the button depicts the interior of the trace filled with a cyan color Pressing a tool button places a description in the title bar of the Tools window Moving into the main window changes the mouse pointer to the tool cursor Most drawing tools also support a precision crosshair cursor Ctrl p Here are some examples of basic drawing tools and their graphical designations The use of the other drawing tools will be described in separate sections in this chapter The Ellipse Tool is used to quickly create circles and ellipses Press and hold the left mouse button while moving the mouse to define the shape Release the mouse button to create the trace To abort the ellipse without creating a trace hit the Esc key before releasing the mouse button The Rectangle Tool is used to generate a closed rectangular trace Drag press and hold the left mouse button to define the rectangle Release the mouse button to create the trace To
49. these values are used to dimension the box in the x y and z directions respectively When the Size parameters are zero or negative the box is sized to the extent of the object Note that the Box object can be used to generate scale bars or cubes for the scene Simply select the Box representation and set the desired Box dimensions with the Size parameters Then select an object defined in the series and add it to the scene The scale Box will be centered on the midpoint of the object extent in 3D If neces sary the scale Box can be shifted to a different location in the scene by changing the Shift Object By parameters see below and re adding the object to the scene BOISSONNAT SURFACE A Boissonnat Surface is generated from a 3D Delaunay triangulation of the traces using the algorithm invented by Jean Daniel Boissonnat Boissonnat 1988 In general terms the surface is constructed of triangular surface patches where an edge of each triangle lies on the traced profile The Boissonnat Surface preserves the vertices created during tracing although additional vertices may be added to the final reconstruction The Boissonnat algorithm attempts to generate a maxi mum solid with no singularity While this representation may not be ideal in some instances the method can handle multiple traces per section in a general way that produces reasonable results for objects that are branched When the Boissonnat Surface is not reasonable there are se
50. window is active shows the name attribute in the title bar The colors and J zNa ze d gt gt El ae el lal lolol ete 7 Figure 8 6 The Trace Palette window The top row of buttons displays the user defined palette entries while the bottom row displays the predefined palette entries Each button shows the default border and fill colors fill style and stamp tool trace shape 92 Reconstruct v1 0 6 1 The Trace Palette fill styles are rendered on the buttons The shape on the palette button is the shape of the stamp trace that will be adopted whenever the Stamp Tool is used see below Define the trace palette entries with the Tracing tab of the Series Options Fig 8 5 Each user definable palette button is shown in this tab Select one and edit the attributes in the Attributes box The palette entry attributes are changed immedi ately an OK in the Series Options dialog is not required To copy the attributes from an existing trace to the palette copy the trace to the trace clipboard and then select the palette entry in the Tracing tab Use the Get Clipboard button to copy the clipboard attributes immediately to the palette entry You may need to close and reopen the Trace Palette to see the changes The palette entries can also be selected and set using the keyboard Table 4 2 even when the Trace Palette window is not open Pressing the number keys 0 through 9 when the main window has the keyboard focus
51. y Reconstruct is a Windows application for montaging aligning iles S tracing measuring and reconstructing objects from serial Photos section images Links 7 Database By subscribing to this group you will receive announcements Polls about future releases and be able to participate in discussions Membars f about using Reconstruct Whether you subscribe or not you Pendir may return to this page to view the latest anouncements and Fencing h gt discussions Calendar J Happy Reconstructing Promote j a Invite A KS JC Fiala 4 15 2004 Figure 3 4 The welcome page for the Reconstruct Users Group at Yahoo Groups History The origins of Reconstruct began in 1995 with the development of a Windows 3 1 program for tracing serial sections WinV8 was developed in the Image Graphics Laboratory at Children s Hospital Boston as a continuation of the DOS based V8 program developed by John Davis Very quickly Win V8 was replaced by a new Win32 application IGL Trace Ver sion 1 0a of IGL Trace was completed in August of 1997 with NIH funding to the Mental Retardation Research Center at Children s Hospital IGL Trace has proven useful for identifying tracing measuring and reconstructing 3D objects in serial sections in a number of scientific studies IGL Trace was designed to operate on aligned sections These can be images obtained from electron or light microscopy For electron microscopy where each section is i
52. 0 all trace points The eigenvector corresponding to the largest eigenvalue of the scatter matrix determines the central axis of the Cylinder The other two eigendirections specify 158 Reconstruct v1 0 6 2 3D Object Representations the directions of the cylinder s diameters These directions are scaled by the eigen values to get the final dimensions of the Cylinder The dimensions of the Cylinder can be explicitly defined using the Size parame ters Size A sets the length of the cylinder Size B sets diameter along the second ary axis while Size C sets the diameter along the minor axis ELLIPSOID The Ellipsoid representation is essentially the same as the Cylinder representation except the object is closed at the ends of the principal axis The axes of the Ellip soid are computed from the eigenvectors of the scatter matrix of the trace vertices and the Ellipsoid is centered at the centroid of the trace vertices The dimensions of the ellipsoid are determined by the scatter matrix eigenvalues unless positive val ues are entered in the Size parameters Information about the calculated axes and dimensions of cylinders and ellipsoids can be obtained by looking at the comments in an exported VRML file for the object Chapter 12 SPHERE The Sphere is centered at the midpoint of the object extent in the x y and z directions The diameter of the Sphere is set to the maximum extent in x y or z In effect the Sphere
53. 6 merons Figure 8 11 An example of using the Wildfire Tool to trace multiple areas To begin the cursor is positioned and the left mouse held down left A rectangle is created by dragging the mouse middle When the left mouse button is released right multiple areas are grown up to the stop criteria of Hue differs by 40 Saturation differs by 50 and Brightness is greater than 200 To prevent creation of small areas a minimum size of 100 square pixels was required for trace creation PROPAGATING WILDFIRES TO ADJACENT SECTIONS Wildfires can be automatically propagated to adjacent sections using the f or b keys The f key propagates the most recent wildfire forward to higher sections while b key propagates the most recent wildfire backward to sections with a lower index Once initiated automatic tracing of wildfires will continue section by section to the end of the series unless the user terminates the process Ctrl t or the wildfire algorithm fails to generate a new region Automatic tracing of wildfires proceeds by using the centroid of the last wildfire as the ignition point of the wildfire on the next section If ignition is successful the wildfire grows to fill an area defined by the stop criteria The area filled is com pared to the area of the previous section If this area differs by more than a certain percentage the wildfire propagation is aborted The percentage value can be spec
54. Action Left mouse drag Rotate scene Right mouse drag Zoom scene Left and right drag Pan scene Note Pressing the right mouse button will pause any on going animation Z Trace List Window The Z Trace List window is opened from the Object menu The window displays a list of all the z traces in the section ordered alphabetically The trace name is dis played along with the section range and the length in series units An additional column can be used to make a notation about the trace The columns of the list can be resized by dragging the borders of the column headers The entire list can be saved to a csv file by choosing the Save menu item A trace in the list can be highlighted with the left mouse button or by using the arrow keys Multiple traces can be highlighted by holding down the Shift or Ctrl 36 Reconstruct v1 0 6 1 Distances List Window keys Pressing Enter or using the Select menu item will cause the highlighted traces to be represented as 3D objects and added to the 3D Scene TABLE 4 12 Keyboard and Mouse Actions in Z Trace List Window Command Action Up amp down arrows Highlight next previous trace in list Page Up amp Down Move through the list by the number of visible traces Home Move to the first trace in the list End Move to the last trace in the list Delete Delete the highlighted traces from the series Enter Put the trace into the 3D scene Shift Highlight multiple traces Left M
55. Changes can be canceled with the Esc key SWITCHING BETWEEN WINDOWS The keyboard focus can be switched between windows of the application by using Ctrl Tab much in the same way as the Alt Tab command is used to switch between active applications on the desktop The window that has the keyboard focus is called the active window An active window usually has a colored title bar while inactive windows often have grayed title bars TABLE 4 1 Basic Keyboard and Mouse Actions for All Windows Keystroke Action Alt Access the menu of the currently active window Alt Tab Switch between applications Ctrl Tab Switch between floating windows of the application Left Mouse Drag Action on title bar Position the window on border Size the window 26 Reconstruct v1 0 6 1 Main Window Title Bar Buttons Main Menu i Tools Window Main Window Status Bar Section 71 Figure 4 1 Reconstruct s initial window layout A main window with a narrow border and a black interior is overlaid with a floating tools window and a status bar Main Window When the application is started the Reconstruct main window appears along with two of the sub windows the tools window and the status bar Fig 4 1 The main window consists of a title bar at the top a set of menus beneath the title bar and a rendering area a k a client area for displaying data At the left end of the title bar is the Reconstruct icon When a series is open
56. Data Precision Image Pixel Size Calibration Grids and Scale Bars Calibrating Traces O O O Drawing Calibration Traces Calibrate Selected Traces Resize Traces Set Pixel Size or Scale Section Apply the Calibration to Other Sections Section Thickness Calibrating Section Thickness Z Distance Status Bar Measurements Domain List Measurements Length Area Midpoint X Midpoint Y Trace List Measurements Length Area Centroid X Centroid Y MinX MinY Max X Max Y Z Position Thickness Object List Measurements Count Surface Area Flat Area Volume Z Trace List Measurements Length Distances List Computation Algorithm e CHAPTER 10 Movements and Alignments O Oo 0 0 0 Transformations Keyboard Increments Movement Type In Dialog Undoing Movements Repeating Movements Propagate Repeating Movement Sequences Alignments Aligning Selected Traces Aligning Entire Sections Evaluating Alignments Undoing and Adjusting Alignments Oo 0 0 0 Repeating an Alignment Montaging Domains by Point Correspondences Aligning By Correlation Tips for Aligning Sections Section the material appropriately Beware of ambiguous alignments Avoid aligning the same features everytime Don t over align Avoid quadratic aligns Choose the starting point carefully One Strategy e CHAPTER 11 Objects O G G O0 0O 0 0 0 0 O The Object List Changing Object A
57. General tab contains parameters for controlling how data is saved and displayed The units description string that specifies what units are used for quantitative values such as measurements All quantities within a series are expressed in these units All non image data is saved as text versus binary for mat The data precision parameter controls how many significant digits are writ ten to represent each number The General tab also contains the default section thickness given to new sections and an option for changing how the section thick ness is used in calculating distances perpendicular to the sections Details on the use of these options can be found in Chapter 9 The hide and unhide options in the General tab determine whether traces and domains images will be displayed on screen when a section is accessed and also whether the traces and domains will be marked as hidden when a section is saved Consequently turning on the Hide all traces option with the Automatically save sections option will hide traces as sections are accessed and these traces will remain hidden even after the Hide all Reconstruct v1 0 6 1 41 Series traces option is turned off To make the traces visible again the Unhide all traces option should be turned on Names Colors The Names Colors tab is defines the default trace parameters i e the name color and other attributes that will be used whenever a new trace is dr
58. Reconstruct Serial Sections User s Manual Updated 3 1 06 John C Fiala e CHAPTER 1 Quick Start oO0O000 0 0 O Getting Started Create a Series Import Section Images Calibrate Pixel Size Align Sections Trace Profiles View Three Dimensional Object About the Rest of the Book e CHAPTER 2 TBD e CHAPTER 3 Software Overview O O Series Sections Domains Traces and Objects Data File Organization XML File Formats Image File Formats System Requirements Operating System OpenGL Hardware Requirements History Credits Source Code Development Tools e CHAPTER 4 User Interface O T O 0 0 0 0 O 0 0 OO Co Windows and Dialogs Resizing and Moving Windows Switching Between Windows Main Window Main Menu Tools Window Status Bar Section List Window Thumbnails Window Domain List Window Trace List Window Trace Palette Window Object List Window 3D Scene Window Z Trace List Window Distance List Window 0 Exiting or Aborting e CHAPTER 5 Series O O 0 0 0 0 O Creating a Series Opening and Closing Series Saving a Series Series Options Exporting and Importing Images Exporting Trace Lists Importing Line Drawing Importing Series Data e CHAPTER 6 Sections Oo 0 0 0 Oo 0 0 0 Oo 0 0 O Section Indexing Creating a Section Saving a Section Displaying a Section Centering Zooming and Panning Paging Sections Blending and Flick
59. Section menu Once additional changes are made the Redo option is no longer available and the Redo item will be disabled Redo can also be used to restore the previous state after a Reset opera tion Thumbnails Thumbnail representations of the sections are displayed in the Thumbnails win dow as described briefly in Chapter 4 Two modes of display are possible Thumb nail buttons can be displayed in a sequential array in which many sections are visible simultaneously Alternatively the thumbnails can be overlaid on top of each other in a flipbook arrangement This mode of display and other parameters of the thumbnails are set in the Thumbnails tab of the Series Options dialog Fig 6 3 The thumbnail size parameters specify the width and height of the images in screen pixels Any size and aspect ratio can be specified but consider that the thumbnail images will all be maintained in memory Thus 100 sections rendered at 128x96 would use about 4 Mb of memory while the same number of thumbnails at 512x384 would require an additional 52 Mb Also rendering of the buttons will take longer for larger image sizes Rendering of the buttons may require nearly as much time as paging the sections depending on how slow the retrieval of image Reconstruct v1 0 6 1 59 Sections data is However once the thumbnails are rendered access will be much faster than paging There is an absolute limit on the amount memory Windows will allocate for b
60. a don t care condition As long as these boxes are not modified these parameters will not be changed on any of the selected traces example the default name string my24 th_ vesicle would result in my25th_vesicle for the next trace created my26th_vesicle for the next and so on When the dollar and plus signs are used together incrementing is restricted to individual sections Each new trace created is given a number higher than all the other traces in the section that have the same leading characters Unlike the previ ous special characters the section is searched for similar trace names every time a trace is created So while a plus sign can generate duplicate trace names within a section dollar plus names tend to be unique For example a default name string vesicle will generate vesicle1 vesicle2 vesicle3 on section 45 and then on section 101 will generate vesicle1 vesicle2 Returning to section 45 would then generate vesicle4 vesicle5 and so on Trace Attributes Every trace has a set of attributes that specify its name color fill style and whether it is closed hidden or simplified Newly created traces are given the cur 84 Reconstruct v1 0 6 1 Trace Attributes rent default attributes see below Trace attributes can be modified at any time by selecting the trace s and then opening the Attributes dialog from the Trace menu Fig
61. a section the pixels are mapped into series units using a magnifica tion factor the pixel size The pixel size parameter tells how big a square pixel of the image is in series units Each image has its own pixel size parameter This allows images obtained at dif ferent magnifications or scanned with different dpi settings to be accurately incor porated into the same series A domain is stored as a pointer to the pixel data file and a pixel size parameter The actual position of the domain on the section is additionally determined by the associated transformation also stored in series units The pixel size of an image can be determined from the magnification factor of the microscope and the dpi used to digitize the optical image into pixels Many com puter controlled imaging systems report the pixel resolution as part of the data so that it doesn t have to be calculated It can just be entered directly into the pixel size input field when importing images or domains The basic formula for calculating pixel size is nit A pixel size dpi magnification inc where units are the chosen series units dpi is dots or pixels per inch and mag nification is the optical magnification from the specimen to the digitizer A reminder of this formula is provided in new domain import dialog chapter 7 For example suppose an electron microscope is used with a known magnification factor of 10 000 from specimen to the
62. acters are gt lt used to label traces when they are drawn m Give new traces these default attributes The attributes of a palette entry can also Name domain be set by pressing one of the palette Border B F Filwhennot selected buttons and then modifying the Attributes Mode of new traces settings Changes to the default attributes and palette entries are C None Darken C Solid Lighten la x CEHZ comment affected immediately The OK and Cancel Get Clipboard Attributes buttons only apply to the simplify settings T Selectormodify palete attributes at the bottom of the tab will fill the dialog fields with the values of the first trace on the clipboard reveal ing the trace name color etc Setting Trace Attributes for Drawing When a trace is drawn it is given the current default trace attributes These are set in the Tracing tab of the Series Options Fig 8 5 The default tracing attributes are saved with the series so that tracing can easily be continued when a series is reopened The Attributes in the Tracing tab are essentially the same as those in the Attributes dialog from the Trace menu described above Absent from the default attributes are Hidden Simplified and Closed checkboxes Drawn traces are always initially unhidden The closed and simplified attributes of new traces are determined by the drawing tool used and the status of automatic simplification Rec
63. ain Image Tool by using keyboard commands or by using one of the other methods discussed in Chapter 10 If the precise arrangement of sections is known in advance e g each image is offset by 10 microns along the 10 axis use the Import Images dialog from the Series menu with particular X and Y offsets to place the domains in the correct locations on the section Reconstruct v1 0 6 1 71 Domain Images fa ali le dialog This dialog allows the attributes pame of a domain to be changed By selecting Source C fiala reconstruct mito _unaligned 11 Cancel no changes are made Format JPEG 8 bits pixel Size 768 x 768 pixels 576 Kb Proxy none Pixel size 0 00269 microns Contrast 1 000 Brightness 0 000 Cancel Multiple domains can be created for an image by importing the image in the sec tion multiple times This allows different pieces of an image to placed in different ways on the section Such a capability can be used to repair the effects of cracks or folds in the section For example an image of a cracked section can be repaired by drawing domains for each piece on either side of the crack and shifting the two pieces back together To do this import the section image select it and redraw the domain boundary around the section on one side of the crack Merge the domain into the section Then import the image a second time and redraw the domain around the other side of the crack Blend the new domain with the section and
64. ain the alignment movement generated from Align Traces on corresponding points Suppose an adjacent section is already montaged Fig 10 4A This can be accom plished by manual movements or by using an image that contains the entire field at low magnification To align domains in the next section to this adjacent section first enter corresponding point traces at places on the domain Fig 10 4B and the adjacent section Fig 10 4A that should be put into alignment This is done on the sections without domains selected rather than on selected domains With the point 140 Reconstruct v1 0 6 1 Montaging Domains by Point Correspondences Q ee se gt We Figure 10 4 Montaging by point correspondence A Adjacent section low resolution field or completed montage B New domain on section with yellow triangles representing correspondences to points in A C Align Traces D Repeat the movement on the selected domain to place image at the correct location on the section traces selected in both sections use Align Traces from the Trace menu to generate the correct alignment movement for the domain This will move the traces from the domain to the correct positions on the section Fig 10 4C To move the domain to match the trace movement select the domain and use Repeat from the Movement menu This will place the domain at the location of the moved traces Fig 10 4D Reconstruct v1 0 6 1 141 Movements and Alignment
65. airs of sec tions can help use more of the whole field Don t Over Align Ignore the urge to make every alignment perfect some local misalignments are to be expected since objects will move relative to each other Don t rely just on blend to evaluate alignment use flicker to see if there is a pattern of motion consistent with a whole field misalignment Reconstruct v1 0 6 1 145 Movements and Alignments Avoid Quadratic Aligns As much as possible use Linear or Rigid Align com mands as these introduce the least distortion Use Quadratic and Deformal align ments sparingly Choose the Starting Point Carefully Don t start the alignment from an obviously distorted section Choose a stationary reference section without folds or cracks and with a uniform high contrast It may be helpful to choose a section in the mid dle of the series to minimize the differences introduced in the series relative to the reference For montaged sections it may be helpful to start from a low magnifica tion section that covers the entire montage area This will allow the montage to be arranged accurately as well ONE STRATEGY One strategy that can facilitate aligning two sections by point correspondence is to begin by picking two points at low magnification that should obviously be near each other These points can be deleted later in favor of more accurate ones With the section unlocked select Align sections gt Rigid Alt t n r to bring the
66. al images underneath a domain or to speed up section display by reducing the amount of image data displayed Hidden domains appear in the Domain List with a red H icon Thus the Domain List can be used to hide and unhide domains All domains can be hidden at the time the section is first displayed This allows all images to removed from display leaving only traces that can be displayed very quickly Hide all domains with the checkbox in the General tab of the Series Options To prevent a permanent change to the section make sure the Autosave checkboxes for sections are unchecked All domains can be unhidden with a Gen eral option as well If both checkboxes are checked simultaneously all hidden domains will be revealed while all visible domains will be hidden This feature can be used to quickly switch between two sets of domains for the series Proxies Proxy images are smaller versions of domain images that can be quickly displayed for low magnification views of the section When proxies are in use the paging of 76 Reconstruct v1 0 6 1 Proxies sections at low magnification and the rendering of thumbnails can be faster because less image data needs to be read from the hard disk Initially no proxy images exist You must explicitly create them Proxy images are created and deleted for all sections from the Proxies tab of the Series Options Fig 7 6 When the Proxies tab is accessed there may be a pause with the h
67. aller say 0 028 the thumbnails could be generated in just a second However at 0 028 our full screen display of sections would slow down again because these proxies are too small to be used PROXY RULE OF THUMB FOR SINGLE DOMAINS If your domain image Domain List gt Modify gt Attributes is at least twice the screen resolution Control Panel gt Display gt Properties gt Settings use this for mula screen height fraction 1 2 P proxy fraction a domain height If your domain image is not twice the screen size set the threshold to less than the domain dimensions and set the proxy fraction for thumbnail display thumbnail height fraction 1 2 proxy traction domain height 78 Reconstruct v1 0 6 1 CHAPTER 8 Traces Traces are the basis for quantitative measurements calibrations and alignments When traces outline the profiles of sectioned objects these data elements can also be used for three dimensional visualizations of object structure Each trace is a polyline or polygon represented as an ordered list of points in series units connected by straight line segments Point order is clockwise for traces with a positive orientation Clockwise traces have positive area and enclose the interior of the object Counterclockwise traces have negative area and enclose holes Creating a Trace Traces are created on sections by drawing with the mouse or pen All traces are drawn and digitized at
68. ally appro priate for compression while maintaining most of the image quality Each image filename is given a leading underscore followed by the string entered in the Series Name box followed by a numerical extension indicating the section number To allow verification of the rendered images using Reconstruct new sec tion files are created along with the image files A new series file is also created with the name entered in the Series Name box All the files are created in the same Reconstruct v1 0 6 1 63 Sections folder as the original series Thus an attempt to overwrite the series by giving the same name in the Series Name box will result in an error message To display only the image data in the section select None in the Traces option box To display traces in the same way they appear in the main window select the As displayed option This will produce a similar effect that seen in the Thumbnails window when the Display traces on thumbnails option is checked Hidden traces will not appear in the exported image and the Series Options to hide unhide all traces will effect whether traces appear Any changes to the current sections must be saved to the section file to be reflected in the exported images This includes traces which have just been hidden or unhidden An alternative method of displaying trace information on rendered sections is the Fill with border colors option With this option traced regio
69. andling the JPEG JFIF image formats Stefan Gustavson Provided the ANSI C port of the complex FFT that is used for image correlations Ju Lu Implemented the section to section propagation of wildfires Anyone may contribute to the next version of Reconstruct by joining the Recon struct Developers Group at http groups yahoo com group reconstruct_developers Members of the Developers Group gain access to the source code and may participate in developing the next version of Reconstruct The developers group also provides additional information and links to the free development tools used for creating the application DEVELOPMENT TOOLS Reconstruct was created using a minimalist approach to development As a conse quence all of the essential tools used to develop Reconstruct are free Reconstruct does not rely on propriety libraries or particular commercial compilers and inte grated development environments The following free resources were the principal development tools used For more information please consult the Reconstruct Developers Group Borland The program was compiled and linked using the Borland 5 5 Free Com mand Line Tools and Standard C libraries http www borland com 22 Reconstruct v1 0 6 0 Credits Jeremy Gordon Borland s free resource compiler did not support all the desired features like animated icons in dialog resources Luckily Jeremy Gordon has a better resource compiler GoRC tha
