SynPAnal User Manual Eric Danielson
Contents
1. File Loginfo Profile Save Profile Syn Dendrites fh wy Eric nN Nanyan SMS Open Change Log Directory Close Data Logging utput Colors gt Red Channel Data Output Type Green Channel Data Output Colors gt Data Output Type Log Puncta Info Per Image i Log Spine Info Per Image 1 Log Generic Info Per Image J Blue Channel J Log Puncta Info Per Dendrite J Log Spine Info Per Dendrite J Log Individual Puncta Info J Log Individual Spine Info lJ Log Individual Generic Info Data Output Colors 1 Before data can be written to a file the user must select the channel from which the data measurements will be collected 0 3 channels can be selected Known bug for spine dimension analysis a color must also be selected File Loginfo Profile Open Data Output Type 2 Before Data will be written to a file the user must select what data will be written Any number can be selected at a time Close Change Log Directory Change Log Directory 3 The default directory where the data will be written is the directory containing oynPAnal jar The user can choose a different directory if desired Auto Data Logging 4 Left clicking the Button 4 will manually write data to a file Generally this is not needed as data is written to a file as the user progresses to the next image This feature was included if only one image was being analyzed2. amp puncta measurements Creates object for measuring spine dimensions Left click writes data To text file Right click toggles auto data writing mode Creates Variable Width dendrite object Removes for measuring puncta Generic Counts puncta Whole Image and spines Object porate Analysis V J PER ININ gt GD ri Za EE im DELS i ear is a AOAO WRK eRe Esc F1 F2 F3 F4 F5 F6 F8 F9 F10 F11 F12 Creates Generic object Removes Manual save for simple intensity spines button measurements Creates Fixed Width Dendrite object for puncta and spine measurements Creates object for counting spines Hides filename for blinded analysis These buttons control 1 The addition removal of regions used for performing measurements 2 Saving and writing data to a file 3 Navigation to and from images Opens previous image Opens a specific image from the original image list a i pSuper GFP_mPSD 95_1 tif Left Right arrow arrow Opens next Image The layout of the buttons is supposed to mimic the F Key layout on a standard keyboard Esc F1 12 and left and right arrows also trigger the button function Thresholding Panel Red threshold controls Green threshold controls Blue threshold controls Red low 256 i Green low 255 1 Blue bw 256 GEA White w high 256 VY White w hgh 255 y White w high 255 N Width 2
3. in the Threshold Panel All fixed width dendrites will have the same width Addition of a dendrite will also trigger the automatic counting of puncta Variable Width Dendrite Clicking on button 2 or pressing F2 will activate Add Variable Width Dendrite mode As with the fixed width mode left clicking and right clicking will add and remove vertices and double clicking will add the final segment After doubling clicking the user will hold down the left mouse button to trace the desired region around the dendrite Double clicking will finish tracing the region Dendrite View Pressing the V key cycles through a solid and open dendrite view mode Dendrite Data Panel Measurements from dendrites il t Right Click Options can be excluded from the Seah eh Holding down the right mouse analysis by ignoring specific Restore all puncta button over a dendrite brings up Spine Dens f dendrites This is done by right various options Ignore aa clicking the desired dendrite invert all puncta all Restore all puncta will gz mr 39 49 row in the Dendrite Data Panel Delete Dendrite exclude include all puncta within 65 49 Ignored dendrites will be the dendrite from analysis aaa colored a dark grey in the data Invert all puncta will swap which panel Left clicking the dendrite puncta are included excluded 6 2253 71 02 row in the Dendrite Data Panel The delete dendrite object will a J wil
4. 0 P x oe EA Y Calibration 0 12726 LUT LUT LUT Channel to be thresholded Threshold value adjustable Red iow 256 White w high 256 alpha LUT L Low threshold slider High threshold slider Threshold overlay transparency slider i Image look up table slider Threshold overlay color Width 20 Fixed Width dendrite width adjustable Caldration 0 12726 Calibration factor microns per pixel adjustable and can be loaded from profile information Data Panel Ctrl Transfected Neuron Data Red Green Ave Puncta Number 235 18 Ave Puncta Intensity 3036 Ave Puncta Area 0 23 Ave Intensity Length 7141 Length Puncta Den o 3569 23518 Puncta Name Puncta Intens 00 2401 O41 02 5817 0 3 BB 73 04 mN Group Tab Panels The tab panels will allow you to switch between groups of objects that belong to each tab lt will also display information about measurements taken by those objects In the left example two tabs have been set up Ctrl and Transfected 1 The user can click on the one of the tabs to highlight it Currently the Transfected tab 1 is highlighted When this tab is highlighted the user can create and modify objects in this tab group e g Dendrite objects added to the image when the Transfected tab is highlighted will be hidden when the Ctrl tab is highlighted Additionally data from each t
