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DSLT_Demo User`s Manual (DSLT_Demo Ver. 1.11)
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1. from the Tools shelf The Watershed settings dialog will appear in the Tools panel This function ignores unselected and undisplayed segments 1 Select all segments Alt A a 2 Set the minimum size of segments on the segment displaying setting dialog 3 Deselect plastid if you want to obtain 3D data for cells This process is not necessary if you want only 3D data for airspaces 4 Switch over to channel 1 5 Click on the Apply button 4 9 Morphological Smoothing Optional Perform morphological smoothing to remove small protuberances Select Segment Dilation and Segment Erosion from the Tools shelf The respective settings dialogs will appear in the Tools panel 1 Select all segments Alt A 2 Erode segments radius 3 clamp checked 3 Dilate segments radius 3 4 Erode segments radius 3 clamp checked 5 Dilate segments radius 3 6 Erode segments radius 1 clamp checked 10 Saving Results File Menu bar gt Save File Menu bar gt Save_Segments Parameter Settings Source ValidTH 1600 y gt r max C mainly affects a segmentation result in the brightest region In this case this parameter mainly affects segmentation results of epidermal cells If max C is too low some over segmentation might occur If max C is too high small cells e g guard cells might be under segmented because the segment validation sy
2. button Perform smoothing and brightness adjustment for improved depth to all channels Select channel gt Brightness adjustment for depth gt Smoothing gt Apply Changes Blightness and Contrast Smoothing Maximum Minimum Filter Type Mean Gaussian Ch 0 Z Gradient Radius Coefident Exp Blightness and Contrast Smoothing Maximum i Minimum Filter Type Ch 2 Z Gradient Radius Coeffident Exp 5 Segmentation Select Segmentation DSLT from the Tools shelf The Segmentation settings dialog will appear in the Tools panel Segmentation DSLT Filter Type Type of kernel for DSLT Filter Type Mean Gaussian Radius Radius Radius of the kernel for DSLT Level Level Z Comect Quality of DSLT Z Correct max 5 Correction factor for DSLT a min E min C Interval Lower limit of Cconst Valid TH max C Closing Upper limit of Ceonst Interval AC onst that each loop iteration will have ValidTH for segment validation The threshold value of the inner structure volume is the sum of multiplying ValidTH and the estimated proper radius of the sphere structuring element in the closing operation of segment validation Closing Radius of the closing operation which is performed after the binarization Check on the Cropping option and set parameters for cropping if you want to specify a region that becomes the object of segmentation You can set the cropping parameters in the Heigh
3. selected segments Radius Radius of dilation Middle Click Select a segment Alt J Join selected segments Alt S Separate selected segments Ctrl Z Undo Alt A Select all segments Alt D Deselect all segments lt Over segmentation gt 1 Select over segmented segments 2 Join selected segments Alt J lt Under segmentation gt 1 Select an under segmented segment 2 Erode the selected segment radius 3 clamp checked 3 Perform segment separation Alt 8 4 Dilate the separated segments radius 3 5 Join fragments The radi of the erosion and the dilation should be the same 7 Extracting Air Spaces Select Segment Selection th from the Tools shelf The Segment Selection th settings dialog will appear in the Tools panel Threshold Threshold value for auto selection of segments If deselect 1s and the mean intensity of a segment 1s higher than this value the segment will be If deselect 1s and the mean intensity of a segment 1s higher than this value the segment will be 1 Switch over to channel 0 2 Set Threshold 3 Click on the Apply button perform auto roughly select airspaces 4 Switch over to channel 2 5 Check deselect 6 Set Threshold 7 Click on the Apply button perform auto deselect plastids 8 Check selection errors and then join the selected segments Alt JJ 8 Watershed Select Watershed
4. DSLT_Demo User s Manual DSLT_Demo Ver 1 11 Before Installing DSLT_Demo Download and install the CUDA Toolkit 6 5 https developer nvidia com cuda downloads Installing DSLT_Demo After downloading the installation package DSLT_setup_x64_111 exe or DSLT_setup_x86_111 exe double click on the file icon and installation will begin DSLT Demo GUI MainWindow File Edit 1 Position Settings sliders 2 Channel Selector 3 Brightness and Contrast Settings sliders 4 Height Map Settings sliders 5 Segment Display slider 6 Apply Changes button 7 Tools shelf 8 Tools panel 9 Orthogonal View Source 10 Orthogonal View Destination 11 View Panel splitter Shift Left Click Zoom in Orthogonal View Shift Right Click Zoom out Orthogonal View View Panel Splitter You can adjust the size ratio of orthogonal views by dragging the view panel splitter horizontally You can reset the size ratio of orthogonal views to the default by right clicking on the splitter and selecting Reset from the menu Segmentation Procedure 1 Opening a 3D image file In the File menu select Open or press Ctrl O to open the image file i This software supports 8 16 and 32 bit TIFF image stacks and ImageJ File Edit TIFF hyperstacks We recommend using Fiji for conversion of a file in a Open cti o different format into one of these formats Load_Segments We use ST_ami Ist adaxial 2 1 tif for demon