70. awn Use this tab to set the values before starting to draw See Chapter 8 for details on trace names and other attributes The Names Colors tab also allows the trace palette entries to be edited The trace palette can be accessed from the Trace menu to quickly switch between trace names and attributes while drawing Note that changes to palette entries are carried out immediately rather than waiting for an OK in the Series Options dialog Such changes cannot be cancelled using the Cancel button Grids The trace parameters for the Grid Tool are specified in the Grids tab Grid traces a re predefined shapes that are draw onto the section in repetitive patterns or arrays For more details on the types of grid patterns that can be generated using these options see Chapter 8 3D The 3D tab specifies how three dimensional representations of objects are generated from traces Chapter 11 These representations determine the size posi tion and appearance of objects in the 3D Scene The 3D tab also controls the sec tion number range from which such representations are created When generating objects from the traces make sure the sections are included in the section number range in this tab Lists The options in the Lists tab Fig 5 2 determine what data values are dis played in the data columns of the list windows The columns of the lists are speci fied by checkboxes for each list These settings are saved with the series The rows of a list are
71. brate operation If multiple traces are selected Calibrate will ask for the length of each trace by name and then com pute the average scale factor or pixel size for these values DRAWING CALIBRATION TRACES The easiest traces to create with a known length are usually line traces but other types of traces can be used First set the default drawing parameters using the Traces tab of the Series Options When drawing multiple calibration traces it may be convenient to use a default trace name to allow the names to be incre mented in the section Chapter 8 When all calibration traces will be of the same length they can all have the same name if desired Select the line tool eft from the tools window and draw a line trace on the calibration grid image Place the ends of the line at the edges of the grid elements Make the trace as long as possible to minimize the effect of Reconstruct v1 0 6 1 113 Calibration and Measurement Calibrate Traces Resize selected trace s Section Range First 1 Last 100 Setpixel size to 0 002691 microns pixel for domain domain1 and optionally modify additional domains Set pixel size to 0 002691 microns pixel A This operation cannot be Undone Cancel Scale entire section by 1 000000 Cancel Figure 9 2 The Calibrate Traces dialog left Three options are provided for adjusting the section data based on the entered trace lengths When the second option is
72. button alone Dragging the cur sor upward with the right mouse moves the viewpoint away from the scene effec tively zooming out Dragging downward zooms in bringing the scene closer As parts of the scene get very close to the viewpoint they will disappear from view This feature can be used to look through the exterior surface of an object to see the inside SPIN An important aspect of viewing scenes is dynamic rotation to allow the 3D struc ture to be fully appreciated on a 2D computer screen As mentioned above the scene may be animated by using the mouse to spin the scene Spin can be com pletely disabled by unchecking the Spin item in the View menu at the top of the 3D Scene window To produce a particular horizontal or vertical spin check the Spin menu item and use the Rotation dialog to set the rotational velocity Horizontal spin can be pro duced by setting the vertical spin rate to zero Likewise purely vertical spin can be realized by setting the horizontal spin rate to zero Setting both rates to zero stops all movement Spin can also be zeroed using the right mouse button in the 3D Reconstruct v1 0 6 1 169 The 3D Scene Scene Window When Spin is completely disabled the Rotation dialog allows a specific rotational increment to be applied to the scene BACKGROUND COLOR The background color of the scene is white by default but can be changed to any color supported by the graphics system To change the ba
73. by using the Open item from the Series menu Use the Open Series dialog to navigate to an existing folder and select the ser file for the series Alternatively a series can be opened by dragging an ser file onto the Reconstruct application icon or filename An open series can be closed by selecting the Close menu item from the Series menu or by exiting the application Within Reconstruct only one series is open at a time Opening or creating another series will close the current one Multiple series can be opened on the desktop by running multiple copies of the application Saving a Series A series can be saved by selecting the Save item from the Series menu This saves only the ser file not the section data files The series file stores the current posi tion in the series z traces palette entries and the series options described below Table 5 1 The series file can be saved automatically whenever the series is closed by turning on an option Automatic series saving is the default for new series To turn it off change the option in the Series Options dialog and then select Save from the Series menu to put the change into the ser file Similarly a Save is needed to turn this option back on Series Options The series file contains a set of attributes that remember the user defined options Table 5 1 These are accessed through the Options dialog from the Series menu The Options dialog contains tabbed pages that separate th
74. can be added to the sections using the Images dialog from the Series Import menu Fig 7 2 This dialog allows a set of images to be added to sequential sections in the series one domain image per section When a candidate list of images is ready for import use the Import button in the upper right of the dialog to carry out the operation To fill the Import Images list with candidate images use the Select button in the upper left corner of the dialog Highlight one or more images in the Select Files dialog Fig 7 3 Use the Open button to close the dialog and add the candidate images to the list Only files that the program can recognize as readable image files will be added to the list Acceptable image file formats are single image file for mats including Windows and OS 2 bitmaps BMP VGA BGA RLE DIB RL4 Reconstruct v1 0 6 1 67 Domain Images RL8 except for CCITT G3 MH Fax compressed and 24 bit RLE files Com puServe GIF files GIF JPEG File Interchange Format files JPG JPEG JPE TIFF files TIF TIFF with 1 4 8 and 24 bits per pixel Truevision Targa Vista bitmaps TGA VST AFI ZSoft PC Paintbrush Image format PCX PCC GEM Raster IMG Portable Greyscale map binary P5 type PGM and Porta ble Pixel map binary P6 type PPM Once the Import Images list has been filled with one or more images specify the first section into which the images will be imported by typing a number in the First Sect
75. ces Don t connect points further than 1 microns Generate V upperand V lowerhorizontal faces emee TRACES The Traces selection in the Generate box Fig 11 2 produces 3D lines corre sponding to the traces defined in the sections The lines are colored using the trace border color and spaced according to the Z distance calculation for each section Chapter 9 For this type of representation only the section range parameters from the 3D Options tab are used TRACE AREAS The Trace Areas representation also shows the traces distributed in space but the traces are filled with a planar surface having the border color Reversed orientation counterclockwise traces appear as holes inside the filled areas For example an object drawn as two concentric circles with the inner circle Reversed using the Trace menu item will appear as an annulus in the 3D Scene with Traces Areas For Traces Areas to generate a reasonable result follow the tracing rules defined below for Boissonnat surfaces In particular traces must be properly oriented with out self intersections This can be most easily accomplished by keeping the Auto matically simplify option turned on when drawing Chapter 8 Reconstruct v1 0 6 2 153 Objects The method used for generating Trace Areas is Delaunay triangulation of the trace vertices followed by boundary completion This is also the first step in the Bois sonnat surfacing al
76. ckground color select the Background item from the View menu Select a color from the Color dialog and click the OK button Custom Colors in this dialog reflect the last custom colors accessed by the program and will not be saved with the series Saving the Scene The scene in 3D Scene window may be saved either as a bitmap image or as a 3D model In neither case is it possible to open the saved scene using Reconstruct The scene can only be exported for viewing or further editing with other programs VRML 2 0 To export the scene as a 3D model use the Export As VRML 2 0 menu item from the Scene menu Select the desired directory and file name Virtual Reality Modeling Language VRML files use the wrl file name extension This file extension will be automatically added to the filename The VRML 2 0 output adheres to the VRML97 file format specification www web3d org All objects are represented as indexed line or face sets and are easily imported into other 3D graphics packages for editing or display The VRML file can be displayed in most web browsers by using a plug in for VRML To find out if a VRML plug in is currently installed direct the web browser to a VRML plug in detector http cic nist gov vrml vbdetect html A number of free VRML plug ins can be downloaded from this web page as well When the scene is opened with the VRML viewer it should appear as it did in the 3D Scene window at the time it was saved The backgr
77. ct is flexible enough to allow the use of this scale bar data but in most instances a calibration grid image is preferred Calibration images can be stored anywhere within the series in a few sections at the end of the data section or in some empty corner of the data sections them selves By tradition a single calibration grid for the series is placed in an empty section at the start of the series in section 0 Section 0 is a special section that is not used to define objects or compute the z distance The thickness of section 0 can be arbitrary A section 0 can be created using New from the Section menu and entering 0 for the file extension In section 0 use New from the Domain menu to import the calibra tion grid image The calibration grid can be imported with any pixel size as this will later be changed to the correct value 112 Reconstruct v1 0 6 1 Calibrating Traces PEPE died ot aah oad ol 3 ted aed ad wed oot r A pa y i Bb l la DE aA g gt DS ip s a zf one D Poper Jy EE E Idaa ded edo oS fon Figure 9 1 Image of calibration grid An electron micrograph of a diffraction grating replica with a known line spacing of 0 463 microns The scale bar at the left under the arrow was generated by the instrument at a nominal length of 1 micron Calibrating Traces The process of calibration in Reconstruct involves drawing traces of known length and then entering that length value using the Cali
78. ct v1 0 6 1 Distances List Computation Finally if fis between zero and e D may be obtained from c if b0 lt 0 if lt 0 then D 2a00 2 b0 c if b0 gt a00 c b0 a00 otherwise all 2 bl c if a01 lt b0 else if g2 e then D h if a01 b0 gt a00 a11 2 b1 c a01 b0 a00 otherwise In the final case where g is also between 0 and e the segments probably intersect but to make sure the full distance is calculated and the magnitude checked x R P f d0 g dl e y R P f d0 g d1 e D x y If this value is very small lt 10 then D is explicitly set to zero So some objects that touch may have small non zero distances but objects that don t touch will never accidently be given a distance of zero The minimum squared x y distance Min D between two traces is determined by repeating the above calculation for every pair of segments in the two traces The actual 3D Distance between the traces is then computed by z ZDistance T ZDistance T Distance T T3 4 Min D z where the z distance of the section of each trace is computed as described above Thus inter object distances will be dependent on the method of z distance calcula tion only when the section thickness is not uniform throughout the series Reconstruct v1 0 6 1 129 Calibration and Measurement 130 Reconstruct v1 0 6 1 CHAPTER 10 Movements and Alignments Data within a section can b
79. ct v1 0 6 2 Renaming Objects menu Scene color changes are forgotten whenever the object is removed from or added to the scene Scene changes are discussed in detail in Chapter 12 The Attributes command permanently modifies all the traces of the selected objects in all sections of the series For example to change the color of an object select the object in the Object List and then open the Attributes dialog The Attributes dialog will appear with a title indicating that 1 object will be changed Fields of the dialog will be grayed to indicate that no changes will be made to these fields unless a change is specifically entered in the dialog Click on the Border but ton and select a color for the object Close the color dialog and click the Apply but ton to change the border color of all the traces of the object in all sections NOTE There is no undo for changing object attributes Changes made to the object traces will not be reflected in the Object List or 3D Scene immediately If a change is made to the color of an object s traces and that object is already in the 3D Scene the object will need to re added to the scene to show the color change Obviously a simpler way to change the color in the scene is to use the Color Transparency dialog from the Object List Scene menu Renaming Objects Objects are renamed by changing the name attribute of the object s traces Unlike the Attribute dialog the Rename dialog all
80. ctions and then start over with Step 3 Step 8 Once the alignment is acceptable you can delete the alignment traces from both sections If you decide to keep the alignment traces for later editing you will need to switch the color and name of future correspondence points to avoid confu sion with the existing ones Switching to a new trace name ensures that traces with the same numbering will be created on both sections To switch correspondence point names simply select the second button on the trace palette Later when you repeat this step after aligning the next section go back to the first palette entry alternating between trace names after each alignment Step 9 Page Up or Down to the next section to be aligned and go to Step 3 TRACE PROFILES Information about sectioned objects is created by tracing the profiles of the objects on the sections To do this return to the first section in the series by using Page Down or by selecting the first section in the list generated from the List item of the Section menu From the Series menu select Options This opens a dialog that will allow you to configure the default parameters for tracing Select the Names Colors options tab at the top the dialog Put the cursor in the Name edit box and type a name for the object to be traced such as myObject This will be the name given to the profiles you draw All profiles with the same name are considered part of the same object Hit Ent
81. d Lum 120 Blue 127 Add to Custom Colors Figure 8 4 The Color dialog This dialog is used for setting trace border and fill colors Colors added to the Custom colors will be saved with the series CLOSED A trace can be either closed or open An open trace does not have the end of the trace connecting back to the start of the trace Generally the open vs closed char acteristic is determined by the choice of drawing tool used to create the trace For example ellipses and rectangles are created as closed traces while lines are open The open vs closed characteristic of traces can be changed by using the Closed checkbox in the Attributes dialog BORDER COLOR The border color is the color of the line segments that are drawn to represent the trace on screen Every trace has a visible border with a color The Border Color button indicates the current color of the selected trace If this button is gray with a diagonal line through it then there is more than one color for the borders of the selected traces The border color can be changed by pressing the Border Color button This opens the Color dialog shown in Fig 8 4 The Color dialog allows a new color to be cho sen from the Basic colors Custom colors or created from the color palette Ini tially the Custom colors are all black These can be modified by using the Add to 88 Reconstruct v1 0 6 1 Trace Attributes Custom Colors button The set of custom trace bor
82. d and Mouse Actions in Domain List Window Command Action Up amp down arrows Move to next previous domain in list Page Up amp Down Move through the list by the number of visible domains Home Move to the first domain in the list End Move to the last domain in the list Delete Remove the domain from the section Enter Select the domain from the section Left Mouse Click Highlight the domain under the pointer Left Mouse Dbl Click Select the domain under the pointer Trace List Window The Trace List Window is opened using the List item from the Trace menu The window displays a list of all the traces within the section ordered alphabetically The trace name is displayed along with a set of measurements in series units The columns of the list can be resized by dragging the borders of the column headers The set of displayed columns can be specified from the Lists tab in the Series Options dialog The entire trace list can be saved to a file by choosing the Save menu item A trace in the list can be highlighted with the left mouse button or by using the arrow keys Multiple traces can be highlighted by holding down the Shift or Ctrl keys Pressing Enter or using the Select menu item will cause the highlighted Reconstruct v1 0 6 1 33 User Interface traces to be selected and the keyboard focus to be shifted to the main window Any traces that were previously selected in the main window are unselected TABLE 4 8 Keyboard
83. d with the keyboard but can be scrolled through using the scroll button on the mouse For more on the configuration of thumbnails see Thumbnails in Chapter 6 TABLE 4 6 Keyboard and Mouse Actions in Thumbnails Window Command Action Tab In flipbook mode stop start shuttle play Enter In flipbook mode page to the section shown Left Mouse Click Page to the section under the pointer Mouse Scroll In flipbook mode move through the thumbnails Domain List Window The Domain List Window is opened using the List item from the Domain menu The window displays a list of all the domains within the section ordered from back 32 Reconstruct v1 0 6 1 Trace List Window to front The domain name is displayed along with the source path to the image data the pixel size and the centroid position of the domain on the section The col umns of the list can be resized by dragging the borders of the column headers A domain can be highlighted in the list with the left mouse button or by using the arrow keys The attributes of the highlighted domain can be edited by selecting the Attributes menu item This action does not close the domain list Double clicking the left mouse button or hitting Enter will cause the Select menu item to be exe cuted on the highlighted domain In this case the domain is selected in the main window and the Domain List Window is closed See Chapter 7 for more on work ing with selected domains TABLE 4 7 Keyboar
84. data columns are selected in the Lists tab of the Series Options In addition to the area Fig 8 1 the Trace List can include comment length centroid extent section thickness and z distance For 82 Reconstruct v1 0 6 1 About Trace Names more on these values see Chapter 9 The entire list can be saved to file using the Save command at the top of the list window Multiple traces can be selected from the Trace List by highlighting trace names with the mouse and then pressing Enter or the Select menu item at the top of the list When traces are selected from the list any traces previously selected in the section are deselected the list traces are selected and unhidden if necessary and control is returned to the main window About Trace Names The name of a trace identifies the object to which the trace belongs Traces with the same name are grouped together to make 3D objects Chapter 11 Trace names are also used to make correspondences between sections for alignments Chapter 10 Trace names are limited to a maximum of 63 characters Certain characters are not allowed in trace names Illegal characters include the quotation marks the equal sign the comma the backslash and the less than lt and greater than gt signs These characters entered in a trace name will be replaced with underscores _ A few other characters have special interpretations These special characters include the
85. der colors is saved with the series These values can also be modified in the Tracing tab of the Series Options dialog FILL COLOR The second color button in the Attributes dialog shows the current fill color if one consistent color is defined among all traces Clicking on the button will open the Color dialog with a separate set of Custom colors for fill colors These values are also saved with the series and can be set as well in the Tracing tab of the Series Options Fill color defines the shading for the interior of a closed trace An open trace is not filled The fill color can be any red green blue combination but the appearance of the fill is affect by both the choice of color and the fill mode FILL STYLE The style of filling can be modified with the checkboxes in the dialog A closed trace can be either filled when it is selected when it is not selected or not filled at all An open trace is never filled Normally the interior of traces are filled when the trace is selected and unfilled when the traces are not selected To turn off the filling of traces completely change the Fill mode to be None The selected traces are filled when they are not selected by checking that box in the Attributes dialog The trace will be given a transparent interior when they are selected This is useful for showing particular regions as highlighted on the sec tions or in the thumbnails The Fill mode determines how the fill color will be comb
86. determined when the data was entered as was the section index of the point In addition the section on which each point was entered was recorded When the Z Trace List is generated these section values are converted to z distances z Z trace lengths can be affected by the method of z distance calculation when section thickness is not uniform see above Distances List Computation Three dimensional distances between objects are listed in the Distances List win dow Be sure to use the limit strings in the Lists Tab of the Series Options to limit the number of distance computations because the computation can be slow All distance are computed after the Distances List window is created You can monitor the progress of the computations by looking at the first line of the list This line shows the objects between which distances are currently being computed To abort the computations close the list window When all distances have been com puted the list will be filled with the results All distances are in series units Algorithm The 3D distance between two objects O and Oy is the minimum 3D distance between any two traces of the object If any two traces intersect on a sec tion then the distance between the objects is zero If traces never intersect then the distance is a positive value Thus if one object is located inside another there will Reconstruct v1 0 6 1 125 Calibration and Measurement still be a distance b
87. e domain transformation from section II Merge the domain back into section II Step 5 Import the domain image into section I using the same pixel size as used when importing it into section II Select the new domain on section I and repeat the transformation recorded from section II by using Ctrl r The domain will now be correctly positioned on section I Go back and delete the domain from section II If desired the deletion can occur in Step 4 immediately after recording the transformation 142 Reconstruct v1 0 6 1 Aligning By Correlation Aligning By Correlation A simple alternative to the point correspondence technique can sometimes be used to align domains or sections This technique uses the By Correlation movement command Ctrl By Correlation computes the peak of the cross correlation func tion between the front section and the back section so two sections or a domain and a section must be available for the operation to do anything You can be sure that two are available if you flicker between them The algorithm determines the translation required to move the front current section or domain to the peak of the cross correlation and immediately applies this shift to the section The entire com putation will typically require several seconds but the end result will be a move ment of the current section into alignment with the previous section provided the current section is unlocked By Correlation only works
88. e 3D representa tion or position of an object can be modified by specifying new parameters and then adding the object to the scene again It is not possible to have two different representations for an object e g traces and ellipsoid in a scene except by gener ating one representation and then renaming the object and generating the second representation without clearing the scene Reconstruct v1 0 6 1 165 The 3D Scene Figure 12 2 The effect of in scene modifications of two objects On the left the scene of an intersecting sphere and cylinder immediately after being added to the scene On the right is the scene after two modifications First the cylinder object was modified to transparency 0 5 and back faces were not filled Where the back faces are visible inside the cylinder they are seen as wireframe Then the sphere was made shiny by increasing the intensity of the specular color Details of the in scene modification options are given below REMOVING OBJECTS Specific objects selected in the Object List are removed from the scene by the Remove menu item in the Object List window s Scene menu All objects can be removed the scene by selecting Clear from the 3D Scene window s Scene menu The objects in the scene are not removed when a series is closed and a new series opened This allows objects from one series to be rendered with objects from another If this feature is not desired simply Clear the scene whenever a ne
89. e 3D surface structure Ambient Intensity The Ambient Intensity specifies how much ambient light a sur face reflects Ambient light is not directional and so comes from all surface orien tations The ambient color of an object is the Diffuse Color scaled by the Ambient Intensity value between 0 and 1 A value of 0 means no ambient light color while 1 gives the maximum reflectance for ambient light Transparency The transparency value determines whether light from other sources passes through the object A value of 1 0 makes the object completely transparent while 0 0 makes the object completely opaque Note that the imple mentation of transparency in the scene is not completely correct although it is often reasonable Fig 12 2 For better transparency export the scene to another application for rendering Specular Color Specular Color and Shininess together determine the specular highlights e g the shiny spot on the sphere in Fig 12 2 The areas of a surface perpendicular to the view direction receive the Specular Color in addition to any diffuse and ambient colors To make a surface shiny choose a whiter more intense Specular Color To make a surface non shiny set the Specular Color to black Reconstruct v1 0 6 1 167 The 3D Scene Shininess Shininess is a real value from 0 to that determines the spread of the specular reflection Lower shininess values produce broad glows while higher val ues result in sharpe
90. e Filename box near the bottom of the dialog This will be the name of the series files created to store the series data Select the Save button in the New Series dialog to create the series IMPORT SECTION IMAGES Import section images into the series using the Import Images item from the Series menu This will open a dialog for creating the section files from a set of image files Fig 1 1 Image files can be in a variety of formats including Windows Bitmaps GIF TIFF and or JPEG Each image file should contain a single image not an image stack representing one section in a series and should use a representation of 8 or 24 bits per pixel Choose image files using the Select button This button will open a file selection dialog In this dialog navigate to the folder containing the image files and highlight the image files using the mouse Multiple files can be highlighted using the Ctrl or Shift keys The filenames will appear in quotes in the Filename box when multiple files are selected When all desired images have been selected use the Open button to transport the filenames into the Import Images list 2 Reconstruct v1 0 6 1 Getting Started Source C fiala RECONSTRUCTitest Import Pixel Size 0 002540 Offset X 00 N o0 IV Copy files to series folder Quit Randomize Sort numerically Start numbering at a and increment by Hl Place Image of Type into Section at Position dendrite1 bmp BMP 1 0 000000