5. Additionally the user can deactivate Auto Data Logging by right clicking button 4 The Auto text will disappear from the button This feature was included so users could go back to previous images without duplicating the data written to a file Analysis Screen File Loginfo Profile em mb pSuper GFP_mPSD 95_1 01 v Ctrl Transfected Red Green Bue c Ave Puncta Number Ave Poncta Intennty Ave Puncta Area Puncta Name Puncta intensity f Red low 256 White iw high 256 c y Width 20 aipha LUT i AEN Calibration 0 12726 Image Panel Draws image threshold overlay and displays region objects where data measurements will occur Zoom Panel Controls image magnification and display of red green and blue color channels Button Panel Contains buttons for creation of measurement tools writing data to a file and for controling navigation to and from images 4 Data Panel Displays current data Thresholding Panel Displays tools for adjusting image thresholding threshold overlay display and image channel look up tables Zoom Panel Toggles Zoom mode Zoom in and out controlled by mouse wheel or and keys Text box displaying zoom factor adjustable Toggles whether red channel is displayed or hidden Toggles whether green channel is displayed or hidden Toggles whether blue channel is displayed or hidden F Key Button Panel Recalculates fluorescent intensity
6. ab group will be calculated displayed and output separately This will allow for separate populations i e Transfected and Non transfected neurons to be analyzed in the same image Color Tabs The user can click on Red Green or Blue sub tabs 2 to display color specific information Data Types The user can click on Neuron Data 3 to cycle between Puncta Info Spine Info and Cell region Info Data Panels The Neuron Panel 5 contains information averaged per image The Dendrite Panel 6 contains information averaged per dendrite The Individual Panel 7 contains the individual puncta spine or generic region object information Adding and Removing Tab Panels New groups can be created by right clicking the group tab and choosing add tab 8 Deleting a tab removes the tab and all regions associated with it This cannot be undone Add Tab A non Delete Tab o Neuronpata _ Dendrite Objects A JA Fixed Width Dendrites Clicking on button 1 or pressing F1 will activate Add Fixed Width Dendrite mode N a Once this mode is activated left clicking anywhere on the opened image will add a vertex to the dendrite object Right clicking will remove vertices The user should use this mode to add vertices to the center of the dendrite to be traced Double left clicking will add the final vertex and will create a fixed width dendrite Width is altered by changing width value
7. ing spine objects The user can continue adding spines until finished To end Add Spine mode the user should left click anywhere outside of a dendrite object This mode adds spines quickly and is exclusively for calculating spine density Measuring Spine Objects Clicking on Button 2 or Pressing F6 will activate Measure Spine mode In this mode 3 segments will be added in this order Spine length Spine Head Width and Spine Neck Width These measurements will be used to categorize the spines into Mushroom Thin Stubby and Filopodia Mushroom spine will be drawn blue thin red stubby pink and filopodia yellow Spine density will also be calculated Punca per spine mode Clicking on Button 4 or pressing F8 will allow the user to automatically remove puncta that do not fall within a specific radius of the spine Pgup and pgdn increase or decrease the radius The number of spines with O 1 2 or gt 3 puncta will be calculated Deleting Spines Clicking Button 3 or F7 activates Remove Spine Clicking on spines during this more removes spines Removing a spine deactivates this mode Spine Data Pane spine Data is averaged per Image per dendrite or individually Clicking on Button 1 or pressing F5 will allow the user 1 o Red Green Blue Ave Spine Nu Ave Spine Nut ooies data mode Ave Spine Head Width 0 6 0 68 14 19 7 95 15 38 Ave Spine Length Mushroom Thi
8. ing the puncta to clean up any errors 5 The user then proceeds to the next image All region information is saved automatically all data is written automatically and the current setting are applied to the next image unless already analyzed 6 The user continues in this fashion until all images are analyzed Following analysis data is copied and pasted from the data text files and into excel for further analysis Introductory Screen Introductory Screen Starting the software will bring up the introductory screen containing ret a menubar with 3 menubar items File Loglnfo and Profile Syn PAnal Ay G arien by Eric i SA aa File Menu y soe 5 Open Allows the user to select what images will be analyzed Change Log Directory Allows the user to select the directory where the data will be written The default directory is the directory that contains SynPAnal Close Exits the software BA SynPAnal Loginfo Data Output Colors Allows the user to select data from what color channel will be output to a file Data Output Type Allows the user to select what type of data will be written to a file The LogInfo section contains more information about these options Profile Calibration information can be stored into profiles and loaded by selecting the desired profile Future versions of the software may contain more complex information in profile files l File Profile