5. range of maximum intensity projections along the z axis The projection results are displayed on the orthogonal views XY If the option Use hmap for Z projection is checked a value for each point on the height map will be used as a z coordinate for the starting point of the maximum intensity projection Generate Button Execute height map generation Clear Button Clear a generated height map If the option Show height map is checked the height map is shown in the orthogonal views YZ ZX as purple curves 1 Set parameters for generation of a height map 2 Click Generate button Blighiness and Contrast 3 Brightness and Contrast adjustment If the option Z Gradient is checked brightness adjustment for improved visualization depth will be activated ao ea aed New intensity values Inew are calculated with the following formula a Inew X Y Z Coefficient ZEP x I x y z Exp Maximum Minimum 1 Check Z Gradient 2 Set parameters for brightness adjustment 4 Smoothing Select Smoothing from the tool shelf and the Smoothing box will a appear in the Tools panel Filter Type Filter Type Radius Type of kernel for the smoothing filter Radius Radius of the kernel for the smoothing filter 1 Set parameters for smoothing 2 Click Preview button Apply Changes To apply the brightness adjustment and the smoothing settings click on the Apply Changes
6. stem cannot detect under segmented cells that have very small inner structure see below min C mainly affects a segmentation result in the darkest region In this case this parameter mainly affects segmentation results of mesophyll cells If min C is too low some over segmentation might occur but the over segmentation should be suppressed if ValidTH is set to a realistic value If min C is too high under segmentation should occur ValidTH mainly affects a segmentation result in regions except for the brightest one In this case this parameter mainly affects segmentation results of mesophyll cells Even if ValidTH is too low it should not affect results significantly when min C is set to a realistic value If ValidTH is too high under segmentation should occur because the segment validation system cannot detect under segmented regions as incorrect segments Correct Incorrect ja plesne H Z AND AND m q Validation of extracted segments Schematics of the validation method showing the major steps by which segments are classified as correct or incorrect based on their extracted inner structures green
7. stration of the save Ctrl 5 segmentation procedure Save_5egments This file includes three channels Exit Channel 0 fluorescence of nile red in silicone oil Blighiness and Contrast Channel 1 fluorescence of nile red in phospholipid bilayers of cells Channel 2 autofluorescence We mainly used Channel 1 for segmentation Note that Channel 1 is used unless otherwise stated in the following procedures 2 Generating a height map to adjust brightness for depth The height map is a 2D image to store surface elevation data of a 3D image The software allows adjustment of brightness for improved visualization with increasing depth from the surface This function enables correction for the reduction in signal intensity with penetration depth of the excitation laser The Height Map panel enables adjustment of the following Height Map parameters a a Filter Type Show Height Map Type of kernel for the smoothing filter that is applied to the Filter Type Mean Gaussian height map oe Radius Smooth Lw Radius of a kernel for the smoothing filter that 1s applied to the height map Threshold Smooth Lv 4 Number of iterations of smoothing for the height map Clear Zi Radius of a kernel for the Z smoothing filter that is applied before surface detection Threshold Threshold value for surface detection The following parameters can be adjusted after height map generation Offset Depth offset of the height map Range Depth
8. t Map settings dialog We skipped this step in this demonstration Upper Upper bound of the cropping region If Use height map is checked the z coordinate of each point on the upper bound is the sum of Upper and the value of the generated height map at the corresponding x y coordinate point Otherwise the z coordinate for each point on the upper bound is set to this value Lower Lower bound of the cropping region If Use height map is checked the z coordinate of each point on the lower bound is the sum of Lower and the value of the generated height map at the corresponding x y coordinate point Otherwise the z coordinate of each point on the lower bound is set to this value Edge X Y edge thickness surrounding the cropping region We recommend that you check the results of the DSLT before segmentation DSLT DSLT Filter Type Mean Gaussian Filter Type Gaussian Radius 14 Lewel zZ Correct Radius Level zZ Correct r pu ar 1 Set parameters for segmentation 2 Click on the Apply button in the Segmentation settings dialog 6 Segment Editing Correct segmentation errors Select Segment Erosion and Segment Dilation from the Tools shelf The respective settings dialogs will appear in the Tools panel Segment Erosion Erode selected segments If Clamp is checked a voxel at an edge of an image will not be eroded Radius Radius of erosion Segment Dilation Dilate
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