91. e Palette Window Command Action Tab Move to next palette entry button Enter Select highlighted palette entry Left mouse click Select the palette entry under the pointer 34 Reconstruct v1 0 6 1 Object List Window Object List Window The Object List is opened or closed by the List item of the Object menu Each time the list is opened the sections are scanned for traces and these are grouped by name into objects If there are large numbers of traces and sections the object list cre ation may be slow Each unique trace name is displayed in the list as an object The list may also display the section range in which the trace name is found a count of the number of component traces that make up the object and the object s surface area and volume Like the other list windows the Object List can be navigated by mouse or key board Columns can be resized at the column headings The window can be resized by dragging the narrow border of the window Multiple objects can be highlighted by holding down the Shift or Ctrl keys Hitting Enter or selecting the Add item in the Scene menu will cause a 3D representation of the objects to be generated and added to the 3D Scene If the 3D Scene window is not open the window is created and the 3D Scene is displayed The Object List can be saved to a csv file using the Save menu item The attributes of the traces or 3D representation of objects can be modified by selecting one of t
92. e arranged by moving image domains and traces in the main window This process of moving data elements in a section is different than panning or zooming the view Changing the view point affects the display of all sections in the same way whereas moving a section or an element within a section changes the structure of the representation Transformations To allow independent movement of data elements the position orientation and scale of each domain and trace in the section is defined by a transformation Every domain and every trace has a transformation associated with it A single domain or trace can be moved within the section by changing its transformation The entire section can be moved by changing all the transformations together Each transformation maps trace points or image pixels into the section using a combination of basis functions in two dimensions Essentially each basis function represents an elementary motion such as translation orientation slant scaling deformation or bending By combining these movement components in different Copyright 2006 John C Fiala Movements and Alignments proportions a complex remapping of the underlying data is possible This remap ping can be used to move an image on the section or to increase the size of a trace for example Keyboard Increments As described in Chapter 4 the keyboard performs diverse functions in the main window but an important additional function i
93. e containing image data at reduced dimensions proxy_scale the dimension of the proxy image divided by the dimension of src Reconstruct v1 0 6 0 15 Software Overview lt xml version 1 0 gt lt DOCTYPE Section SYSTEM section dtd gt lt Section index 7 thickness 0 05 alignLocked true gt lt Transform dim 6 xcoef 0 240299 0 999416 0 0182671 0 0 0 ycoef _ 0 0104287 0 0297386 0 997894 0 0 0 gt lt Image mag 0 002691 contrast 1 brightness 0 src _unaligned 7 gt lt Contour name domainl hidden false closed true simplified false border 1 0 1 111 1 0 1 mode 11 points 0 0 768 0 768 768 0 768 wy lt Transform gt lt Transform dim 0 xcoef 0 10 0 0 0 ycoef 0 010 0 0 gt lt Contour name a4 hidden false closed true simplified false border 1 0 5 0 fill 1 0 5 0 mode 13 points 0 377196 0 55957 0 377196 0 555605 381161 0 55164 385125 0 55164 38909 0 555605 38909 0 55957 385125 0 563534 0 381161 0 563534 wy lt Transform gt CAO lt Section gt Figure 3 3 An example XML section data file Section number 7 consists of one image domain domain1 and one trace a4 16 Reconstruct v1 0 6 0 Data File Organization image The Contour element paired with an Image element is the set of points defining the boundary of the image domain A Contour element is the set of points defining a trace on the section
94. e editor for serial section microscopy J Micros copy 218 52 61 Fiala JC Harris KM 2001a Cylindrical diameters method for calibrating section thickness in serial electron microscopy J Microscopy 202 468 472 Fiala JC Harris KM 2001b Extending unbiased stereology of brain ultrastructure to three dimensional volumes J Amer Med Informatics Assoc 8 1 16 Fiala JC Harris KM 2002 PC based alignment and reconstruction of serial sec tions Microscopy and Analysis USA Edition 52 5 7 Howard CV Reed MG 1998 Unbiased stereology Three Dimensional Measure ment in Microscopy Springer Verlag New York Schneider PJ Eberly DH 2003 Geometric Tools for Computer Graphics Morgan Kaufman Publishers Amsterdam Copyright 2005 John C Fiala 173 pavoqday uo skay uoyoUun ZI A I 4 4240 adv gq Saul payjop uo mp UO PIUAN BUIJAS ASV 01 114 NOYJIM JULI WIOOZ ISP PUY 197020 swop Pod sven opun avedsyseq suono s OSed UMOG dj ISed ysenuoy ssouyysiig SUOT ODS YOUMS SMOPUIM YOUMS qQBL I Anus ayoyed jos 6 TO 1ID Anuo yod 3 6 10 Mwao 93W YS WOWIDAOW ews YD bo 4 Zd bd a Zd kld Old 64 ha 8h 84 44H 9A GA Ce PS See Sa bo ee EEEE ayes LIS ayes ayes ques URIS yueTS ques 3910Y 3WIOA IANI ZIOH Jeon EIA ZIOH ZIOH JEDIA TEOMA ZIOH 006 dija E 5 see Se
95. e group of traces is centered at the cur sor using the midpoint of the x and y extent of the set of clipboard traces The group is then repeated at each point specified by the Distance and Number param eters By selecting the grid Number to be 1 only 1 copy if the clipboard traces will be produced and the Grid Tool acts like a Stamp Tool for multiple traces Grid elements created with the Grid Tool are given the name and color attributes of the default tracing parameters Each grid element is given the default trace 96 Reconstruct v1 0 6 1 Open Point by Point Drawing name allowing for interpretation of any special characters Thus using the default name grid will result in elements being named grid1 grid2 grid3 and so on The sampling frame element is given a fixed color pattern Fig 8 8 where exclusion lines are colored red and inclusion lines are colored green The Clipboard trace s grid element does not use the default tracing name or col ors Rather the grid elements replicate the clipboard attributes for each grid ele ment Also unlike the other grid types the Clipboard traces are not resized using the Element size parameters The Element size parameters are simply ignored and Clipboard traces are reproduced at their actual sizes Open Point by Point Drawing This tool is used for drawing an open trace as a sequence of line segments Select the tool and begin drawing in the main windo
96. e modified then open the dialog Enter the desired contrast and brightness values and select OK It may be helpful to first determine appropriate values by use the key commands on a selected domain and then checking the final values in the Domain Attributes dialog Reinitializing a Domain When a domain is selected the Reinitialize submenu becomes accessible on the Domain menu This submenu provides a way to reset all of the changes in contrast and brightness the domain boundary and in the positioning of an image CONTRAST The contrast item of the Reinitialize menu clears the contrast setting to 1 and the brightness setting to 0 These are the values when the domain image is first imported Reconstruct v1 0 6 1 75 Domain Images BOUNDARY Reinitializing the boundary sets it to a rectangle that borders the entire image The last boundary can be recovered by Undo Ctrl z until the section is saved and removed from memory TRANSFORM By reinitializing the transform all positioning and alignment information will be cleared from the domain and it will be set to the origin of the section The pixel size information will not be changed but any magnification information stored in the transform will be removed as well Ctrl z Undo can be used to recover the transform while the section is still in memory Hiding Domains A domain can be hidden from view even though it still exists in the section This can be used to reve
97. e modified at once even though the traces have different names The asterisk notation can also be used to modify a set of trace names Any text after the asterisk is appended to all the existing trace names For example suppose traces named d1 d2 and d3 were selected prior to invoking the Attributes dialog The Name box would have an asterisk inside because the names are different Changing the Name box string to spine would change the trace names to d1spine d2spine and d3spine Leaving the Name Reconstruct v1 0 6 1 85 Traces 66399 box string as would leave the trace names as d1 d2 and d3 Replacing the asterisk with spine would result in all traces being named spine COMMENT A comment string of up to 127 characters can be associated with each trace This comment can be displayed in the Trace List window The rules for comment strings are similar to those for trace names Illegal characters lt gt are replaced with underscores The special characters are not interpreted for comment strings HIDDEN A trace can be hidden from view in a section by checking the Hidden checkbox in the Attributes dialog A hidden trace will not be drawn in the main window but will appear in the trace list with a special H icon Fig 8 1 To unhide a trace simply select it from the Trace List or use Unhide from the list Modify menu
98. e options into categories 40 Reconstruct v1 0 6 1 Series Options wx oe Figure 5 1 The Series Options Dialog Movements Proxies Thumbnails AutoTracing Th h s f General Names Colors Grids 3D Lists e General tab has options or automatically saving the series and as All option settings are saved with the series sections files and the units description P TAR and default section thickness Any Series units description microns changes will be accepted with the OK button and rejected with the Cancel button To ensure that the change persist Data precision is significant digits V Automatically save series when closing JV War before deleting unsaved sections V Automatically save sections before deleting after the series is closed save the series I Beep when deleting before closing it Shown are the default I Beer teenie feil settings for a new series Hide all traces when loading section Unhide all traces when loading section Hide all domains when loading section I Unhide all domains when loading section Use absolute paths for new domains Default section thickness 0 05 Computer z distance to middle of section instead of top To change options set the desired values in the pages and then select the OK but ton at the bottom of the dialog Selecting Cancel will cancel the changes in most cases The options are saved when the series is saved General The
99. e ordering of the selected traces Some non overlapping traces may be deleted while overlapping ones are merged Another possibility is that none of the traces will be changed REVERSE Reverse changes the orientation of the selected traces Normally traces are gener ated as clockwise boundaries of the exterior surface of an object Reverse allows a clockwise trace to be redefined as the surface of an interior hole by changing its orientation to counterclockwise Likewise reverse changes a counterclockwise trace to a clockwise one SIMPLIFY Simplify provides on demand simplification of selected traces provided the Sim plified attribute of the traces is false If a trace is already simplified then the Sim plify command has no effect If no traces are selected that have the Simplified attribute clearer then the Simplify operation will generally report Nothing to sim plify To enable further simplification on an already simplified trace use the Trace Attributes dialog to first uncheck the Simplified attribute Simplify applies a simplification process similar to that normally used when com pleting a drawing operation see the Simplified attribute section above The trace is re pixelized and then reduced to a set of line segments that match this path If the trace is closed all loops are removed and the trace is reoriented to a clockwise direction Reconstruct v1 0 6 1 101 Traces ax op i ae Figure 8 10 The Au
100. e the Object List window is opened Since each section file in the series is opened and scanned for object traces generating the Object List may be slow if there are large numbers of sections or access to the section files is slow In addition if a new object is created by tracing in the sections it will not immediately appear in the Object List Rather the Object List window will need to be closed and reopened from the Object menu in order for the new object to be recognized Multiple objects can be selected in the list by using the control or shift keys in combination with the left mouse button Pressing the Enter key or double clicking Copyright 2006 John C Fiala Objects Figure 11 1 The Object List window Objects are x listed along with the range of sections in which traces of the object were found The Scene and Sone SNE oy Cree Modify menus appear above the list The object Object Start End D21 has been highlighted in the list The object D10 1 94 D12 has been added to the 3D Scene as indicated 012 1 94 by the shaded icon with the cyan background None 215 1 94 of the other objects are in the scene as denoted by oa i z the yellow icon with lines representing section 1 94 profiles The rows of the list were limited to objects with names that consist of the letter D followed by 2 digits as indicated by D in the title bar the mouse causes the selected objects to be added to the sc
101. e to the middle of the section z t to t3 t _4 t 2 The other method computes the distance to the top of the section z ty tot tg t _4 t middle of section top of section z distance 118 Reconstruct v1 0 6 1 Status Bar Measurements thickness offset in z values The method of z distances calculation can be selected in the General Tab of the Series Options Physical gaps in the series can be represented by empty sections of an appropriate thickness For example suppose a series was cut at 50nm section thickness but only every fifth section was imported The sections were numbered 1 6 11 16 to reflect that every fifth section was used Assuming a uniform section thickness d these sections could be given thickness 50nm New sections could be created at 2 7 12 with thickness 200nm to represent the gaps To get accurate object mea surements with this method traces would need to be created in the gap sections so that the missing thickness is accounted for in the surface area and volume calcula tions see below Alternatively only sections 1 6 11 16 could be created but with thicknesses 50nm 250nm 250nm 250nm reflecting the gaps between these sections The z distances to the tops of these sections would then be 50nm 300nm 550nm 800nm etc No additional gap traces would be needed in this case to get accurate surface areas and volumes Status Bar Measurements Traces are quantif
102. e top of the Domain List window NAME The current domain name is shown in the Name box A new name for the domain may be typed in the box The rules for domain names are the same as for traces Domain names are limited to a maximum of 63 characters Illegal characters lt gt are replaced with underscores The special tokens are interpreted for domain names entered in the Attributes dialog These tokens allow domain names to be automatically numbered just as for trace names but the token is interpreted independently for domains and traces See Chapter 8 for details SOURCE The source path is a string of up to 255 characters that defines the location of the image file This path can be an absolute path or a path relative to the series folder Fig 7 4 When a new domain is created by default the program stores the relative path from the series folder to the image in the section file Thus if the image file is copied into the series folder the domain path to the image simply the file name By checking the Use absolute paths for new domains option in the General tab of the Series Options the absolute path including both the folder name and the file name will be stored as the domain path This option would allow the series folder to be moved around while the image files remain in a fixed location Reconstruct v1 0 6 1 73 Domain Images Information about the source image file is also included in the attribu
103. east 100 pixels on the screen Fig 8 11 Z lraces Most traces lie only within a section These traces lie wholly in the section on which the trace was started no matter what sections were visited during tracing Z traces on the other hand are traces that span multiple sections Z traces are not stored or displayed on sections They are stored in the series file and are listed in the Z Trace List opened from the Object menu Z traces will be saved with the series file not with the sections The position and structure of a Z trace can be visualized by adding it to the 3D Scene Note that if the 3D Scene Window is open when Z trace is added it may be out of the field of view and so will not appear in the window The 3D Scene may 106 Reconstruct v1 0 6 1 Z Traces need to be rotated or reset in order to see the new Z trace Another important fea ture is that only one object in the same can have the same name Creating a Z trace with the same name as an existing object will preclude them both being visualized together in the same scene Consequently Z traces should be uniquely named as well The special character can be used in the default name when repeatedly drawing Z traces to create unique names Important Every Z traces should have a different unique name The Z Trace Tool resembles the letter Z The tool is used just like any of e the other point by point tracing tools but each point can be entered on a different
104. ecting the differs by relation with a large 255 value For example to ignore color dur ing region growing set Hue differs by 255 Since this can never be true a change in hue will never stop a region from growing In addition to the stop criteria Wildfires can be contained by creating firebreaks with the right mouse button This is typically used to separate two regions that are connected by a small gap Such gaps are often discovered after having created a Wildfire that spread more than expected To correct the spread of the Wildfire delete the bad trace and place the cursor at one edge of the gap Press and hold the right mouse button The cursor will change to an extinguished match Drag the cursor across the gap leaving a small black line Release the mouse button to create the firebreak Use the left mouse button to repeat the original Wildfire The region growth will not pass through the firebreak although it can still grow around it if additional gaps remain During Wildfire region growing the cursor will change to an hourglass This should last for only a second but during this time region growing cannot be aborted by the Esc key The drawing of a firebreak with the right mouse button can be aborted by hitting Esc before releasing the button 104 Reconstruct v1 0 6 1 Wildfire Region Growing Progam amp Section 1 Poston 43 25 216 808 mesons T Section 1 cf Secton 1 Trace ce Postion 216 25 15 307
105. ecting Save from the Section menu Since the filename is already specified there is no dialog the save operation will be exe cuted immediately Displaying a Section Images and traces within a section are defined in a local coordinate system and transformed into position on the section by a general nonlinear transformation To display a section each image data file is read from disk and transformed pixel by pixel into position on the section s display bitmap To support color images with different color formats the display bitmap uses 24 bit color After all images are rendered the line traces are transformed into position and drawn using the win dows graphics device interface Traces are drawn with or without a colorized fill to highlight the interior of the object Finally the rendered bitmap is copied to the cli ent area of the window The process of rendering a section can be slow depending on how much data is to be displayed and the media on which the data is stored When a section is being rendered for display the cursor will change to the wait busy cursor typically an hourglass When rendering is complete the section will be displayed and the cursor will be restored CENTERING The set of images that comprise the section can be centered in the main window by selecting the Center item from the Zoom submenu of the Section menu Pressing the Home key will accomplish this quickly To center a section that contains only trace data
106. ed sections can be changed simultaneously using the Modify menu at the top of the Section List window The Modify operations allow highlighted sections to be locked unlocked renumbered deleted or given particular pixel size or thickness attributes Chapter 6 Reconstruct v1 0 6 1 31 User Interface Each list window has a Save menu item that allows the list to be saved as a Comma Separated Values csv file This file is an ASCII text file that can be readily imported into spreadsheet programs such as Excel The Save dialog allows the filename and folder for the csv file to be specified The list columns and rows will be saved in the file just as they appear in the window Thumbnails Window Thumbnails images of the sections can be viewed by selecting Thumbnails from the Section menu Each thumbnail image is placed on a button in the thumbnails window and the buttons are displayed in one of two ways In the default display mode the thumbnail buttons are arranged to fill the window starting in the upper left hand corner Resizing the window results in rearrangement of the buttons to fill the window Putting the pointer over a thumbnail button for a certain amount of time results in display of the section number in the title bar when the thumbnails window is active Selecting a thumbnail with the left mouse button results in the main win dow being activated and paging to the section The thumbnail buttons cannot be navigate
107. ene Selected objects can also be added to the scene by using the Add item from the Scene menu When an object has been added to the scene the object s icon in the Object List is changed to a shaded figure with a cyan colored background Fig 11 1 By this mechanism the Object List indicates which objects are in the 3D Scene This allows particular scene objects to be selected and removed Selected scene objects are removed from the scene using the Remove menu item from the Scene menu The Object List window can be made to include 3D measurements of objects such as the volume and surface area or counts of the number of component traces of the objects This is accomplished by checking the appropriate boxes in the Lists tab of the Series Options before opening the Object List These values are all calculated only when the list in opened and so the list must be closed and reopened whenever there are changes to the trace data For details on the volume and surface area cal culations see Chapter 9 Changing Object Attributes Changing the attributes of objects changes the attributes of every trace of the object in all the sections This change is performed on the currently highlighted objects in the Object List by using the Attributes dialog of the Modify menu Note that color of an object can be temporarily changed in the 3D Scene without modifying the traces This is done by the Color Transparency dialog of the Scene 148 Reconstru
108. ene as denoted by the red Z icon example renaming a list of Z traces to length05 would produce length06 length07 length08 etc 162 Reconstruct v1 0 6 2 CHAPTER 12 The 3D Scene The 3D Scene window displays the 3D representations of objects generated from the Object List Fig 12 1 The window can be opened or closed by selecting 3D Scene from the Object menu The 3D Scene window will be automatically opened whenever an object is added to the scene from the Object List The 3D Scene window can be resized and positioned arbitrarily on the desktop but it cannot be minimized or moved behind the main window To hide the scene tem porarily close the 3D Scene window This can be done by selecting Hide from the 3D Scene window s Scene menu or clicking on the X box on the right side of the window s title bar Closing the 3D Scene window does not erase the scene Reopening the window will show the same scene although the viewing angle will be reset to the default position OpenGL Rendering The 3D Scene is rendered using OpenGL www opengl org The OpenGL librar ies provide a software interface to the computer s graphics hardware These librar ies must be properly installed on the computer to open and view the 3D Scene Copyright 2006 John C Fiala The 3D Scene OpenGL is shipped standard with every Windows 95 98 2000 NT XP computer and should work with Reconstruct without
109. epresentation and editing of objects is discussed as well as the methods for generating 3D shapes The 3D Scene window and the rendering of reconstructed objects is covered in Chapter 12 10 Reconstruct v1 0 6 1 CHAPTER 3 Soft tware Overview Reconstruct was developed from years of experience working with high magnifi cation serial section images of brain tissue In this work brain tissue is embedded in resin and serially sectioned on an ultramicrotome Sections are mounted on coated slot grids and photographed in the electron microscope EM Since each section is imaged separately in the microscope the images must be brought back together to understand the structure of the whole tissue Reconstruct aids in the extraction of three dimensional 3D information from the serial section images Series Sections Domains Traces and Objects Serial section microscopy consists of an ordered set of sections representing the sequential pieces of the tissue block Data in Reconstruct are organized around this concept An ordered set of sections is called a series Reconstruct operates on one series at a time Within a series data are grouped by section and each section is given an index indicating its position within the series Each section in the series contains the data related to one physical section of the tissue The section data includes images of the section and traces drawn on these images The traces are later grouped toget
110. er or select the OK button to close the Options dialog The Tools window Figure 1 2 should be visible at the top of the main window Use the cursor to select the tool button that looks like a pencil The cursor will change to a pencil when the cursor is moved back into the main window Reconstruct v1 0 6 1 7 Quick Start Figure 1 4 Drawing a trace with the freehand drawing tool eft Start of the tracing Th pencil is positioned at the profile to be traced and the left mouse button pressed and hel down middle Just before release of the mouse button The pencil left a trail where it was moved with the mouse from the start position right Right after release of the mouse button The trace is closed and filled with color Place the tip of the pencil on the edge of the profile to be traced as shown in Figure 1 4 Press and hold down the left mouse button Drag the tip of the pencil around the profile and release the mouse button when the tip gets near the start of the trace The trace will be completed closed and filled with a color provided the interior of the profile is not too dark Press the Page Up key to advance to the next section The trace on the previous section will be saved automatically as long as the default save options were not changed in the Series Options dialog Repeat the process of tracing the profile with the mouse Continue paging and tracing until several sections have been traced Use the Page Down bu
111. ering Sections Listing all Sections Editing Section Attributes Movement Lock Thickness Section Numbering Deleting Sections The UNDO Stack Thumbnails Exporting Section Images e CHAPTER 7 Domain Images O OO 0O Gs 0O 0 Creating a Domain Importing Multiple Images Listing Domains Selecting a Domain Merging a Domain Moving and Montaging Domain Images Domain Boundary Domain Attributes Name Source Pixel Size Contrast and Brightness Reinitializing a Domain Contrast Boundary Transform Hiding Domains O Proxies Proxy Rule of Thumb for Single Domains CHAPTER 8 Traces O Os O O 0T O OG O O O Creating a Trace Ellipse Tool Rectangle Tool Line Tool Selecting Traces Selection Tool Deselecting Traces Zooming to Selected Traces Trace List About Trace Names Trace Attributes Name Comment Hidden Simplified Closed Border Color Fill Color Fill Style Trace Clipboard Setting Trace Attributes for Drawing The Trace Palette Stamping Predefined Shapes Creating a Grid of Traces Open Point by Point Drawing Closed Point by Point Drawing Freehand Drawing Editing Traces Scissors Tool Scalpel Tool Merge Reverse Simplify Wildfire Region Growing Propagating Wildfires to Adjacent Sections Generating Wildfires Throughout a Region Z Traces CHAPTER 9 Calibration and Measurement O O O Units of Measurement
112. etween them that reflects the minimum distance between their boundaries Distance O O2 Min Distance T T V T O and T O3 where the 3D distance between any two traces T and T gt is the minimum 3D dis tance between any two line segments of the traces Distance T T Min Distance S S V S T and S T Since all the segments of T and T lie on two parallel planes we can defer dealing with the z component of the distance and first compute the minimum squared x y distance The squared x y distance between two line segments S from point P to point Q and Sp between points R and T is computed by first computing the vectors a 0 T R dl Q P d R P then using the x and y components of these vectors computing a00 d0 d0 d0 d0 all d1 d1 d1 d1 a01 d0 d1 d0 d1 bO d0 d d0 d bl dl d d1 d c d d d d e d0 d1 d1 d0 f a0l b1 all b0 g a00 bl a01 b0 h a00 2 a01 al11 2 50 2 bl c 126 Reconstruct v1 0 6 1 Distances List Computation The segments are parts of two lines The squared x y distance between the lines is given by Schneider amp Eberly 2003 a00 R 2 a01 t t a1l 2 b0 2 b1 t the minimum of which occurs at the values t and t where a00 t a01 t b0 0 all t a0l t b1 0 The correct values of tg and t must take into account the end points of the line seg ments This constrains the solutions of tg and t
113. ey on an unlocked section will move the section relative to the rest of the series Chapter 10 Therefore it is a good idea to maintain the movement lock at all times The lock unlock attribute is reflected in the icon on the left end of the sta tus bar The lock unlock attribute of multiple sections can be changed by one command from the Section List Open the Section List and highlight the sections to be changed Select Lock or Unlock from the Modify menu at the top the Section List The changes will be immediately reflected in the Section List icons A red closed 56 Reconstruct v1 0 6 1 Editing Section Attributes Renumber Sections Shift section numbers by D AN This operation cannot be Undone AN This operation cannot be Undone Cancel Cancel Figure 6 1 Dialogs for modifying attributes of sections from the Modify menu of the Section List All the sections which are highlighted in the list at the time the dialog is invoked will be changed and this change cannot be globally recovered with an Undo command Section Thickness l Setthickness in microns to 0 067 padlock appears by sections that are locked while unlocked sections display a clear open padlock THICKNESS The section thickness is changed by selecting the Thickness item from the Section menu and typing in a new value The section thicknesses of many sections can be changed to the same value using the Modify gt Thickness
114. f different objects will likely be different and oriented randomly with respect the plane of sec tioning Avoid Aligning the Same Features Every Time To minimize the effect of ambigu ous alignments it is also a good idea to choose distinct objects for correspondence points on each pair of sections Repeatedly aligning the same profiles is more likely to produce a pattern of shift or scaling that builds up to high levels over many sections For example two objects can be moving towards each other as the 144 Reconstruct v1 0 6 1 Tips for Aligning Sections i m Figure 10 4 Objects with ambiguous alignments and the resulting distorted reconstructions top A circular cylinder on the left is sectioned obliquely so that on the sections it appears as a sequence of shifted elliptical profiles By mistakenly aligning the elliptical profiles an elliptical cylinder is reconstructed instead of a circular one bottom A conical object on the left appears as circles of increasing diameter on sections If sections are scaled to align these circles then the cylinder on the right would be erroneously reconstructed rather than the original cone Many other types of locally ambiguous alignments are possible and these may combine e g a cone sectioned obliquely to produce complex distortions sections are ascended By aligning only these objects an artifactual scaling may be introduced Randomly distributing correspondence points as you align p
115. for pure translational offsets between two sections but it can sometimes be used iteratively with keyboard commands to get more general alignments A protocol for aligning By Correlation could be as follows Step 1 Go to a section which will serve as the stationary reference and lock it Ctrl L Then page to the adjacent section that will be aligned to the stationary one Unlock the section if necessary Blend the two sections spacebar Step 2 Align the sections By Correlation Ctrl Step 3 Inspect the sections to determine if the alignment is acceptable Use flick ering as needed If alignment is acceptable go to step 1 with the current section as the new stationary reference Step 4 Estimate the rotational adjustment that would improve the alignment Blend spacebar and then rotate the section F3 or F4 Go to step 2 By Correlation uses only image intensity information visible in the Main Window to compute the correlation Color and trace information is not used Color images will be converted to grayscale intensities for the correlation computation You can restrict the region used for the alignment by sizing the window and or zooming Reconstruct v1 0 6 1 143 Movements and Alignments into a subregion of the section For example to align based on just a single object zoom in until that object fills the Main Window flicker to update the back view and then use By Correlation Note that the object to be aligned
116. gions To avoid loops all tracing should be performed with the Automatically Simplify option Chapter 8 turned on In addition traces should be drawn with one of the tools designed to produce closed profiles rather than drawing open traces and then changing the trace attribute to closed Do not intersect traces of the object Traces within a section must not overlap as depicted in Fig 11 3 B Such traces also produce ambiguity about interior and exterior regions of the object However traces of one object may overlap traces of another object without problems because Boissonnat surfaces are generated inde pendently for each object ACOS Domain Error If this error message appears when generating a Boisson nat surface then probably the traces of the object contain intersections on one or more sections Check for overlapping and or multiple copies of traces Use the section range parameters in the 3D tab of the Series Options to narrow down which section s contain the intersecting traces CYLINDER A Cylinder may be generated to represent an object The midpoint center of the Cylinder is given by the centroid of the trace points _ il Xo Yo Ze A n gt x y z all trace points where n is the total number of trace vertices and the vertices of the traces are at the positions x y z in 3D space The axes of the Cylinder are determined from the scatter matrix of the trace points scatter matrix D 25 942 2 Wo Vo Z to Yo Z
117. gle object highlight all the vesicle names in the Object List and again use Modify gt Attributes gt of Traces Change the Name string to VESICLE and press Apply Now all vesicles are back to being a single object In general anytime there are name changes to objects the Object List will have to be reopen to reveal the changes Object renaming can also be used to divide an object into its component sections In this case rename the object using the special character to add the section num ber Chapter 8 For example rename myObject to myObject to create a set of objects myObject1 myObject2 myObject3 etc Each of these objects will correspond to one section of the original object Now it is possible to rename a group of sections into one subpart For example myObject1 through myObject9 could be renamed to myObject_part1 to create an slice of the orig inal object from sections 1 through 9 Copying Objects Selecting Copy from the Create menu of the Object List will duplicate all the traces for every highlighted object in the list The duplicate traces have the prefix Copy of added to the name While there is no undo for this multi section opera tion the copied object can be easily deleted see below The Copy command will create a duplicate set of traces on the sections These traces will exactly overlay the existing traces of the object so be careful about selecting and edit
118. gorithm so the output of Trace Areas represents the input to the surfacing process Traces Areas can therefore be used to evaluate the quality of the input to the surfacing process Problems evident in Trace Areas will likely result in defects in the Boissonnat surface of the object TRACE MIDPOINTS To represent Trace Midpoints a single triangular face is centered on each trace of the object Each triangle lies at the Z distance of the section and is centered at the midpoint of the trace extent in the x and y directions within the section The width and height of each triangular face is equal to the width and height of the corre sponding trace provided the Size A and B parameters are zero or negative If Size A is positive the width of each triangular face is set to Size A If Size B is positive the height of each triangular face is set to Size B The representation is a single indexed face set object with each disjoint face cen tered on each trace This can be used to represent trace positions in 3D But more important the object can be used as a distribution object when exported to 3D ren dering software like 3D Studio MAX 3D Studio MAX allows an arbitrary 3D shape to be centered on each face One application of Trace Midpoints would be to place a small sphere at each trace If each trace was used to identify the location of a cell or vesicle say the sphere distribution would visualize the cells or vesicles in 3D There are two rea
119. he Attributes items from the list Modify menu Similarly highlighted objects can be deleted from the series with the Delete operation TABLE 4 10 Mouse and Keyboard Actions in Object List Window Command Action Up amp down arrows Highlight next previous object in list Page Up amp Down Move through the list by the number of visible objects Home Move to the first object in the list End Move to the last object in the list Delete Delete all traces of highlighted objects from the series Enter Generate the 3D objects and add to the scene Shift Highlight multiple objects Left Mouse Click Highlight the object under the pointer Left Mouse Dbl Click Generate and add object under the pointer Reconstruct v1 0 6 1 35 User Interface 3D Scene Window The 3D Scene Window displays the 3D representations of objects using OpenGL OpenGL libraries must be available in order to view this window The window can be opened or closed from the Object menu or by creating a 3D object representa tion from the Object List Window Details of the 3D scene manipulations using the mouse are described in Chapter 12 but are briefly summarized in Table 4 11 Note that if the scene or menus flicker setting the animation mode to None in the Scene menu may remedy the problem The animation performance may also be affected by having other win dows open in the application or on the desktop TABLE 4 11 Mouse Actions in 3D Scene Window Mouse
120. he Recon struct user community The Reconstruct Users Group is available to everyone at http groups yahoo com group reconstruct_users Development of the software has also continued through the suggestions of users and the donation of program ming time by Dr Fiala A Reconstruct Developers Group has been established at http groups yahoo com group reconstruct_developers to provide a forum for collaborative open source development Credits The development of Reconstruct was funded in part by the National Institutes of Health under grants P30 HD 18655 R01 MH DA 57351 R01 EB 002170 ROI NS024760 and ROI MH057414 A number of different people contributed ideas for the user interface and behavior of the software In addition a number of free programming resources were used in the production of Reconstruct as acknowl edged below Reconstruct v1 0 6 0 21 Software Overview SOURCE CODE The majority of Reconstruct was designed and written by John Fiala fiala bu edu Other contributors are listed in the License dialog of the Help menu The contributions of these individuals are as follows Andy Key and the Independent JPEG Group Image input and output was real ized by incorporating the Generalized Bitmap Module GBM library written by Andy Key http www nyangau fsnet co uk These libraries incorporated the source code of the Independent JPEG Group http www ijg org written by Tho mas G Lane for h
121. he main window while windows release control to each other The exception to this is that some message dialogs such as the Version dialog from the Help menu release control back to the main window Dialogs cannot be resized but can be moved Windows float on top of the applica tion They can be resized and moved but not minimized independently of the application A special case is the status bar that is not moveable but remains docked at the bottom of the window Copyright 2006 John C Fiala 25 User Interface RESIZING AND MOVING WINDOWS Windows can be resized by placing the pointer on the narrow border of the win dow pressing the left mouse button and dragging the border The pointer should change to resize arrows when over the border of a realizable window The main window can also be resized by dragging from the resize area in the lower right hand corner of the status bar Both windows and dialogs can be moved by dragging the title bar with the left mouse button Windows that display an icon in the title bar namely the main window and the Series Options window can be moved with the keyboard Selecting the icon with left mouse button opens the system menu To resize or move the window select the menu item and then use the arrows keys to adjust the position of the window Holding the Ctrl key down allows finer control of the movements Release the key board mode by hitting Enter or by pressing the left mouse button again
122. he series to get correct 3D distances Creating a Section A new section can be created by selecting the New item from the Section menu Sections can also be created by importing sections images or lines into multiple sections using the dialogs from the Series Import menu The New Section dialog asks for the number of the new section The default value is the section number after the current section After a number is entered and the OK button pressed a section file with default attributes is created with that index If a section with the index already exists no new section is created and an error message is displayed After creating the new section the main window switches display to it Initially the new section contains no data A section without images or traces displays as black Data can be added to the section by importing domain images Chapter 7 and by drawing traces Chapter 8 The new section is given the default section thickness defined in the Series Options General tab Saving a Section By default section data is automatically saved to the section file before it is deleted from memory This can be changed in the General tab of the Series Options When sections are not automatically saved the user will be warned before any changes are deleted provided the Warn before deleting unsaved sec tions checkbox is set 52 Reconstruct v1 0 6 1 Displaying a Section A section can be manually saved by sel
123. he third axis of Ellipsoids and Cylinders In all cases the size parameters can be disabled by entering a negative number in these boxes When 160 Reconstruct v1 0 6 2 Z Trace List sizes are negative objects are sized according to the traces as described above for each object type Boissonnat Surfaces The parameters in the Boissonnat Surfaces box are only used by the Boissonnat surfacing algorithm The algorithm attempts to connect vertices in one section to the nearest vertices in the adjacent section You can limit these connections by setting a positive value in the Don t connect points further than box When the value is negative or zero no limits are applied A negative value is recommended although unwanted connec tions can sometimes be eliminated by a positive value that is not so small that it interferes with reasonable connections Faces closing the ends of the object are generated by checking the two check boxes at the bottom of the dialog Unchecking the boxes will leave open ends but may also leave open some horizontal faces in the middle of the object Z lrace List Open the Z Trace List window by selecting Z Traces from the Object menu If any Z traces have been defined for the series a list will appear showing the Z traces arranged alphabetically by name Fig 11 5 Depending of the settings in the Lists tab of the Series Options the range of sections and the length of each Z trace can be disp
124. her across sections to form 3D objects Copyright 2006 John C Fiala 11 Software Overview domain boundaries Transformation for domain1 Transformation for dendrite Trace dendrite Figure 3 1 The mapping of data elements onto the section The contents of this section are two domains domain1 and domain2 and one trace named dendrite composed as shown in the upper left Each trace and domain is associated with an independent transformation that determines the size and location of the element on the section In this example each domain has a rectangular boundary that defines the area of the image to be displayed Reprinted from Fiala 2005 Each image within a section is called a domain reflecting that it has a defined boundary and independent position within the section Multiple image domains can be placed side by side within a section to make a montage a composite picture of the section from many small images Images are entered into the computer by digitizing photographic prints negatives or by direct digital imaging with a digital camera For example the negatives on EM film are typically scanned at 1000 dpi using a conventional desktop scanner These files are saved as Bitmaps or TIFFs from the scanner software usually with a number in the filename indicating the section number The image files are then imported into Reconstruct as domains within the sections 12 Reco
125. hese files Reconstruct v1 0 6 1 49 Series 50 Reconstruct v1 0 6 1 CHAPTER 6 Sections Sections form the main data elements of a series A section is composed of a set of images domains and a set of polylines traces The domains and traces for each section are stored in a separate file the section file Chapter 3 Section Indexing Since serial sections have an intrinsic order the position of a section in this order is used as the file extension of the section filename Sections must be numbered with non negative integers in the range of 0 through 2147483646 The section with the smallest section number is referred to as the first section while the highest num bered section is the ast section The section index or number is also stored internally in the section file The inter nal index and the file extension must be the same Leading zeros and spaces should be avoided in the section extension since these will not be maintained when saving the section file Copyright 2006 John C Fiala 51 Sections Sections do not have to be numbered sequentially Gaps in section numbering are generally ignored But note that gaps may require careful interpretation for 3D reconstructions and measurements The position of a section in the volume is determined by the combined section thicknesses of lower numbered sections see Z Distance in Chapter 9 so the section thicknesses must account for any gaps in t
126. ich the calibration lines were traced Right The next dialog allows this same pixel size to be applied to every image in every section of the series When the lengths for all selected traces have been entered a Calibrate Traces dia log will appear Fig 1 3 offering three options Select the second option to set the correct pixel size for the current section When the Set Pixel Size on All Domains dialog appears set the First Section to the first section of the series and the Last Section to the last section Verify the value in the Set pixel size to box and click the OK button This will set the pixel size of all images in the series to the value determined from the calibration image The pixel size for any image can be verified by using the Domain List while viewing the section in the main win dow Note that after calibration of pixel size the calibration lines are no longer accu rately matched to the calibration image on which they were drawn This is to be expected Since these calibration lines are no longer useful they can be deleted from the calibration section An additional check on the calibration would be to redraw one or two calibration lines and verify that their length is now correct ALIGN SECTIONS Tissue that has been physically sectioned and then imaged must be realigned for accurate 3D measurements and visualizations Section images can be manually moved into relative alignment using keyboard commands
127. ick visual reference in the sequential mode Limiting the range of sections for thumbnails or skipping sections can speed up thumbnail generation When traces are displayed on the thumbnails they are drawn using the normal trace attributes for color and filling By choosing the flipbook style thumbnails will be overlaid rather than arrayed sequentially Thus the thumbnail window will be sized to display only one thumb nail button Resizing the window is not possible Selecting which section is visible in the flipbook can be done by using the scrollbar at the bottom of the window or by using the mouse scroll button Clicking the scrollbar with the left mouse button 60 Reconstruct v1 0 6 1 Exporting Section Images a ax Figure 6 3 The Thumbnails tab of the ati L aas Series Options dialog These options allow the thumbnail size and style to be changed Changes are accepted by selecting the OK button and rejected by the Cancel button Changes will appear the next time the amona ae thumbnails window is opened WARNING Large thumbnails deplete memory resources Thumbnails are small versions of the sections displayed in a separate window for quick access Make thumbnails resemble current view C Fitwhole section to each thumbnail First Section 1 Last Section 16 Display every 1 th section v Display traces on thumbnails v Overlay thumbnails to make flipbook Shuttle rate 5 Hz will begin
128. icker Backspace Delete last trace point entered when drawing Esc Abandon current drawing or tool operation 0 1 9 Switch default trace attributes to palette entry Ctrl 0 ctrl 1 ctrl 9 Set the corresponding palette entry to the current default attributes and stamp tool shape F1 F12 keys Section domain and trace movements Arrow keys Section domain and trace movements Decrease brightness of selected domain Increase brightness of selected domain Decrease contrast of selected domain Increase contrast of selected domain Mouse input May depend on the currently active tool Main Menu The main menu consists of Program Series Section Domain Trace Object and Help open menus Submenus of these menus are indicated by an arrowhead on the right hand side of the item Items that open a dialog requiring further user input are indicated by a trailing These operations can generally be canceled in the dia log Other menu items execute immediately The mouse pointer can be used to select items in the main menu but menu items can also be selected with the keyboard Keyboard control of the menu is activated by pressing and releasing the Alt key When the menu is under keyboard control certain letters in the menu items will be highlighted These items can be selected by tapping the key for the highlighted letter For example pressing and releasing the Alt key and then the T key will open the Trace menu The L key will the
129. ied in various ways and these values are reported in the lists as discussed below In addition the status bar reports incremental measurements related to the position of the mouse pointer on the screen When no tool is active the status bar reports the name of the trace nearest the pointer and the position of the pointer on the section in series units When a selected domain is displayed instead of a trace name the domain is reported along with the position of the pointer in image pixels When a drawing tool is active the reported value depends of the particular tool in use R F O Ow These tools create a rectangular region when dragging with the left mouse button depressed The status bar reports the area of this rectangle in series units squared except in the case of the ellipse For the ellipse the rectangular area is multiplied Reconstruct v1 0 6 1 119 Calibration and Measurement by 7 4 giving the area of the ellipse that would be created when the mouse button is released kx For the Panning and Domain Selection Tools the status bar reports the total dis tance moved by a left mouse drag When a line is being drawn with the Line Tool the status bar reports the length of the candidate line on the section If the section is changed during drawing the 3D length is not reported For a dynamic length measurement that takes into account the section thicknesses use the Z Trace Tool s For both the open and closed
130. ined with the underlying image or other trace data to produce the final display A Fill mode of Solid will color the interior with a solid color no image data will be visible A Fill mode of None means that no filling will be applied to trace regions Reconstruct v1 0 6 1 89 Traces Darken fill mode sets the display to a combination of the colors common to both the fill color and the underlying image such that a fill color of white would result in a completely transparent fill while a black color would result in a solid black fill A pure blue 0 0 255 fill on a grayscale image would result in shades of blue replacing the grayscale Lighten uses a combination of the fill color and the image color such that the opposite effect is produced Black fill results in transparency while white fill color produces a solid white interior The basic colors that have either 0 or 255 for the red green and blue channels work best for fill modes using Lighten or Darken Trace Clipboard The trace clipboard is an internal memory of the most recent set of traces copied or cut from the series This clipboard is distinct from the Windows clipboard and can not be used to paste data into another application It can be used to paste trace data between sections within a series or even between series Put the selected trace on the trace clipboard by selecting Copy from the Trace menu Ctrl c This item is only enabled when one or more traces have been
131. ing common hardware graphics capabilities It also allows the scene to be saved as a general indexed face set in VRML format for export to many other programs Composing a Scene 3D representations of objects are added and removed from the scene using the Scene menu in the Object List window Chapter 11 The type of 3D representa tion generated is determined by the parameters set in the 3D tab of the Series Options The positions of objects in the scene can also be modified by using the Shift Object By parameters Changes made in the 3D Options tab will only be realized after the object is regenerated for the scene ADDING OBJECTS Objects are added to the scene from the Object List window When an object is added to the scene the display parameters of the scene are not modified This allows objects to be added to and or removed from a particular view or rotating scene But is also possible that the added object is outside of the current view To see objects newly added to a scene it may be necessary to zoom out or reset the scene view point using the Reset item in 3D Scene s View menu WARNING Closing the series while objects are being added to the 3D Scene will cause Reconstruct to crash When an object is added to the scene it replaces any existing scene object with the same name Any modifications to the object s in scene attributes color transpar ency etc will be lost when the object is re added to the scene Th
132. ing matching of names follows the same rules as for limit string in the Lists tab of the Series Options above WARNING Importing series elements cannot be undone Checking the Copy from Sections box enables the importation of section data from the series Data will be imported only from existing sections within the range specified Importing domains or traces will add these elements to sections in the current series If a corresponding section does not exist in the current series then the section will be created The matching strings in the Import Series dialog can be used to select which domains or objects to import This trace matching string works just like the limit string in the Object list So if you can limit the Object list 48 Reconstruct v1 0 6 1 Importing Series Data to just the objects desired to be exported from one series using that same limit string in the Import Series dialog will ensure that only those objects are imported Be aware that importing an existing object will create multiple copies of the object traces in the current series To merge an object from two series first rename the object in the current series then import it from the other series and per form a section by section comparison of traces to decide which ones to keep The Import Series dialog can be used to copy and rename an entire series This includes copying the actual image source files but it does not facilitate renaming t
133. ing nor mals will result in an unrecognizable 3D shape because there will be no shading Face normals generally produce a faceted appearance to the 3D object Vertex nor mals produce a smoother shading transition between faces Consequently vertex normals are best except when the object really does have sharp corners e g the Box object Faceting Density Faceting density refers to the number of faces generated for the object A low faceting density number means fewer faces in the object representa tion A high density in combination with vertex normals will produce a smooth surface representation Faceting density only applies to Cylinders Ellipsoids and Spheres For a Cylin der it specifies the number of facets around the circumference For instance a value of 4 produces a 4 sided cylinder Each facet is made up of 2 triangular faces For Spheres and Ellipsoids the total number of faces is fx f 2 where f is the face ting density Size The size parameters allow you to specify the dimensions of the Trace Mid points Box Cylinder Ellipsoid and Sphere objects Size A specifies the width in the x direction for Boxes and Trace Midpoint faces For Cylinders Ellipsoids and Spheres Size A specifies the length or diameter along the principal axis Size B specifies the height in the y direction for Boxes and Trace Midpoint faces and the diameter of secondary axis of Ellipsoids and Cylinders Similarly Size C specifies the diameter of t
134. ing traces of copied objects as changes can easily be made on the wrong object 150 Reconstruct v1 0 6 2 Hiding and Unhiding Objects Hiding and Unhiding Objects The Hide command in the Object List Modify menu allow all the traces of high lighted objects to be be hidden from display when paging through the section The Unhide command has the opposite effect Be aware however that in either case the result can be overwritten by the Hide Unhide flags in the General Tab of the Series Options Simplifying Objects Selecting Simplify from the Modify menu of the Object List will apply the opera tion immediately to any highlighted objects in the list Every trace of an object that is not already marked as simplified will have the Simplify operation applied to it as described in Chapter 8 The Simplify on demand resolution specified in the Series Options will be used to determine the new traces for the object When traces are already marked as simplified as is typically the case for most objects the Simplify command will have no effect Therefore to simplify an object you must first uncheck the simplified flag on all traces This can be done by opening the Object List Attributes dialog and clearing the simplified box WARNING There is no undo for simplifyng objects Deleting Objects All the traces associated with a set of objects can be deleted from the series by using the Delete key on the object list o
135. ion box The target sections for the images in the list will numbered sequen tially from this first section If the first section is negative the parameter is ignored and an attempt is made to extract the desired section numbers from file names This works when the file names already have the desired section number as the file extension for instance The order of image files in the list can be modified by using the Sort numerically checkbox This option treats the file name as having an embedded numerical value and resorts the list accordingly in ascending numerical order The Randomize but ton puts the image files in random order It is not possible to reorder individual list items To get finer grain control over the ordering of the images just select one image at time The Pixel Size box specifies the width of an image pixel in series units Chapter 10 All domains created by Import Images will receive this pixel size attribute The pixel size of a domain can be changed later by editing the domain s attributes or calibrating the pixel size Images can be tiled within a section to create a montage A montage is created by importing multiple domains and placing them at the correct positions within the section The initial position of each domain can be set in the Import Images dialog by using the Offset X and Offset Y values Multiple domains can be added to a section positioned at different offsets by filling the list and clicking the Impor
136. irst Section is 1 the numbers in the image file names are used as the desired section numbers To specify different section numbers change this 1 to a positive value Creating a Domain Domains can be created within a section by selecting Import image from the Domain menu This will open an Import Image dialog for selecting the image file for the domain Specify the estimated pixel size of the image Chapter 9 at the bottom of the dialog before selecting Open Backing up or transferring series can be facilitated by keeping all the component image and series files in one folder For this reason it is recommended that the Copy file to series folder option is used When a domain image is imported it is placed at the origin i e x 0 and y 0 of the section If the current view does not include the origin you may not be able to see the new domain image Use the Home key to center the view of the entire sec tion including the new domain 66 Reconstruct v1 0 6 1 Creating a Domain Select Files Lzix Lookin E test x cf Fav q fth gradient jpg _J100CX 1893 proxy E _unaligned 14 w calgrids 1895 fe j fuck _gradient proxy jpg _J100CX 1894 E _unaligned 15 calgrids ser we fi _3100CX 1885 E _J100CX 1894 proxy _unaligned 16 E debugContour b My Recent _3100CX 1885 proxy AESSR E _unaligned_aligned 2 dendrite1 bm
137. it To 7 Not v ote Area a 7 Midpoint cal Festi V Length D will list rows D01 D02 and D99 but not Da1 D100 or Den drite Movements The Movements tab contains the options related to keyboard move ments described in Chapter 10 The initial values contained in this tab may not be appropriate for a given data set since this depends on the magnification used For example the initial value for translational movements using the arrow keys are one full series unit So if your data is examined at high magnification 1 unit may move more than the whole screen width resulting in objects disappearing when ever an arrow key is pressed Adjust the size of the arrow key movements by edit ing the values in the Translate X and Translate Y boxes Proxies The Proxies tab contains options for the use creation and deletion of proxy images on the section described in detail in Chapter 7 When this tab is opened the program searches through all the sections to determine how many domains have image proxies This can be slow if there are a lot of sections during which time the program might become unresponsive When the process is com plete the percent of domain images that have proxies is displayed When creating Reconstruct v1 0 6 1 43 Series or deleting proxies the program again may become unresponsive while is pro cesses all the section The Cancel button
138. it can be applied using Repeat or Propagate Alignments One of the principal features of Reconstruct is the ability to align serial section images Bringing data on adjacent sections into alignment facilitates analysis and improves accuracy of measurements Alignment of adjacent sections can be real ized by moving one section in relation to another with the keyboard This process can be difficult and time consuming so an alternative methods are provided Sec tions may be aligned By Correlation from the Movement submenu of the Section menu or by trace correspondence using the Align submenu of the Trace menu 136 Reconstruct v1 0 6 1 Alignments The Align submenu provides four alignment operations Table 10 2 The align ment operations adjust different numbers of basis functions in the transformation Using more basis functions in a transformation allows more degrees of freedom but requires more data points for the Align computation TABLE 10 2 The Align Submenu Commands Command Basis functions data pts Movement potential Rigid 1 x y 2 Rotations and translations Linear 1 x y 3 scaling and skew slant Deformal 1 x y xy 4 deformations Quadratic 1x yxy xy 6 bending ALIGNING SELECTED TRACES The Align submenu commands can be used to put the centroids of two or more traces into alignment with the centroids of corresponding traces in another section When multiple traces are to be aligned they must have unique
139. ity The brightness and contrast of a domain can also be changed in the main window when a domain is selected This is done with the hyphen equal sign and bracket keystrokes as described in Table 4 2 Pressing the hyphen key decreases the bright ness while the equal key increases the brightness Contrast is increased by the right bracket and decreased by the left bracket To return to the original brightness and contrast values select Contrast from the Domain Reinitialize menu 74 Reconstruct v1 0 6 1 Reinitializing a Domain pa oere me OES AN tel z Options dialog The use of proxy images General Tracing Grids 3D Lists Movements Proxies Thumbnais can be turned on or off by the checkbox The proxy images can also be created or Speed tion display atl ification b ati ae ees se eas E T deleted by the buttons Note that creating Banca proxies may be very slow and the program will be disabled until the v Use proxy images when displaying sections j operation is complete Threshold size for creating proxies 2048 x 1536 pixels 0 ofimages bigger than this threshold have proxies Re Create Proxies atfraction 0 2500 offull dimensions Delete All Proxies coe The brightness and contrast can also be changed for all the domains contained in a range of sections by using the Modify gt Brightness Contrast dialog from the Sec tion List First highlight the range of sections to b
140. l number of sections the object spans Reconstruct v1 0 6 1 117 Calibration and Measurement used to compute 3D values from traces The z distance for any section is computed by summing the thicknesses of preceding sections The section indices are not used in the Z distance calculation Section indices only indicate the order of sections not the relative locations of sections in space Section locations and their relative distances are entirely determine by the z distance The z distance is computed rel ative to the section with the lowest index greater than zero Changing section indi ces without altering the order does not affect z distance However adding sections with lower indices will change the z distances Two methods are provided for the computation of z distances Fig 9 4 One com putes the distance to the middle of the section while the other computes the dis tance to the top of the section The former method gives more accurate z distances and 3D representations when sections are not uniform in thickness Setting z dis tances to the tops of sections provides an easy way to represent gaps of missing sections by giving a larger thickness to the sections at the top of the gaps When section thickness is uniform the only difference between the methods is a half section n Figure 9 4 Two methods are available for computing the z distance of section n One computed the distanc
141. layed in the list In addition each Z trace may be given a descrip tive annotation of up to 127 characters The entire list can be saved to a comma separated values csv file using the Save command at the top of the list win dow This file format can be readily imported into any spreadsheet application The Modify menu allows the color name or annotation of highlighted Z traces to be changed These changes permanently alter the attributes of the trace and the changes are immediately reflected in the 3D Scene window if it is open Z traces can be added or removed from the 3D Scene using the Scene menu of the Z Trace List Note that all Z traces should be given unique names so they can be indepen dently displayed in the 3D Scene A group of Z traces in the list can be renamed using the special character to get a sequence of uniquely named traces For Reconstruct v1 0 6 2 161 Objects E E E window Every z trace in the series is z listed along with the range of sections Sae Brem e which each trace spans The Scene Z Trace Note Start End Length and Attributes menus appear above Z D10length 1 94 4 6204 the list The z trace D21length is i apie jir poco highlighted The z trace D18length Z D20length 1 04 5 88029 has been added to the 3D Scene as 1 94 5 40342 indicated by the shaded icon with the Z D3length 1 94 5 10665 cyan background None of the other Z D6changed 1 94 4 61658 z traces are in the sc
142. le Option Type Default s Description index integer 0 current section number units string microns units of measurement viewport floats 0 0 0 00254 position and magnification of view defaultThickness float 0 05 default section thickness zMidSection boolean false select top or middle for z distances autoSaveSeries boolean true automatic series saves autoSaveSection boolean true automatic section saves warnSaveSection boolean true warn if section save needed beepDeleting boolean true beep when item is deleted beepPaging boolean true beep when paging fails hideTraces boolean false hide all traces when paging unhideTraces boolean false unhide all traces when paging 44 Reconstruct v1 0 6 1 Series Options TABLE 5 1 Series Options Stored in the Series File Option hideDomains unhideDomains useAbsolutePaths thumbWidth thumbHeight displayThumbContours useFlipbookStyle fitThumbSections firstThumbSection lastThumbSection skipSections flipRate useProxies widthUseProxies heightUseProxies scaleProxies defaultBorder defaultFill defaultMode defaultName defaultComment listSectionThickness listDomainSource listDomainPixelsize listDomainLength listDomainArea listDomainMidpoint listTraceComment listTraceLength listTraceArea listTraceCentroid listTraceExtent listTraceZ listTraceThickness listObjectRange listObjectCount listObjectSurfarea listObjectFlatarea listObject Volume listZTraceNote listZTraceRange
143. list is open the current section is highlighted A different section can be displayed in the main window by double clicking the new section number in the Section List Window The section list will be closed once a new section is selected The paging operation can also be executed by selecting the Page To menu item at the top of the Section List Window in which case the section list is not closed The Section List Window can also be closed by selecting the List item from the Section menu in the main window Navigate the section list with the keyboard using the up and down arrow keys the Page Up and Page Down keys or the Home and End keys Use Enter to execute the Page To operation for the first highlighted section TABLE 4 5 Keyboard and Mouse Actions in Section List Window Command Action Up amp down arrows Move to next previous section in list Page Up amp Down Move through the list by the number of visible sections Home Move to the first section in the list End Move to the last section in the list Delete Delete the highlighted sections from the series Enter Page to the first highlighted section Ctrl or Shift Highlight multiple sections for Modify operations Left Mouse Click Highlight the section under the pointer Left Mouse Dbl Click Page to the section under the pointer Multiple sections can be selected in the section list by holding the Ctrl or Shift key down while selecting sections in the list with the mouse The highlight
144. ly available after the domain is selected from the section Repeating Movements An individual movement can be repeated by using the Repeat item Ctrl r from the Movement submenu This allows a movement made on one section to be repeated on other sections in the series Similarly a movement made on one trace or domain can be repeated on other selected traces or domains The movement to be repeated is the last one made by keyboard input or the Type In dialog In addi tion a movement sequence or an alignment command may also be repeated The entire transformation of a selected element can be applied to another object using Repeat To copy a transformation use Record from Selected in the Move ment menu This will set the transformation of the selected trace or domain into the buffer that stores the last movement Selecting a new trace or domain and using Repeat will apply the buffered transformation to the selected item Note that new transformation will be a combination of the old one and the buffered one not just the buffered one alone PROPAGATE A repeated movement can also be applied to a range of sections within the series by using the Propagate command from the Movement submenu This is particu larly useful for repeating a change made to one section on the rest of an aligned series Such an operation allows a misalignment at one point in the series to be eas ily fixed For example suppose a small offset existed in the middle of the
145. made carefully Some computations are dependent on discretizing the data and this is sometimes done at relative frac tion of 1 unit Thus if the objects to be reconstructed are about 1 micron in size use microns as the units rather than millimeters or angstroms A good rule of thumb is to choose units so that the image pixel size is a small fractional value e g 0 01 Copyright 2006 John C Fiala 109 Calibration and Measurement All input and output values are consistently in series units For example if the units are declared to be microns then all values entered for section thickness movement values image pixel size etc should be in microns A section thickness value of 50 nm would be entered as 0 05 microns A deformation would be entered as microns Trace area would be reported in the Trace List window as square microns Similarly the cursor position would be given on the status bar as the x and y position on the section in microns DATA PRECISION Section data is displayed and stored in series units For example trace points are entered as screen pixels and these values are immediately transformed into the cor responding series units on the section The traces are stored in the section file as a sequence of x y points in series units These points are stored as ASCII strings with a fixed number of significant digits The default number of significant digits 6 would allow values between 999 and 999 to
146. maged separately it is necessary to realign the images for accurate reconstructions IGL Align also developed in the Image Graphics Laboratory was a first attempt at aligning grayscale images Funding from NIH s Human Brain Project to Children s Hospital allowed the development of a more sophisticated alignment program sEM Align in 1999 sEM Align was developed to allow on 20 Reconstruct v1 0 6 0 Credits screen alignment of larger images than was possible through the use of IGL Align This is accomplished by displaying and moving scaled versions of the original images on screen Images can be incrementally moved using keyboard input or alignments can be computed from a set of feature correspondences specified by the user To use the alignments computed by sEM Align a new set of aligned images are rendered for input into IGL Trace Most of the functionality of sEM Align and IGL Trace is incorporated into Recon struct By combining alignment and tracing into one program Reconstruct allows images to be processed more efficiently Tracing can be done directly on the trans formed images and alignments can be easily modified Converters are available to convert existing sEM Align and IGL Trace series to ones compatible with Recon struct See the synapses bu edu website for details Reconstruct development was funded through April 2004 by NIH s Human Brain Project A Users Group was created at that time to continue to support t
147. measured from minimal folds in the section when these are present Fiala amp Harris 2001 An alternative method for estimat ing average section thickness can be used when there are longitudinally sectioned cylindrical objects such as mitochondria Fiala amp Harris 2001 This method is applicable to series of calibrated pixel size and aligned Chapter 10 sections A cylinder sectioned longitudinally has its diameter appear in the series in two directions Fig 9 3 First in the section passing through the middle of the cylin der the diameter d can be measured in the plane of the section The diameter also extends orthogonal to the plane of sectioning causing the cylinder to appear across several sections By counting the number of sections s through which it appears section thickness can be estimated as the ratio d s Taking the average of many such measurements distributed throughout the series results in an estimate of the average section thickness This value can be applied to every section in the series using the Thickness dialog from the Modify menu of the Section List Z Distance The z distance of a section is the distance along an axis perpendicular to the plane of sectioning from the start of the series to the section Fig 9 4 This z distance is Figure 9 3 The method of cylindrical diameters involves computing the ratio the diameter of a cylindrical object at its widest point on a section arrows to the tota
148. menu item from the Section List Fig 6 1 Warning There is no Undo for Modify gt Thickness from the Section List SECTION NUMBERING Sections can be renumbered using the Modify gt Numbering command from the Section List Fig 6 1 The Renumber Section dialog allows a shift to be applied to the section numbers of all section highlighted in the list The integer value entered is added to each section number Thus a positive value shifts the section numbers up and a negative value shifts the sections numbers down Only constant shifts of section numbers are performed and existing sections cannot be overwritten When a section would overwrite another section renumbering is terminated Reconstruct v1 0 6 1 57 Sections Delete Sections AN The sections will be permanently deleted Delete all image files referenced from these sections This operation cannot be Undone Continue Figure 6 2 The Delete Sections dialog invoked whenever the user requests to delete sections from the Section List Warning There is no Undo for Modify gt Numbering from the Section List Deleting Sections Sections can be deleted from the series using the Section List Highlight the sec tions in the list to be deleted and select the Delete item from the Modify menu or use the Delete key This will delete the section files permanently from the series Warning There is no Undo for Delete Sections Section files refer to
149. mplify will use a resolution set by the user in the Series Options while simplification during drawing is done relative to the current screen pixel size Note that the simplification process applied to an open point by point trace will very likely add points so that the trace more closely matches the option specified resolution Closed Point by Point Drawing The closed point by point tracing tool works in the same way as the open one but the candidate completed trace is shown along with any applicable fill color To avoid the fill color while tracing select None for the fill mode in the default attributes This attribute can be easily changed after tracing To accept the candidate trace press the right mouse button Use Backspace to remove segments and Esc to abandon the trace altogether If automatic simplifica tion is enabled the final trace is given a positive orientation and all internal loops are removed Thus a trace with a single closed path is always generated Freehand Drawing The freehand drawing tool has a cursor that looks like a pencil The tool is used much like a pencil in that a single drag operation by the mouse gen erates the closed trace Pressing and holding the left mouse button starts the trace The path of the trace follows the mouse movement The drawing opera tion can be aborted by using the Esc key When the mouse button is released the trace is closed and simplified if enabled to produce a single
150. much smaller trace in series units than tracing at lower magnification Shapes of a fixed size in series units can be created using the Grid Tool R The Stamp Tool allows creation of traces with a predefined shape to be The right mouse button allows panning the section while using the Stamp Tool Press and hold the right button and drag the cursor to the edge of the main window The section will scroll as long as the cursor is near the edge and the right button is held down Stamp shapes can be selected from the Trace Palette by selecting one of the palette buttons or equivalently by using the number keys A stamp shape can also be selected from the section by clicking the left mouse button while holding down the Ctrl key on the keyboard The trace closest to the cursor point replaces the current stamp shape This shape can replace the shape in a palette entry by using the Ctrl number key sequence to update the palette Stamp shapes can be either open or closed depending on how the shape was originally drawn or how its attributes were set prior to selecting it To stamp shapes consisting of more than one trace use the Grid Tool Creating a Grid of Traces J The Grid Tool is used to create a repeating pattern of traces by placing ham multiple copies of a set of traces at a certain distance apart Primarily the Grid Tool is designed to generate grids for facilitating stereological mea surements on sections but it can also be used to c
151. n Thickness The section s thickness in series units Object List Measurements The trace list column data can be used to compute 3D quantities by utilizing the length and area columns in combination with the section thickness column Since these quantities depend only on the section thickness and not the z distance they are not affected by changing the method of z distance calculation A few common computations are done automatically in the Object List The columns that are dis played in the object list can be saved to a csv text file using the Save As item at the top of the list window Reconstruct v1 0 6 1 123 Calibration and Measurement All values are computed at the time the Object List window is created To make sure that the values are up to date close the Object List window and reopen it after any changes to the traces are made The area and volume measurements are com puted only if these columns are displayed in the list so removing these columns can speed up the creation of the list Count The count is the total number of traces in all sections that have the object name Surface Area Each trace of an object contributes to the surface area an amount equal to the product of the trace length as computed in the Trace List and the sec tion thickness Surface Area gt length x thickness all sections Flat Area The enclosed areas of closed traces in the object are summed with the surface areas of the
152. n open the Trace List Alternatively the arrow keys can be used to browse through the 28 Reconstruct v1 0 6 1 Main Menu menus The highlighted menu item can be selected by using the Enter key The menu selection process can be aborted by using the Esc key Some menu items have special keystrokes associated with them that allow them to be rapidly executed without using the menu selection process Table 4 3 For example pressing the S key while holding down the Ctrl key when the main win dow is active will select all the traces in the section Many of the menu items may be grayed or disabled if they are not currently appro priate For example if no series is open then the menus for manipulating sections domains and traces are not applicable Likewise the domain and trace menus are mostly disabled when no domain or trace has been selected since most operations apply to selected data elements TABLE 4 3 Keyboard Accelerators for the Main Menu Keystroke Action Space Blend the front and back section images Esc Abandon current menu selection activity Ctrl Move front section By Correlation with back section Ctrl A Edit the attributes of selected traces Ctrl B Paste attributes from clipboard traces to selected traces Ctrl C Copy the selected traces Ctrl D Deselect all the selected traces Ctrl H Hide the selected traces Ctrl L Toggle the movement lock on the section Ctrl M Merge the selected traces Ctrl O Open the
153. nce a trace has been simplified the Simplified attribute is checked to prevent fur ther unnecessary simplification The possibility of further simplification can be reestablished by unchecking the Simplified attribute Automatic simplification can be disabled by unchecking the option in the AutoTracing tab of the Series Options Disabling simplification will allow the cre ation of traces with internal loops Fig 8 3 Do not create traces with loops The behavior of many subsequent measurements and operations are adversely affected by traces with self intersections In addition simplification can reduce the number trace points and speed up further processing of traces NOTE Keep automatic simplification on at all times for accurate results Loops are not eliminated from open traces by the simplification procedure There fore traces with loops can be created even when automatic simplification is enabled A closed trace with loops can be created by drawing an open loop trace and then converting it to closed by changes its attributes Doing this does not elim inate the loops even when automatic simplification is turned on To eliminate the loops in a closed trace originally drawn as open apply the Simplify command from the Trace menu Figure 8 3 The closed trace on the left has loops while the closed trace on the right does not Reconstruct v1 0 6 1 87 Traces 2x Hue 220 Red 255 Sat 240 Green 0 ColorlSoli
154. ne and then removing them To reset the bounding box after removing objects it is necessary to clear the scene add any desired objects and then reset the scene ROTATING PANNING AND ZOOMING To rotate the scene press and hold the left mouse button in the scene window Drag the cursor across the scene in the desired direction of rotation The scene will begin to rotate at a speed proportional to the speed of the cursor movement across 168 Reconstruct v1 0 6 1 Viewing a Scene the window Releasing the mouse button results in the scene continuing to rotate at this speed in spin animation To stop this rotational spin press the right mouse button Scene rotation with the mouse is in two directions left to right horizontal rotation or up and down vertical rotation The final motion can be a combination of these rotation directions but not a rotation in the plane of the scene window To roll the scene in the plane of the scene window it is necessary to use a combination of rota tions First rotate the scene 90 degrees horizontally then apply the desired vertical rotation and horizontally rotate the scene back to the original viewpoint To pan the scene press and hold both left and right mouse buttons simultaneously Then drag the cursor to shift the scene in a particular direction To avoid activating rotational spin release the left button before releasing the right button The scene can be zoomed by using the right mouse
155. ng IMPLIED points SMFVec2f IMPLIED gt lt ELEMENT Image EMPTY gt lt ATTLIST Image mag SSEFFloat WA oO contrast SSEFloat ws brightness SFFloat wor src SSFString proxy src SSFString T proxy scale SFFloat 1L 0 gt lt ELEMENT Transform Image Contour Contourt gt lt ATTLIST Transform dim SSFInt32 eN xcoef SMFFloat 0 100 0 0 ycoef SMFFloat o OL 0 0 O gt lt ELEMENT Section Transformt gt lt ATTLIST Section index SSFInt32 Sp thickness SFFloat 70305 alignLocked SSFBool false gt Figure 3 2 The Document Type Definition of the section data file section dtd 14 Reconstruct v1 0 6 0 Data File Organization XML FILE FORMATS Reconstruct uses XML eXtensible Markup Language www xml com for repre senting series and section data XML files are in plain ASCII text and are therefore readable by text editors XML can also be validated allowing errors in the files to be automatically detected A Document Type Definition DTD specifies the for mat for a valid file For Reconstruct there are two DTDs one for the section data file and one for the series data file By including a reference to the DTD in the header of the XML data file an XML validator can determine whether the syntax of the data file is correct The section DTD Fig 3 1 specifies the XML file format for all section data files The basic data types are defined by the ENTITY descriptions referenced late
156. ns will be colored with the border color using the Colorize algorithm Colorize replaces only the most intense most white colors in the region with the border color Using very light pastel colors for the border colors will result in colored regions in which much of the underlying contrast is still visible as shown in Fig 6 3 This algorithm also allow coloring with subtle shades that would not give nice fills in the main win dow Like all fills the Colorize fills may give different results for a different trace orders Fig 6 3 A trace can be moved to the top of the implicit ordering by first selecting it then deselecting it and then saving the section After the OK button is selected in the Export Sections dialog the sections begin being rendered and the new files created A small dialog box appears showing the progress of the rendering The operation can be aborted by using the Abort button in the dialog or the Ctrl Pause key sequence Aborting may be slow but additional sections will not be processed after the Aborting message appears in the dialog When all sections have been rendered a message is displayed indicating that the operation completed The new images may be reviewed by opening the new series from the Series Open menu item 64 Reconstruct v1 0 6 1 CHAPTER 7 Domain Images Domains are image regions defined by an image file bmp jpg etc and domain boundary outlining the region to be displayed on the
157. nstruct v1 0 6 0 Data File Organization Reconstruct is designed to facilitate image cropping scaling and alignment Crop ping is achieved by drawing a new domain boundary that specifies the region to be displayed on the section Image scaling is specified by a pixel size magnification parameter Alignment is realized by a non linear transformation associated with the image All this information is stored with in the section In addition to the images and transformations polygons within the section specify object profiles on the image data These traces are added to the sections by draw ing with a mouse or a pen on a Tablet PC All the traces that share the same name belong to the same 3D object This convention allows the 3D objects to be extracted from the section files by grouping together all the traces with the same name Objects are thus represented by the traces in the sections Objects can be split apart or combined together simply by renaming the traces Data File Organization The data for each section images and traces is saved to a file indexed by the sec tion number as the filename extension A series with 100 section would have 100 section data files numbered sequentially 1 through 100 For a series named myseries the section data is stored in files named myseries 1 myseries 2 myseries 3 and so on to myseries 100 All the section data files are stored in the same folder or direc
158. oblem with the traces used for correspondence Undo the alignment and carefully check the trace names to make sure that the desired correspondences were correctly specified Undoing and Adjusting Alignments If the alignment produced by one of the Align commands is not adequate it can be modified or undone To undo it first make sure the section number in the title or status bar is the one on which the alignment was done i e the section that was moved Then use Ctrl z Undo to return the section to its previous state If you plan to try the same Align operation again after modifying or adding traces for correspondence points it is not necessary to first undo the alignment Points can just be added to each section and the Align operation repeated If a quadratic or deformal alignment is followed by a rigid or linear alignment it will be necessary to clear the nonlinear components from the transformation by Ctrl z since they are not cleared by executing a subsequent linear or rigid alignment After a linear alignment an Undo is not necessary prior to adding points and trying again with a linear deformal or quadratic Align Repeating an Alignment An Align command can be repeated and propagated just like any other movement Use the Repeat item from the Movement submenu to apply the same adjustment to subsequent sections Use Propagate to apply the same adjustment to many sec tions Remember that Repeat can be undone but Propagate cann
159. onstruct v1 0 6 1 91 Traces The default trace attributes can be filled from the first entry on the trace clipboard by selecting the Get Clipboard button This provides a way to copy the attributes of an existing trace and use them for subsequent drawing of new traces This also provides a mechanism for copying attributes of existing traces to the Trace Palette see below Values entered in fields for the default trace name colors fill style and comment will be given to all traces subsequently created Unlike other most other values in the Series Options dialog default trace attributes and palette entry changes are made immediately when the value is entered Selecting the Apply button is not required However if a change is made to a palette entry and you also wish to use this as the default tracing attributes select Apply rather than Cancel to exit the Series Option dialog The Trace Palette A set of predefined default trace attributes can be quickly accessed using Palette from the Trace menu The Trace Palette window Fig 8 6 has ten predefined pal ette entries and ten user definable palette entries A new series begins with user entries set to the predefined values To use the trace palette simply select a button in the palette window using the mouse The default trace attributes will be immediately set to the name colors and fill mode defined for that palette button Holding the mouse over a button when the palette
160. open traces to get the total flat area Flat Area l gt length x thickness by area all sections open traces closed traces The area is computed using the Green s theorem formula as in the trace list Nega tive areas detract from the total flat area This measurement is useful for objects that are known to be flat but may sometimes appear in the plane of section as well as perpendicular to it The flat area can also be used to include end caps on sur faces Volume Volume is computed using the Cavalieri formula Volume y area x thickness all sections Each trace contributes the product of its enclosed area as computed in the Trace List times the section thickness Traces with negative areas diminish the volume and open traces contribute nothing 124 Reconstruct v1 0 6 1 Z Trace List Measurements Z Trace List Measurements Lengths of z traces are reported in the Z Trace List window The columns that are displayed in the Z Trace List can be saved to a csv text file using the Save As item at the top of the list window All values are computed at the time the Z Trace List window is created To make sure that the values are up to date close the Z Trace List window and reopen it after traces are added All values are in series units Length The length of a z trace is the sum of the distances between points length 641 22 O41 W Gin 21 it The x and y positions of the points were
161. order of domains can be changed by selecting and merging the domains to either the front or rear of the domain order Merge Front puts the domain at the end of the domain list where it will be painted on top of all other domains Merge Rear puts the domain at the start of the domain list where it will be painted over by all other domains When the Select Domain Image Tool is being used the Merge Front operation can be carried out by clicking the right mouse button Moving and Montaging Domain Images Each domain in a section is associated with a transformation that places the image on the section at a particular location and orientation Chapter 10 To change the position of an image on the section pick up the Select Domain Image Tool see below and click on the image using the left mouse button The domain image will be selected and displayed by itself inside its domain boundary The position of the domain relative to the rest of the section can be seen by blending the domain with the section Spacebar Translate the domain image by using the arrow keys or by dragging the domain with the left mouse button When done moving the domain merge the domain with the rest of the section by clicking the right mouse button Up to 254 images may be arranged within a single section to create a montage Fig 7 1 Each image is imported as a separate domain see above and then moved into position with the other images by dragging it with the Select Dom
162. ormance is free RAM equal to a least 3 times the total amount of data on a section Thus if a section contains 40 Mb of image data 40 40 40 120 Mb of RAM would be needed for running the program Allow for an additional 128Mb for the Windows XP operating system and desktop along with 20 40 Mb for each major application Acrobat Outlook Word Excel etc to used simultaneously with Reconstruct 512Mb of memory was adequate for all data used to test the software The greatest performance enhancer is a fast hard drive Note that hard drive perfor mance is not necessarily the transfer rate given in the hard drive specifications This Data Transfer Rate is usually a burst rate for the interface to the system mem ory not the actual speed of data access from the drive Look for drives with the highest speed of rotation RPM and the most sectors per track Real sustained data transfer rates are roughly proportional the product of rotational speed and sectors per track For example a 15 000 RPM SCSI drive will outperform a 5 400 RPM IDE ATA drive resulting in faster loading and paging of sections in Reconstruct A 7 200 RPM IDE ATA drive was used during development and testing of the software A 7200 RPM drive gives satisfactory performance on images less than 15 Mb Reconstruct v1 0 6 0 19 Software Overview YAHOO Groups 5 Ree Home Description Messages Welcome to the Reconstruct Users Pending Group Post Chat
163. ot in general Reconstruct v1 0 6 1 139 Movements and Alignments section 101 section 102 section 103 section 104 section 105 gt LY OW WY amp Figure 10 3 A series with a misalignment between sections 103 and 104 All the lower sections 1 through 103 are aligned to each other All the upper sections 104 200 are also aligned but a misalignment between sections 103 and 104 spoils the complete series alignment Sometimes a misalignment is found in the middle of an aligned series creating the scenario depicted in Figure 10 3 To fix such an alignment mistake Align the sec tion on the upper side of the mistake section 104 in the figure to the section on the lower side section 103 Keep undoing the Align command and fixing the points until completely satisfied with the Align result Only the last Align com mand can be propagated so using multiple Align commands without undos between them will not work in this case Once 104 is aligned to 103 select Propa gate from the Movement submenu and enter the range for the remaining sections on the upper side sections 105 200 The result will put the entire series in align ment Montaging Domains by Point Correspondences Chapter 1 gave a protocol for aligning sections using point correspondences The point correspondence technique can also be applied to the problem of montaging multiple images within a section This is done by repeating on the selected dom
164. ound and object colors 170 Reconstruct v1 0 6 1 Saving the Scene including transparency are preserved However the Front and Back faces wire frame setting will not be retained The VRML file is a simple text file Comments in the file indicate the version of Reconstruct used to generate the file the series name and units Each object in the scene is delineated with comments at the start and end of the object description The starting object comment in addition to the object name and type contains the following information Traces shifted by x y z Trace Areas shifted by x y z Trace Midpoints size ab shifted by x y z Box size a b c shift by x y z Boissonnat Surface shifted by x y z Cylinder center x yz axes X yz xyz x yz size lwh shifted by x yz Ellipsoid center x y z axes X y Z Xyz x yz size lwh shifted by x y z Sphere center x y z size a shifted by x y z These comment values record the settings of the Shift Object By and Size settings of the 3D Options tab In the case of the cylinder and ellipsoid however the 1 w h values reflect the Size parameter settings only when these are positive Other wise the actual eigenvalues calculated from the scatter matrix Chapter 11 are reported with 1 the diameter of the principal axis w the diameter of the secondary axis and h the diameter of the minor axis The axes are the eigenvectors of the scatter matrix given in the same order The center is
165. ourglass displayed at the cursor During this pause the program is exam ining all the domains in the series and determining how many domains have proxy images When the examination is complete the information will be reported in the dialog The use of proxies can be turned off by unchecking the Use proxy images when displaying sections Warning Re Create Proxies may be slow and it cannot be aborted Proxy images can be created by pressing the Re Create Proxies button A proxy image is then created for every domain image in the series for which the domain image dimensions equals or exceeds the threshold dimensions A dimension of the proxy image is the fraction of the domain image dimension as specified in the dia log For a domain image 4000 pixels wide by 2000 pixels high a fraction value of 0 25 would produce a proxy image of 1000x500 pixels so that whenever the domain is displayed at a screen resolution of 1000x500 or smaller the proxy image would be accessed and displayed instead of the full domain image For proxies to speed up section display you must carefully specify the fraction and threshold prior to creating the proxies For example suppose you want to work with the Main Window sized to full screen 1600x1200 and you want to display quickly the entire section 732204840 in the full screen window First calculate the fraction dimensions of the section relative to the full screen width fraction Scteenwid
166. ouse Click Highlight the trace under the pointer Left Mouse Dbl Click Put the trace under the pointer into the scene Distances List Window The Distances List window displays a table of distances between objects in the series For more information on the distance computation see Chapter 9 The object names and calculated distance in series units are displayed for each pair of objects that matches the list s limit strings To limit the number of distances that are calculated and displayed set the limit strings in the Series Options BEFORE selecting Distances from the Object menu The list display may seem to hang for 65 535 or more distances but displaying even a few thousand distances may be slow If more than 65 535 distances are calculated a warning message offers the opportunity to cancel the action before the list is displayed There are no special keyboard or mouse operations on the Distances List It is a plain vanilla list view As with all list views the columns of the list can be resized by dragging the borders of the column headers The entire list can be saved to a csv file by choosing the Save menu item Exiting or Aborting Most windows in Reconstruct can be closed by choosing the x in the upper right hand corner of the window This also typically aborts the operation being done by Reconstruct v1 0 6 1 37 User Interface the window For example if too many distances are being computed in
167. ows the operation to be applied to a subset of the object s traces The operation is carried out over a range of sections specified in the dialog The default section range will apply the name change to all traces of the object To limit the name change to part of the object entering a nar rower section range in the dialog Note that when an object is renamed its entry in the Object List is no longer valid Furthermore such name changes are not immediately reflected in the list The old object name is deleted from the list but the list has to be regenerated manually by closing and then reopening it to see the new object names For this reason after Rename is complete a message will appear suggesting that any name changes will only be revealed by reopening the Object List Reconstruct v1 0 6 2 149 Objects By changing an object s name attribute the object can be broken into its compo nent traces Suppose for example a set of vesicles were labeled with point traces all given the name VESICLE These traces would form a single object in Recon struct You can make each vesicle be its own object by using the Attributes gt of Traces dialog to change the Name string from VESICLE to VESICLE This will rename the individual traces to VESICLE1 VESICLE2 VESICLE3 and so on Close the Object List and reopen it and you ll see a separate object for each vesicle To group the vesicles back into a sin
168. p Documents li _J100CX 1886 ei _J100CX 1895 proxy N 2rings bmp N dendrite2 bmp _J100CX 1886 proxy amp _UNALIGNED O N 3rings bmp N dendrite3 bmp G li _J100CX 1887 E _unaligned 1 N 4rings bmp N dendrite4 bmp _3100CX 1887 proxy E _unaligned 2 ras 16 jpg A dendrite5 bmp Desktop _J100CX 1888 E _unaligned 3 fth 16 proxy jpg N dendrite6 bmp _J100CX 1888 proxy E _unaligned 4 E calgrids 1885 N dendrite7 bmp li _J100CX 1889 E _unaligned 5 ei calgrids 1886 N dendrites bmp eS _3100CX 1889 proxy E _unaligned 6 E calgrids 1887 N dendrite9 bmp L4 BETERI E _unaligned 7 calgrids 1888 N fig3a bmp My Documents eS _3100CX 1890 proxy E _unaligned 8 E calgrids 1889 A fig4 bmp li _J100CX 1891 E _unaligned 9 ej calgrids 1890 3 gradient 1 e _3100CX 1891 proxy _unaligned 10 3 calgrids 1891 E gradient 2 or g EB year E _unaligned 11 calgrids 1892 gradient 3 E _J100CX 1892 proxy _unaligned 12 calgrids 1893 gradient 4 My Computer J100CX 1893 E _unaligned 13 3 calgrids 1894 E gradient fm File name _J100CX 1895 _J100CX 1885 _J100CX 1886 _ 7 Files oftype Jan Files v Cancel A Figure 7 3 The Select Files dialog This dialog is invoked by the Select button in the Import Images dialog Multiple image files have been selected from the folder test These files will be placed in the Import Images list by clicking the Open button IMPORTING MULTIPLE IMAGES Multiple images
169. photographic film The film is then devel oped and scanned at 1000 dpi If the series units were chosen to be microns then the pixel size would be 25400 microns 0 00254 J 1000 10000 inch Reconstruct v1 0 6 1 111 Calibration and Measurement While this estimate is a useful first approximation magnification factors of instru ments are rarely known precisely Accurately determining the pixel size from the data is the main goal of calibration If no image data is entered or calibrated the initial screen resolution is assumed to represent a pixel size of 0 00254 in the default series units microns Note that the final size of an image pixel in a section is determined by a combina tion of the pixel size and the transformation that maps the image to the section Chapter 10 If this transformation contains scaling the pixel size parameter will be different than the actual pixel size in series units For this reason it s possible to calibrate the image by a combination of transformation scaling and pixel size but the preferred mode will be a fully calibrated pixel size Calibration Grids and Scale Bars The pixel size can be accurately determined from calibration images These can be scales or grids of a known physical size that were imaged at the same magnifica tion and digitized at the same settings as the serial sections With some imaging systems a scale bar is imprinted on the section data Fig 9 1 Reconstru
170. point by point drawing tools the status bar reports the position of the pointer on the section This allows points to be entered at partic ular locations When a z trace is being drawn the status bar reports the length of the candidate next segment including the difference in the z distances of the start and end sec tions length OA T Wisi 7y Zi 1 z i 120 Reconstruct v1 0 6 1 Domain List Measurements Domain List Measurements Two dimensional measurements on the domain boundary including length area and the x and y position of the midpoint are reported as columns in the domain list window The columns are selected by checkboxes in the Lists tab of the Series Options The columns that are displayed in the domain list can be saved to a csv text file using the Save As item at the top of the traces list window All values are computed at the time the Domain List window is created To make sure that the values are up to date close the Domain List window and reopen it after any changes to the domains are made All values are in series units Length The length of the domain boundary is the sum of the distances between boundary points Area The area is the size in square units of the region enclosed by the boundary Note that this area is computed using a formula based on Green s Theorem 1 area 5 Ois Xj4 i i for each segment from x y to X 1 yj in the boundary The accuracy of this compu
171. ps yahoo com group reconstruct_users files Run the downloaded file and a dialog should appear asking you whether you want to install Reconstruct Select Yes and proceed through the installation dialogs until the process is com plete To test your installation start Reconstruct and select Open from the Series menu If this is the first time you ve installed and used Reconstruct a series file called example ser should be visible in the Open dialog Open the example series When Copyright 2006 John C Fiala Quick Start the series opens you should see some text on the screen Follow these text instruc tions to get familiar with the basic operations of Reconstruct This chapter only covers a small fraction of Reconstruct functionality To get a bet ter idea of the full functionality review the Key and Mouse commands listings available on the Help menu Then open the Options dialog from the Series menu and review the contents of all the Options tabs This will give you an idea of what features of the program can be reconfigured to suit your purpose Details about these features can be found in subsequent chapters CREATE A SERIES Start the Reconstruct program and from the Series menu select the New menu item A file open dialog will appear Navigate to a folder on the hard drive where the new series files can be written If desired you may create a new folder from this dialog The word newSeries should appear in th
172. r by the sign SFBool defines a single field Boolean either true or false SFColor is an RGB color defined by 3 floats where 0 0 0 is black and 1 1 1 is white SFFloat is a single 32 bit floating point value MFFloat is an array of float ing point values separated by white space SFInt32 is a single 32 bit integer value SFString is a string of characters excluding the characters lt and gt MF Vec2f is an array of pairs of floats specifying x y coordinates on the section The rest of the DTD defines the basic ELEMENTS of a section and their attributes A section contains one Section data element The Section has three attributes an index specifying the position of the section in the series a thickness specifying the section thickness and an alignLocked flag that specifies whether the entire section can be moved The Section may contain one or more Transform elements The Transform element contains the coefficients of the linear combination of the bivariate polynomial basis and a dim parameter specifying how many of these terms are to be used Each Transform element contains either an Image element and boundary Contour or a set of Contour elements The Image element is the reference to an image file Image attributes are mag the width of a square pixel in series units src the path to the file containing the image data contrast and brightness operate on a virtual intensity range of 0 1 proxy_src the path to a fil
173. r highlights Filled or Wireframe Faces By default the triangular surface patches are filled with the color of the object both when seen from the front and when seen from the back Generally speaking the front surfaces are those seen from the exterior of the object while the back surfaces are those exposed to the interior of the object Fig 12 2 To display the front or back surfaces as colored wireframes uncheck the Fill checkboxes in the Change Scene Object dialog Viewing a Scene The viewing parameters of the scene are modified by using the mouse to rotate pan or zoom or by using the View menu at the top of the scene window The ini tial or Reset view position is from a point beyond the last section of the scene look ing down toward section 0 In effect looking in the z direction For an object oriented perpendicular to the plane of sectioning the Reset view will be looking at the end of the object from the highest section number The Reset view places the box that bounds all of the objects in the scene in the center of the window It may be desirable to reset the scene when objects are added The center of the scene s bounding box also determines the point around which rotations are performed Adding objects to the scene expands the bounding box but removing objects from the scene does not reduce the bounding box This allows arbitrary centers of rotation to be defined by adding appropriately posi tioned objects to the sce
174. r selecting the Delete item from the Modify menu Deleting objects is permanent the deleted traces cannot be recovered For this reason any request to delete objects will be challenged by a warning dialog WARNING There is no undo for deleting objects Reconstruct v1 0 6 2 151 Objects Calculating the Distances Between Objects A distance between two objects is defined to be the minimum 3D distance between all the line segments of all the traces of the objects Chapter 9 If object traces intersect the distance is zero This distance does not take into account whether one object is inside another rather the distance between boundaries of objects is com puted Three dimensional distances between objects are listed in the Distances List win dow Chapter 4 Distances are calculated only when the Distances List is opened and all distances are computed before the list filled with entries The list needs to be regenerated whenever there are changes to the data Object names and calculated distance in series units are displayed for each pair of objects that matches the list s limit strings To limit the number of distances that are calculated set the limit strings in the Lists tab of the Series Options BEFORE selecting Distances from the Object menu With the default settings the dis tances between every object in the series and every other object in the series will each be computed twice Once with the object in the left
175. reate lines rectangles and circles of a specific size The left mouse button creates a grid on the section The right mouse button can be used to scroll the section by dragging at the edge of the main window 94 Reconstruct v1 0 6 1 Creating a Grid of Traces The type of grid generated by the Grid Tool is determined by the settings in the Grids tab of the Series Options Fig 8 7 Five predefined grid types consist of rectangles crossed lines ellipses sampling frames and cycloids Fig 8 8 The number of elements in the grid is specified in the x and y directions along with the size of each element and the distance between elements For rectangular and sampling frame grids the left mouse button places the upper left corner of the first grid element For cycloidal grids the left mouse button places the left most point of the first grid cycle For the remaining grid types a left mouse button press places the center of the first grid element Additional grid elements are added to the left x direction and below y direction the first element Arbitrary grid elements can be defined using one of the two remaining element types Stamp shape or Clipboard trace When the stamp shape type is selected the Grid Tool centers the current stamp shape at the grid positions The stamp shape can be selected from the Trace Palette or by using the Stamp Tool to pickup a trace from the section The stamp shape is scaled to the Element size parame
176. rom the Tools window Click on an image in the section with the left mouse and the domain will be selected The boundary of a domain is visible only when it is selected In addition this boundary can be edited only when it is selected see below A selected domain is removed from the section and displayed alone in the front display The back display contains the remaining components of the section So the same techniques for comparing sections can be used to compare a domain to its section or to an adjacent section Use the Blend from the Section menu Spacebar to overlay the domain and the section To flicker between the domain and the rest of the section use the key A domain can also be selected from the Domain List as mentioned above Only one domain can be selected from the section at a time In order to select a different domain the currently selected must be merged back into the section A selected domain can be permanently deleted from a section by using the Delete key when the domain is displayed or by using the Delete item from the Domain menu when anytime the domain is selected 70 Reconstruct v1 0 6 1 Merging a Domain Merging a Domain To return a selected domain to the section i e to deselect it use either the Merge Front or Merge Rear commands Domains have a implicit order that determines which image will be displayed on top of and occluding other images This order is reflected in the Domain List The
177. rs 1 1 number of elements in x and y gridSize floats 1 1 size of elements in x and y gridDistance floats 1 1 separation between elements in x y hueStopWhen integer 3 wildfire hue stop criterion hueStop Value integer 50 wildfire hue stop value satStopWhen integer 3 wildfire saturation stop criterion satStopValue integer 50 wildfire saturation stop value brightStopWhen integer 0 wildfire intensity stop criterion areaStopPercent float 999 0 wildfire autotrace area change areaStopSize float 0 0 wildfire minimum region size brightStopValue integer 100 wildfire intensity stop value tracesStopWhen boolean false wildfires stop at traces if true mvmtIncrement floats 0 022 1 1 1 01 sizes of keyboard movements 1 01 0 02 0 02 0 001 0 001 ctrlIncrement floats 0 0044 0 01 sizes of keyboard movements 0 01 1 002 when Ctrl key is held down 1 002 0 004 0 004 0 0002 0 0002 shiftIncrement floats 0 11 10 10 sizes of keyboard movements 1 05 1 05 0 1 when the Shift key is held down 0 1 0 005 0 005 contours various various palette contour attributes and stamp shapes 46 Reconstruct v1 0 6 1 Exporting and Importing Images Exporting and Importing Images Sections contain a set of transformed images domains and traces Importing mul tiple images into sections can be carried out by opening the Import gt Images dia log from Series menu This dialog is documented in Creating a Domain in Chapter 7 Entire sets of section data can be exported
178. rt button store the DXF data as traces on the corresponding sections The DXF data is assumed to be in the same units as the series Reconstruct v1 0 6 1 47 Series Figure 5 3 The Import Series Series Cala RECONSTRUCT iestimiio ser dialog The Series name is filled by selecting a series file with the v Importthe Series Options settings Browse button When copying JV Importthe Trace Palette section data be sure to set the ImportZtracesnamed FSS section range Use the limit strings to select a subset of the data for Copy from sections 0 through 16 import Check the Copy image files box to copy the image data Jv domains named into the same folder as the current series Without this the current eS ANEA Foo series will create a relative path to the images in the original folder V Copy image files into series folder WARNING This operation cannot be Undone Import Cancel Importing Series Data Elements from an existing series can be copying into the current series using Import gt Series dialog from the Series menu Fig 5 3 Use the Browse button to navigate to a folder and select an existing ser file to copy from Then select what elements are to be copied by checking the appropriate boxes Importing the Series Options or Trace Palette will overwrite the values in the current series Importing z traces will add to the current series only the z traces with names that match the string Str
179. s The technique can be repeated with each domain to align each element of a mon tage separately Just make sure that a unique trace name is selected for entering trace points on each domain so that the traces for each domain can be distin guished A variation of the above method can be used to add and align a domain to an exist ing section Assume that goal is to import a new domain image into section I and align it to the existing part of section I instead of to an adjacent section Step 1 Import the domain to a different section II This section is arbitrary but it may be convenient to use an adjacent section for paging Select the domain and move it to the approximate desired location on the section Merge the domain into the section Step 2 Enter correspondence points on sections I and II that would align the domain on section II with desired features on section I Then use Align Traces from the Trace menu on section I This will move the correpondence points into alignment which can be verified by flickering Step 3 Select the domain from section II Use Ctrl r to repeat the alignment that was applied to the traces The domain will now have the correct transformation on section II that it should have on section I If desired the alignment can be verified by merging and flickering however at the end of this step the domain should be selected Step 4 From the Movement submenu use Record gt from selected to capture th
180. s in the series by using the Repeat or Propagate commands from the Movement submenu To rescale one other section unlock the section and select Repeat or Ctrl r To rescale a range of sections use Propagate and speci fied the range of section to modify Be careful not to rescale the current section again because this section was already rescaled by the Calibrate Traces dialog If the first calibration option Resize selected trace s was chosen in the Calibrate Traces dialog then no multi section application of the calibration is possible Section Thickness Section thickness is critical for determining the 3D representation and for getting accurate 3D measurements If section thickness is known in advance for all sec tions it can be set in the General tab of the Series Options This value is applied to new sections If a default section thickness is not specified a value of 1 20 of a sec tion unit will be used for new series The section thickness of individual sections can be set at any time using the Thick ness item from the Section menu The thickness can be set over many sections using the Thickness dialog from the Modify menu of the Section List 116 Reconstruct v1 0 6 1 Z Distance CALIBRATING SECTION THICKNESS Thickness is a property of a section that needs to be calibrated or estimated Micro tome settings can be used as an estimate but accuracy may be an issue The thick ness of individual sections can be
181. s making movements Table 10 1 summarizes the movements that can be performed by keystrokes This information is also available by selecting Key commands from the Help menu A keyboard overlay Appendix can also be used as a quick reference Generally all keyboard movements can be applied to either traces domains or entire sections If a trace or domain is selected then keyboard movements apply only to the selection Selected elements are always moveable locking the section does not prevent movement of selected elements If no elements are selected and the section is unlocked then keyboard movements are applied to the entire section including all traces and domains TABLE 10 1 Movement keystrokes in the Main Windows Keystroke Action F1 Flip horizontally F2 Rotate 90 degrees clockwise F3 Rotate counterclockwise increment F4 Rotate clockwise increment F5 Slant deform horizontally in counterclockwise direction F6 Slant deform vertically in counterclockwise direction F7 Slant deform vertically in clockwise direction F8 Slant deform horizontally in clockwise direction F9 Scale to shrink horizontally F10 Scale to shrink vertically F11 Scale to enlarge vertically F12 Scale to enlarge horizontally Up Arrow Move upward Down Arrow Move down Left Arrow Move to left Right Arrow Move to right 132 Reconstruct v1 0 6 1 Keyboard Increments F5 F6 F7 F8 Deformations l TAQ oo Figure 10 1
182. sections closer together Now more precise correspondence points will be easier to deter mine Use Blend to compare the sections Some parts of the blended sections will be blurry if the alignment is inadequate Use the apparent motion produced by flicker ing to decide whether there are scaling differences between sections that require a Linear alignment for correction Now zoom in to one of the blurry regions to add more correspondence points at high magnification Select the Stamp Tool and add a pair of points to this region Drag near the edge of the window with the right mouse button depressed This allows rapid panning to the next area of the section that needs correspondence points without changing tools After panning and adding points to several areas use the End key to return to the low magnification display Select Align section gt Rigid or Linear if required Compare the sections and continue adding points until the alignment is satisfactory 146 Reconstruct v1 0 6 1 CHAPTER 11 Objects An object is the set of all traces in sections with index greater than zero that have the same name Thus an object is defined by a trace name not by the color or other shared attributes of traces Traces with different names belong to different objects The Object List The list of all objects in the series is reported in the Object List window Fig 11 1 This list of objects is regenerated from the section data every tim
183. shuttle play of the thumbnails in the flipbook In shuttle play the thumb nails are displayed first in one direction and then in the other continuously The rate of switching images is determined by the Shuttle parameter in the Thumbnails Options tab Shuttle play can also be started and stopped by the Tab key when the thumbnails window has the keyboard focus Exporting Section Images Sections can be exported to image files in much the same way they are rendered on screen or on thumbnail buttons This provides a way to export aligned montages created with Reconstruct to other programs for further analysis To create new image files from the sections select the Render Sections item from the Series menu The Render Sections dialog Fig 6 2 allows the range of sections to be selected By default all sections in the series will be rendered Reconstruct v1 0 6 1 61 Sections When the dialog is opened the series is scanned for all sections and the image dimensions necessary to contain all the series data is determined The output images will be sized accordingly so that every section is completely visible The images will be given the same orientation and position as they appear when dis played in the main window so aligned sections will be aligned in the output images The sections can be rendered at any resolution by adjusting the pixel size The pixel size is the number of section units per pixel Chapter 9 By decreasing the pi
184. sons to use the Trace Midpoints distribution method rather using Sphere representations directly First Trace Midpoints representations are much smaller an important consideration when dealing with hundreds of spheres Secondly a single object name can be used for all spheres whereas if a Sphere representation were to be generated for each trace each trace must have a unique name and be a unique object Box The Box is centered at the midpoint of the object s extent in the x y and z direc tions The sides of the box are parallel to the axes If Size A B or C parameters are 154 Reconstruct v1 0 6 2 3D Object Representations see leo Figure 11 4 Examples of 3D representations of one object The object is a dendritic spine about 0 5 microns tall reconstructed from serial section electron microscopy All images were saved from the 3D Scene at the same magnification and viewing angle A Traces B Trace Areas C Trace Midpoints with a Size A and B set to 0 25 microns D Box E Boissonnat surface with no normals The front faces were drawn as wireframe instead of filled F Boissonnat surface with face normals G Boissonnat surface with vertex normals H Cylinder with the length Size A set to 0 5 microns l Ellipsoid J Sphere with a faceting density of 4 and face normals K Sphere with a faceting density of 32 and vertex normals Reprinted from Fiala 2005 Reconstruct v1 0 6 2 155 Objects positive
185. t but ton repeatedly each time with a different offset specified To learn how to mon tage domains using the point correspondence method see Chapter 10 68 Reconstruct v1 0 6 1 Listing Domains Section 12 domains x Select Save Modify Domain Source Pixel size domaini C fiala reconstruct mito _unaligned 11 0 00269 mito _unaligned 12 0 00269 Figure 7 4 The Domain List window Section 12 has two domains with names domain1 and domain2 Domain has an absolute source path while the source path for domain2 is relative to the series folder Domain2 is highlighted in the list The limit string appearing in the title bar ensures all domains are listed When all domains have been imported exit the Import Images dialog by selecting the Quit button If not already displaying a section in the main window use Page Up and Home to display the first section Listing Domains The Domain List window is opened by selecting List image domains from the Domain menu Every domain in the section is included provided the domain name matches the limit string in Lists tab of the Series Options The limit string appears at the end of the text in the title bar Fig 7 4 When changes are made to domains in the section e g by importing images or a change is made to the limit string in the Lists tab the list may need to be updated by closing it and opening it again Each domain is listed by name in the
186. t position 0 0 in series units This movement center can be changed by using the Type In dialog as described below The exception to this is the horizontal flip F1 and the quarter revolution F2 These movements are always made with respect to the center of the image or section being flipped or rotated Reconstruct v1 0 6 1 133 Movements and Alignments While there is no vertical flip key command the F2 F2 F1 sequence of two 90 degree rotations followed by a horizontal flip yields a vertical flip Likewise F2 F2 will produce a 180 degree rotation and F2 F2 F2 will produce a counterclock wise 90 degree rotation These movements can also be produced directly using the Movement submenu of the Section menu Movement Type In Dialog The dialog invoked by selecting Type In from the Section Movement submenu can be used to specify a movement of selected traces or domains or of the entire section As with keyboard movements the entire section is only moved when it is unlocked AND nothing is selected in the section The Type In dialog allows input of an incremental rotation translation scale skew and or deformation that will be executed when the OK button is selected This movement is made in the section coordinates relative the specified movement center The center of the movement can be automatically set to the center of the selected elements by using the Set to Center button This movement center point is used for all subsequent
187. t was freely available http www godev tool com GoRC was used to compile the resource file prior to linking with the Borland linker Microsoft Microsoft development tools were not used the only exception being the Microsoft Help Workshop compiler for converting rtf files into Windows Help files Open Watcom Another useful resource was the publicly available suite of devel opment tools from Sybase http www openwatcom org The Open Watcom Image and Resource editors were used to produce the cursors icons and many of the other resource elements Subversion For source code version control Reconstruct development is now using Subversion http www subversion org This software allows multiple developers to coordinate their changes through an internet accessible source code repository residing on the synapses bu edu server Jordan Russell Finally thanks to Jordon Russell for his wonderfully well designed InnoSteup http www jrsoftware org used to create the final installa tion file Reconstruct v1 0 6 0 23 Software Overview 24 Reconstruct v1 0 6 0 CHAPTER 4 User Interface The user of Reconstruct interacts with a set of screen elements using the mouse or pen and keyboard This chapter describes the basics of this graphical user inter face Windows and Dialogs Two kinds of screen elements will be distinguished windows and dialogs Dialogs must be completed prior to returning to t
188. take full advantage of the software be sure to read the remaining chapters as well Note Spending an hour skimming through all the chapters could save you many hours of frustration Chapter 3 gives an overview of how data is organized and stored and includes technical details on the system requirements and file formats This chapter intro duces the basic data elements series sections domains traces and objects Chap ters 5 8 describe how these data elements are created edited saved and deleted Chapter 4 describes the user interface and includes important information on what particular keystrokes and mouse buttons do in the various program windows This information is also available from the Key and Mouse Commands help menu items Chapter 9 explains in detail how to set and calibrate the units of measurement used in Reconstruct This chapter explains the meaning of the image pixel size parame Reconstruct v1 0 6 1 9 Quick Start ter and how its value can be accurately determined Chapter 9 also explains how to get quantitative measurements of the traces created on serial sections Chapter 10 covers movements of section data including all the operations that aid in aligning sections Chapters 4 9 are required reading to understand how to use the full capabilities of the software Chapter 10 is necessary only if sections or traces will be moved or aligned Chapters 11 12 cover 3D object visualization In Chapter 11 the r
189. tation is dependent on the boundary being free of internal loops and the val ues x y not being too large Since 64 bit floating point values are used for all such calculations the second factor should not be an issue for most series Midpoint X This column reports the x position of the midpoint of the extent of the boundary in series units The calculation is max x min x 2 Midpoint Y This column reports the y position of the midpoint of the extent of the boundary calculated by _ max y min y 2 Reconstruct v1 0 6 1 121 Calibration and Measurement Trace List Measurements Two dimensional measurements are reported for traces within a section in the Trace List window These include trace length area and the x and y position of the traces These values are reported as columns in the trace list window The col umns displayed are determined by Trace Data Columns checkboxes in the Data Values tab of the Series Options The columns that are displayed in the traces list can be saved to a csv text file using the Save As item at the top of the traces list window All values are computed at the time the Trace List window is created To make sure that the values are up to date close the Trace List window and reopen it after any changes to the traces are made All values are in series units Length The length of a trace is the sum of the distances between trace points length gt vei a i y
190. ter the images have been imported you can repeat the process to add more images to your series When all images have been added use the Quit button to close the Import Images dialog To view the sections open the List from the Section Menu and double click on one of the listed sections This will move the display to that section in the series Press the Home key After a brief delay the image of the first section will be cen tered in the main window Use the Page Up and Page Down keys to advance sec Reconstruct v1 0 6 1 3 Quick Start Figure 1 2 The Tools window This window should be visible at the top of the Reconstruct aiala IN main window Use the left mouse button to select a tool The Line Tool used for calibration lines is the 8th button The Pencil Tool is the 10th button The Wildfire tool button is depressed and so a description of the tool appears in the title bar tion by section through the series Verify that the images were imported as expected with the correct section numbers The number of each section appears in the title bar of the main window CALIBRATE PIXEL SIZE An important parameter during image import is the pixel size which we ignored in the previous section This parameter specifies the size of image picture elements pixels in terms of the units of measure of the series Chapter 9 If the pixel size in the Import Images dialog was accurate no further calibration is required How e
191. ters in the Grids tab This allows the Grid Tool to be used to stamp shapes with a fixed size unlike the Stamp Tool which draws in pixels When the Element size parameters 2x Movements Proxies Thumbnails AutoTracing General Names Colors Grids 3D Lists Specify shape to use as grid element along with element size separation distance and quantity Element type Rectangle C Cross C Ellipse Oycloid C Samplingframe C Stampshape C Clipboard traces Element size A uo 1 ee Figure 8 7 The Grids tab of the Series Distance xf 1 xf 1 mios Options dialog The Element type ee m z determines the shape to be drawn as shown in Fig 8 8 The shape is sized in the x and y directions according to the Element size boxes Shapes are repeated Number times and at Distance intervals The settings are used only when OK is a selected at the bottom of the tab Reconstruct v1 0 6 1 95 Traces C D Figure 8 8 Some types of predefined grids A A 2x2 grid of rectangles B A 2x2 grid of cross points C A 2x4 grid of cycloids D A 2x2 grid of sampling frames are zero or negative the stamp shape is drawn in pixels just as with the Stamp Tool A group of traces can be used as the grid element by using the Clipboard trace s option When this option is selected in the Grids tab the Grid Tool copies the con tents of the Traces Clipboard to the grid Th
192. tes dialog This information included the image file format the number of bits per pixel and the file size The size is given both as width and height dimensions in pixels and as the number of kilobytes of memory the image requires when decompressed The Attributes dialog also reports the name of the proxy image file if one exists and the dimension reduction factor of the proxy image These values can only be changed in the Proxies tab of the Series Options see below PIXEL SIZE The pixel size specifies the size of the image picture elements pixels in terms of the units of measure of the series Chapter 9 This can be changed for a domain by typing a new value in the Attributes dialog The pixel size can also be changed by using the Modify gt Pixel Size dialog from the Section List or by calibrating traces It is best to calibrate the pixel size at the time images are imported For more information on the pixel size parameter and its calibration see Chapter 9 Note Changing the pixel size of a domain will effect the alignment of the domain image with traces and with other images CONTRAST AND BRIGHTNESS The contrast and brightness of the image can be adjusted by typing in new values in the Domain Attributes dialog The intensity of each pixel is adjusted by first multiplying it by the contrast value A contrast value of 1 indicates no change Then the brightness value is multiplied by 255 and added to the contrast adjusted intens
193. th _ _ 1600 _ 02185 section image s width 7322 height fraction Sereemheight _ _ 1200 _ 0 2479 section image s height 4840 Reconstruct v1 0 6 1 77 Domain Images The fraction value in the Proxies tab must be set to a value higher than both the width and height fractions in order for proxies to be used at this magnification For the example numbers given a fraction of at least 0 28 is required for proxies images to be loaded during full section display Without proxies each 7322x4840 section would require a long time to load and display After creating proxies at fraction 0 28 the image loading time would decrease by a factor of 4 or 5 But if you zoom in to the section the display would slow back down because the whole image must loaded to display the data at the higher magnification In addition to section display in the Main Window proxies are also used when sec tions are displayed on thumbnails or exported as images If desired you could set the fraction parameter to a much smaller value to increase the speed of thumbnail generation For the 7322x4840 sections suppose the full image load without proxy requires 800ms Generating 100 thumbnails at a size of 128x96 might therefore require more than a minute without proxies Creating proxies at 0 28 of full dimensions would allow the 100 thumbnails to be generated in 15s and speed up the full screen display of sections as well By making the proxy fraction even sm
194. the series name is displayed in the title bar along with the current section number On the right end of the title bar are the usual minimize maximize and close buttons Selecting the minimize button by clicking the left mouse button when the pointer is over the button hides the window The hidden window can be reopened by selecting it on the task bar or by using Alt Tab pressing the Tab key while holding the Alt key down Alt Tab can also be used to rapidly switch between Reconstruct and other applications running on the desktop The maximize button resizes the main window to fill the screen The main window cannot be manually resized when it is maximized Unmaximize the window by the selecting the maximize but ton again A number of keystrokes and mouse actions can be used in the main window Table 4 2 These are mostly for navigating between sections in the series and creating or Reconstruct v1 0 6 1 27 User Interface manipulating section data Mouse actions in the main window may depend on the which tool has been selected from the tools windows These actions are discussed in more detail in Chapters 5 8 However moving the mouse wheel has the same effect as Page Up and Page Down in the main window TABLE 4 2 Keyboard and Mouse Actions in the Main Windows Keystroke Action Page Up Display next higher section in series Page Down Display preceding lower section in series Switch front and back sections Hold to fl
195. the centroid of the trace points For the sphere the center is calculated midpoint of the object extent and the size is the diameter of the sphere specified by the Size A parameter setting Reconstruct v1 0 6 1 171 The 3D Scene VRML 1 0 Since some older programs accept VRML 1 0 input but not VRML 2 0 a provision is provided to Export As VRML 1 0 Output is similar in all respects to the VRML 2 0 except the wrl file created adheres to the 1 0 format specification www web3d org This only means that the Background color will not be saved with the model DXF Some even older programs accept AutoCAD s DXF input but not VRML For this reason support is provided to Export As DXF but this representation is less rich than VRML The dxf output does not include the scene view point or color and shading information An Object consisting of line data will be output simply as a series of LINE entities while an Object consisting of triangular faces will be out put as a series of 3DFACE entities BITMAP A simple method of capturing the Reconstruct display as an image is to use the Print Screen key on the keyboard This copies the entire screen to the clipboard where it can imported into image editing software for refinement The Export As Bitmap operation expedites this process by storing just the image in the 3D Scene window The image data is stored in a Windows Bitmap file JPEG The Export As JPEG operation stores the
196. the entire perimeter of the trace by a distance equal to the resolution This is because the simplification process ensures a clock wise orientation without loops by tracing around the outside of the existing trace This expansion can be reduced by checking the box next to Shrink back after sim plifying in the AutoTracing tab When this box is checked the clockwise outer expansion is followed by a inner clockwise traversal around the interior of the trace Shrink back should produce a reasonable result for convex traces but trace that are highly concave with narrow interior regions exhibit loops after shrink back For this reason the shrink back procedure should be used with caution Wildfire Region Growing vat The Wildfire Tool has a cursor that looks like a match Pressing and releasing the left mouse button can be thought of as igniting a fire that spreads in all directions from cursor point When the spreading stops at the boundary of a region a trace is created that surrounds this boundary After the trace is created it is added to the selected traces and drawn on the section If the trace is unacceptable it may be deleted by using the Delete key but be careful that no other traces are also selected f With the default settings the Wildfire tool ignites on bright regions and stops spreading at black regions Ifa black region is completely surrounded by the bright region the wildfire will grow completely around it and the
197. the next lower section number If no higher or lower section is found for Page Up or Page Down respectively a warning beep may be issued depending on the Series Options settings Blending and Flickering Sections Reconstruct maintains two sections in memory the currently displayed section and the section previously displayed It is therefore possible to rapidly switch the main window display between these two sections This can be done with the key Pressing and releasing the key will switch the display back to the previous sec tion If no previous section is available a warning beep will be issued provided the Beep when paging fails option is turned on Holding the key down will flicker between to the two memory sections Flicker ing sections is sometimes useful to detecting misalignments by observing apparent motion of objects in the images Chapter 10 Note that first after paging may require rendering of the previous section Since this operation may be slow the hourglass will appear briefly during the first toggle of sections Subsequent switch ing will be very fast since both sections will be fully rendered in memory The two memory sections sometimes referred to as the current and previous sec tions can also be blended together by selecting Blend from the Section menu Once in blend mode all displays will be the blending of the last two sections Thus Page Up or Page Down can be used to sequentially compare
198. ting Traces The Resize selected trace s option will only modify the selected traces The transformations of the traces are scaled so that the traces will be redrawn with the specified new lengths Each trace is scaled independently rather than to an average rescaling value as in the other options The domain on which the selected traces are centered is listed in the second option If the calibration traces are not centered on a domain then this option will be dis abled The correct pixel size of the domain will be set by selecting the second option This pixel size value is computed by transforming the selected traces into the domain and computing their lengths on the image in pixels The sum of the entered lengths in units is divided by the sum of lengths in pixels to get the cor rect pixel size for the calibration grid This pixel size can be subsequently applied to all domains as discussed in the next section Selecting the second option will only change the pixel size of domains not of traces in the section If the section has lots of traces and domains it may be advan tageous to chose the final option instead This option computes a scaling factor based on the ratio of the sum of the entered lengths divided by the sum of the cur rent lengths The scaling factor is applied to the transformations of all domains and traces in the section effecting the calibration by scaling rather than setting the pixel size It desirable to ha
199. to image files using the Export gt Images dialog from the Series menu This dialog is described in Export ing Section Images in Chapter 6 Exporting Trace Lists The Trace List window provides a listing of traces and trace measurements from the current section see Chapters 4 and 8 These individual Trace Lists can be saved to a file and then imported into Excel by using the Save menu item at the top of the list window The Series Export of all Trace Lists provides a method for collating all the individual section lists in one output file Choose the filename in the Save As dialog and the export operation will automatically generate and save the Trace Lists for all the sections in the series The output is constrained by the options selected in the Lists tab of the Series Options so the export can be limited to a subset of trace names and a subset of measurements The output is a Comma Separated Values file csv identical to the format for an individual Trace List with the addition of a section number field at the beginning of each row Importing Line Drawing Line drawings in DXF format can be imported into a series using the Lines item from the Series Import menu The format of the line importing dialog takes the same form as the images import dialog DXF files are selected into the dialog and then section numbers are assigned to these files using the dialog parameters When section numbers are correctly specified the Impo
200. toTracing tab of the General Names Colors Grids 3D Lists Movements Proxies Thumbnails AutoTracing Series Options dialog Automatic simplification can be disabled by Simplify finds the clockwise boundary of your drawing and unchecking the option but this effect is eliminates loops Wildfire region growing finds the boundary of t g an image region from an interior point only temporary When ever a series Is Automatically simplify new closed traces after drawing reopened Automatic simplification will be Simplify on demand to a resolution of 0 00254 microns turned back on This is because Shrink back after simplifying May recreate loops simplification is important for eliminating loops that reduce the accuracy of Stop wildfires when D tue atey H 0 282 measurements and prevent proper 3 or Saturation differs by eH 50 0 252 surfacing of the traces The Stop or Brightness is less than 4 100 0 252 Wildfires box specifies the Hue r e Soe Saturation and Brightness HSB criteria for halting region growing Valid HSB are between 0 and 252 By setting a criterion to differs by 255 that criterion is disabled and will not affect region TR growing The default settings are shown Stop wildfire autotracing when area changes by 999 Ignore regional wildfires of less than 0 square pixels The resolution of the re pixelized line segments is determined by the resolution parameter for Simplify on demand
201. tory Information about the whole series e g option settings is stored in a separate series data file in this same folder The series data file has a ser file extension e g myseries ser Thus the data files associated with a series reside in the same folder and share the same filename Only the file extension is different ser for the series data file and a positive integer for each section data file The image data itself is not stored in the section data file For each domain in the section there is a reference to the corresponding image file The actual image data can be stored anywhere on the computer but generally it is best to store the image files in the same folder as the section data files This allows the entire series to be easily off loaded to CD ROM or other archival media and restored at a later date Reconstruct v1 0 6 0 13 Software Overview lt ENTITY SFBool true false gt lt ENTITY SFColor CDATA gt lt ENTITY SFFloat CDATA gt lt ENTITY MFFloat CDATA gt lt ENTITY SFInt32 CDATA gt lt ENTITY SFString CDATA gt lt ENTITY MFVec2f CDATA gt lt ELEMENT Contour EMPTY gt lt ATTLIST Contour name SSFString unknown hidden SSFBool false closed SSFBool true simplified S SFBool false border SSEColor my Oo 2b fill SSEFColor E OLIT mode SSFInt32 wom comment SSFStri
202. tton to go back and verify that the data was saved correctly VIEW _THREE DIMENSIONAL OBJECT Select List from the Object menu to see the list of objects defined by traces in the series Use the left mouse button to double click the object name in the list or use a single mouse click and select Add from the Scene menu at the top of the list win dow A three dimensional view of the object traces will appear in a 3D Scene window after a brief delay You can rotate the object representation by placing the mouse in the 3D Scene window and pressing and dragging with the left mouse button You 8 Reconstruct v1 0 6 1 About the Rest of the Book can zoom in or stop the object s spin by pressing the right mouse button Pressing both mouse buttons together allows you to pan the scene To surface the object open the Series Options dialog and select the 3D tab In the 3D options change the Generate option from Traces to Boissonnat surface and click OK Go back to the Object List window and again double click on the object name The object will be surfaced and replace the trace representation in the scene Note that traces must conform to certain criteria for Boissonnat surfacing to work correctly For example traces of the same object within a section cannot overlap For a detailed discussion of tracing rules related to Boissonnat surfacing see Chap ter 11 About the Rest of the Book This concludes the Quick Start tutorial To
203. ttributes Renaming Objects Copying Objects Hiding and Unhiding Objects Simplifying Objects Deleting Objects Calculating the Distances Between Objects 3D Object Representations Traces Trace Areas Trace Midpoints Box Boissonnat Surface Cylinder Ellipsoid Sphere 3D Parameters Shift Object By Surface Normals Faceting Density Size Boissonnat Surfaces Z Trace List e CHAPTER 12 The 3D Scene O O OpenGL Rendering Composing a Scene Adding Objects Removing Objects Changing Object Appearance Diffuse Color Emissive Color Ambient Intensity Transparency Specular Color Shininess Filled or Wireframe Faces o Viewing a Scene Rotating Panning and Zooming Spin Background Color o Saving a Scene VRML 2 0 VRML 1 0 DXF Bitmap JPEG o Scene Information e CHAPTER 13 TBD e Bibliography e Appendices o Keyboard Overlay CHAPTER 1 Quick Star t This chapter gives the steps for reconstructing serial sections You will learn the basics of using Reconstruct to import a series of images view and align the sec tions trace profiles and generate a 3D view of the traces Getting Started The only requirements for getting started are that the Reconstruct application is installed and that a set of image files is available for input To install the software download the latest version from http synapses bu edu or from the Users Group http grou
204. uld be clockwise to delineate the exterior of the object Use Reverse to fix the orientations case open traces will be partially filled in the clockwise part of the traces In gen eral however the results with open traces will be difficult to control Straight line traces will be ignored altogether since these are not capable of forming a solid con nection with the rest of the object Use clockwise traces to define outer boundaries Clockwise traces represent the exterior of the object Clockwise traces must be wholly outside of all other clock wise traces of the same object in the section The exception to this rule is when the clockwise trace is completely contained within a counterclockwise trace A clock wise trace can occur inside another clockwise trace only if it is fully enclosed by a counterclockwise trace of the same object Use counterclockwise traces to define holes Counterclockwise traces represent an interior hole boundary Traces representing holes must be wholly contained within clockwise traces Fig 11 3 C When traces are automatically simplified during tracing they are also made clockwise To change a simplified trace to coun terclockwise use the Reverse command from the Trace menu Trace without loops or self intersections As depicted in Fig 11 3 A traces with self intersections do not produce a valid surface because of the ambiguity between Reconstruct v1 0 6 2 157 Objects interior and exterior re
205. use button zooms the section Drag upward to decrease the magnification and downward to increase the magnification A particular point in the section can be zoomed by putting the cursor over the point before pressing the right mouse button ay The Pan and Zoom tool can be used to dynamically adjust the section e Zooming in will ultimately lead to a pixelized image where each pixel of the origi nal data appears as a square on the screen This is normal But zooming in while proxy images are used for low magnification Chapter 7 may result in premature pixelization To make sure the display is at the maximum resolution of the data page through the sections to force a load of the full image instead of the proxy A section reload can also be accomplished by selecting the section from the section list Note that panning and zooming is entirely different from moving a section Chap ter 10 Panning and zooming temporarily changes the view point for the whole series All sections are affected in the same way The relative position of a section is not changed by panning or zooming Moving or aligning a section permanently changes the relationship of the section to the rest of series 54 Reconstruct v1 0 6 1 Paging Sections Paging Sections Sections in the series can be accessed sequentially by using the Page Up or Page Down keys Page Up changes the displayed section to the next higher section num ber while Page Down changes to
206. using the right mouse button of the Scalpel Tool D The final product after deleting the unwanted piece with the Delete key when the trace has a clockwise positive orientation On a reversed trace the Scalpel Tool will add a piece to the trace rather than divide it The left mouse but ton press must occur near the trace to be divided and not closer to another trace The trace closet to the starting point is the one that will be divided The division path should be perpendicular to the trace at the points where it crosses it Under some circumstances the Scalpel Tool will fail to find a suitable division and will return fewer pieces than expected This is usually due to the fact that the division line is parallel to the trace at the point where it crosses The Scalpel Tool is often useful for quickly removing an external evagination of a trace but it does not allow an internal invagination to be sliced off To remove an internal invagination select the trace and use the Line Tool to make a new trace 100 Reconstruct v1 0 6 1 Editing Traces with the same trace name that would produce the desired cut Then use Merge from the trace menu to merge the line trace and original trace effectively remov ing the invagination MERGE The Merge operation attempts to combine all selected traces into one closed trace Traces to be merged should be closed and should overlap If some traces do not overlap the effect of merge will depend on th
207. utton Warning Unsaved changes to the current and previous sections or to the series options will be lost by aborting Reconstruct from the Task Manager 38 Reconstruct v1 0 6 1 CHAPTER 5 Series A series consists of set of files in a single writable folder although the image data can reside in read only files in another folder or drive This chapter describes how to create and manipulate a series Manipulation of sections within the series are described in the next chapter Creating a Series A new series can be created by selecting New from the Series menu The New Series dialog will appear Navigate to an existing folder or create a new one for the series Type a name for the series in the Filename box Each series in a folder must be given a unique name following the usual rules for filenames given by the oper ating system The maximum length for the series name is 255 characters The extension ser will be appended to the filename if it is not typed in the box After selecting the Save button the ser file will be created with default settings WARNING Any existing series file with the same name will be overwritten Copyright 2006 John C Fiala 39 Series The next step would be to add sections to the series This can be done by using the Import Images menu item Chapter 7 or by creating new sections from the Section menu Chapter 6 Opening and Closing Series An existing series can be opened
208. utton images This value is independent of the amount of physical memory installed in the computer If Windows runs out of memory for button images the first part of the thumbnail series will have sections images but the remaining buttons will all be gray This is more likely to occur when you ask for large thumbnail images Note that this problem may be remedied in future versions of the Windows operating system Thumbnail buttons are created initially as gray buttons without images The images are added sequentially by a background thread that runs at a lower priority to the main window Thus you can continue working while the thumbnail images are being rendered When the thumbnails window is closed all of the thumbnail images are deleted from memory and they will need to be recreated the next time the thumbnails window is reopened Changes to the thumbnail options will only be realized when the thumbnails window is opened and the thumbnails are recreated Two ways of rendering the thumbnails are selectable Thumbnails can be made to resemble the current view or the section can be centered on the button In the former case the thumbnail images will maintain the existing section to section alignment and can be zoomed into particular parts of the section While in the later case the entire section will be visible in each thumbnail Making the thumbnails resemble the current view is most useful for flipbooks while the other mode can be used for a qu
209. vailable for a variety of operating systems including GNU LINUX FreeBSD and Solaris Users have verified WINE for running Reconstruct under Mandriva Linux w KDE 3 3 2 In addition the Reconstruct binary may run on Virtual PC Apple s Pentium Windows emula tor but this has not been tested OPENGL OpenGL libaries are required for previewing and saving 3D objects The OpenGL libraries provide a software interface to the computer s graphics hard ware OpenGL is shipped standard with every version of Windows 95 98 2000 NT XP and should work with Reconstruct without any additional configuration For more information on OpenGL see Chapter 12 18 Reconstruct v1 0 6 0 System Requirements HARDWARE REQUIREMENTS Performance of Reconstruct is determined by three main hardware components the graphics display adaptor the amount of RAM and the speed of the hard drive A high quality graphics display adaptor that accelerates OpenGL is highly desir able for 3D Scene operations Most modern game adaptors are satisfactory for this purpose The software was tested on a Radeon VE adaptor with 32 Mb of memory This device gave excellent OpenGL performance Reconstruct is not a very demanding application from a memory standpoint because the data from only two sections is held in memory at one time Thus a very long series of hundreds of sections will not require any more memory than a short one A minimum requirement for good perf
210. ve the pixel size parameter be accurate for later reference since the scaling factor will not be visible to the user So the second option should be used in the Calibrate Traces dialog for the initial calibration With this second option existing traces will no longer be correct and existing alignments between domains will be disrupted This is not a problem if calibration is carried out before any domain movements section alignments or tracing However the calibration traces will be incorrectly sized after a pixel size adjustment These calibration traces may be simply deleted To verify the calibration redraw the calibration traces and check their length to see that they match the know lengths in the calibra tion image Reconstruct v1 0 6 1 115 Calibration and Measurement APPLY THE CALIBRATION TO OTHER SECTIONS The pixel size set by the Set pixel size option can be subsequently extended to other domains in the series When this option is chosen the calibration automati cally opens the Set Pixel Size on All Domains dialog box Fig 9 2 This dialog displays the same pixel size value as in the Calibrate Traces dialog and allows a range of sections to be selected All domains in the included sections will be set to the pixel size value Note This operation cannot be undone when executed over many sections The rescaling performed by the third option of the Calibrate Traces dialog can be applied to other section
211. ver in most cases the pixel size should be adjusted using the image of a calibra tion grid or scale that was photographed with the series This is best done before any alignments or tracing since changing the pixel size later can invalidate these adjustments The process of calibration in Reconstruct involves drawing traces of known length and then entering that length value into the program Select the Line Tool Fig 1 2 from the tools window and draw a line trace of known length on a section contain ing a calibration grid image For more details on calibration grids and lines see Chapter 9 After the calibration trace is drawn select the Calibrate item from the Traces menu A dialog box will appear for each calibration trace displaying the current length of the trace Enter the actual length of the trace in place of the current length 4 Reconstruct v1 0 6 1 Getting Started Calibrate Traces Resize selected trace s Set Pixel Size on All Domains i Section Range First Last 16 Set pixel size to 0 002691 microns pixel A This operation cannot be Undone Cancel Set pixel size to 0 002691 microns pixel for domain domain1 and optionally modify additional domains Scale entire section by 1 000000 Cancel Figure 1 3 The Calibrate dialogs as filled out for a global calibration of the pixel size of images Left The first dialog requests that a pixel size calibration be done for the image on wh
212. veral things to try to improve the result First and foremost make sure that the traces used to generate the surface follow the rules outlined below Commonly counterclockwise traces or traces with internal loops cause the algorithm to generate invalid triangles out side of the objects intended surface Since a 2D Delaunay triangulation of the traces is the first step in the surfacing algorithm the Traces Areas output can be used to see whether the problem lies with the traces or with the surfacing algorithm itself If Trace Areas does not produce a filled representation similar to Traces locate and correct the traces that are invalid Fig 11 3 It may help to limit the section range in the 3D object to narrow down which traces are problematic Draw closed traces Boissonnat surfacing is designed to surface closed traces however the algorithm will attempt to incorporate open traces as well In the best 156 Reconstruct v1 0 6 2 3D Object Representations PE A B C Figure 11 3 Examples of invalid object traces for Boissonnat surfacing A A trace with an internal loop is invalid Avoid this scenario by using the Automatically Simplify option when tracing B One trace intersecting another trace for the same object is invalid Merge traces to eliminate this problem C A counterclockwise trace without a surrounding clockwise trace is invalid The inner trace should be counterclockwise to mark the hole boundary and the outer trace sho
213. w by pressing and releasing the left mouse button A candidate line segment is shown as the mouse is moved When the line segment is as desired click the left mouse button again to add the segment to the trace Segments are sequentially added in this man ner until the right mouse button is pressed to complete the trace During the entering of segments preceding segments can be removed by using the Backspace key The starting point cannot be removed using Backspace To aban don the trace and start over use the Esc key It is possible to change sections while drawing point by point The trace will be created in the section in which the trace began even if points were added or the trace completed while other sections were displayed This is true of both open and closed point by point drawing and the Line Tool To create a trace that actually spans multiple sections use the Z Trace Tool below Traces created by open point by point drawing are not automatically simplified on completion The trace may consequently contain loops that will adversely affect the interpretation of the trace These loops can be eliminated by closing the trace and using the Simplify menu item see below Drawing an open point by point trace closing it by changing its attributes and then using Simplify is essentially Reconstruct v1 0 6 1 97 Traces the same as using the closed point by point tracing tool in the first place The main difference is that Si
214. w series is opened Scenes are not preserved when the application is terminated Closing the program requires the scene to be recreated by adding the objects again CHANGING OBJECT APPEARANCE After an object has been added to the scene certain aspects of the object s appear ance can be modified by selecting the In Scene item from the Modify gt Attributes menu in the Object List window Fig 12 1 Only the highlighted objects in the Object List will be modified The number of highlighted objects is indicating in the 166 Reconstruct v1 0 6 1 Composing a Scene title bar of the Changing Scene Objects dialog Every object will receive the same changes specified in the dialog when the Apply button is selected Diffuse Color The parameters in the Change Scene Objects dialog determine object color and reflectance properties Diffuse Color is the main reflective color value Surfaces perpendicular to the view point strongly reflect with this color while surfaces pointing away from the view point reflect very little of the Diffuse Color and instead take on the Emissive and Ambient colors Emissive Color Emissive Color is emitted from the object in the absence of reflected light The object simply glows this color Emissive color lacking vari ations in reflectance does not give a sense of the 3D shape of the object Emissive color is used by default for Traces Trace Areas and Trace Midpoint representa tions that do not hav
215. xel size more pixels will be used to render the data A larger pixel size will result in fewer pixels being used to represent the same image data An arbitrary positive number can be typed in the Pixel Size box to specify the number of pixels used in the output For convenience two different pixel sizes are automatically provided for full and current view resolutions Full resolution uses the smallest pixel size detected among all domains in all sections Essentially every pixel in the original data will have at least one full pixel in the rendered output provided there are no scaling transformations applied to the data For any pixel size the resulting width and height in pixels of the output files is given in the dialog Fig 6 2 i Save each section as a single image file Figure 6 4 The Render Sections dialog The Pixel Size parameter determines the output file size Output will be a 24 bit Windows Bitmap or JPEG image file The output image can include a representation of the traces if desired Each section in the specified section range will be rendered to a file with the Series Name and the section number as the file extension C Make images resemble current view Pixel Size 0 002690 Save whole sections at full resolution Image size 1188 x 874 pixels 2 97 Mb uncompressed r File Format Bitmap C JPEG Quality 1 100 80 r Traces None Asdisplayed Fillwith border colors First Section 2
216. y using the Esc key but this will completely delete the trace being edited Use Undo Ctrl z to recover the original trace 7 Existing traces can also be quickly sliced into separate pieces using the Scalpel Tool Fig 8 9 The tool is used to divide a closed trace into two or more pieces based on a separation line drawn with the cursor To divide a closed trace place the cursor outside of the trace near the point where the trace is to be divided Press and hold the left mouse button and drag the cursor through the interior of the trace A temporary line will show the intended path of the division When the cursor reaches the outside of the trace again release the left mouse but ton and the trace will be divided along the path If the division path is wrong press the Esc key before releasing the mouse button to abort the operation After a suc cessful division the middle mouse button can be used to select one of the pieces for further processing such as deletion or attribute changes Note that the division of a trace may not always succeed Carefully observe the result to make sure it is as intended The Scalpel Tool only works as advertised Reconstruct v1 0 6 1 99 Traces Figure 8 9 The process of the dividing a trace using the Scalpel Tool A A left mouse drag is executed along the line shown B When the left mouse button is released the trace is divided into three pieces along the line C The middle piece is selected by

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