9. l bring that dendrite into view permanently delete the dendrite in the main image and all puncta Puncta Objects Counting Puncta Clicking on Button 1 or pressing Esc key will automatically add puncta objects within drawn dendrite objects A yellow border will be drawn around all puncta objects and the puncta lable drawn adjacent to the puncta 2 Puncta are labeled as X Y X indicates which dendrite the puncta belongs to and Y indiciates the order in which the puncta was found Puncta cannot be deleted but can be excluded from the measurement by right clicking the puncta object or the puncta row in the puncta data panel To count puncta dendrite objects must be present and the image must be thresholded for the desired color channel Puncta Data Panel Puncta data is displayed averaged per image 3 per dendrite 4 or for each individual puncta 5 Ignored puncta rows are colored dark gray Values from ignored puncta do not contribute to the final totals and are not output to a file Cta Red Green Blue Neuron Data Ave Puncta Number Ave Puncta Intensity Ave Puncta Area Ave Intensity Length Length Puncta Den Spine Objects Right Click Options Holding down the right mouse button over a spine will allow you to manually specify the spine type auto type mushrrom thin stubby filopodia Counting Spine Objects to add count
10. n Stubby 28 66 26 66 22 79 Length Spine Dens a 26 02 M Generic Regions Add Generic Region Clicking on Button 1 or pressing F3 will activate Add Generic Region mode The user will left click and hold down SS ea the left mouse button to trace the desired region Releasing the mouse button will finalize the region This mode will stay Ave Intensity 1648 95 active until the user double clicks on the image After tracing Integrated Intensity 246942 2 the average intensity per image will be automatically Ragin Nonii 5 calculated Changing the threshold settings will require Button 3 to be pressed to update the calculation Remove Generic Region Clicking on Button 2 or pressing F4 will activate Remove Generic Region mode Left clicking on any cell body object Dendrite Info will permanently delete it Generic Region Data Data for each color is displayed in the data pane Data is averaged per image or displayed individually Integrated intensity is the sum of pixel intensities for all pixels above threshold within the designated area of the region Ave intensity is the integrated intensity divided by the area of the region Ctrl Red Green Blue Whole Image Analysis Add Whole Image Analysis Region Clicking on Button 1 or pressing F12 will add a dendrite object that encompasses the entire width so the entire image can be analyzed at once Like dend
11. rite objects the image must be thresholded for puncta to be detected The length of this region will be 0 and cannot be changed Puncta Density A Puncta number Area Clicking on Button 2 will recalculate puncta density Remove Generic Region The region can be removed by right clicking and selecting Remove Dendrite Generic Region Data Data for each color is displayed in the data pane Data is averaged per image or displayed individually Integrated intensity is the sum of pixel intensities for all pixels above threshold within the designated area of the region Ave intensity is the integrated intensity divided by the area of the region Puncta Density L will be infinite due to length 0 Puncta Density A is Puncta number divided by the area Puncta Intensity Length is infinite Puncta Intensity Area is the Integrated intensity divided by the area Puncta Density L oO Puncta Density A 1 4698 Puncta Intensity 3158 Puncta Area 0 21 Puncta Intensity Length 0 Puncta Intensity Area 4 64E2 Dendrite Length Dendrite A 0 jo 16981 8 Puncta Intens lt 0 0 2617 3810 4240 2335 04 0 5 J374 2 3 oe jao o7 je os jso ag AT ooo je onmo jsa orz jooss 400 Right Click Options for Image Panel Restore all Puncta Hide Dendrites Invert Ignored Puncta Auto Ignore Criteria Auto Ignore Criteria Overlaping Set Dendrite Puncta Limit Clear All Regions set minimum red puncta si
12. synPAnal User Manual Eric Danielson and Sang H Lee Medical College of Wisconsin Installation Instructions Make sure the most recent version of JAVA is installed and is functioning Unzip the SynPAnal_Installation zip file into a new directory A SynPAnal jar file and directories ext and profiles should be created Double click the oynPAnal jar file to run the software Software Overview synPAnal is designed to aid the quantification of puncta from within the dendrites of neurons The steps of image analysis are as follows 1 The user starts the software and opens all images to be analyzed All images should be stored in the same directory and should be 8 bit RBG color TIFF files SynPAnal can open 16 bit images but will convert them to 8 bit images 2 The user should choose what data should be written where the data should be written and from what color the data should be collected 3 The user should then set the desired threshold Once the threshold is set it will be saved and reloaded The current threshold setting will be applied to all subsequent opened images unless the images have been thresholded previously in which case the saved threshold will be loaded 4 The user will then choose what regions will be analyzed by drawing either dendrite objects or general region objects onto the image Puncta will automatically be quantified within the dendrite objects Undesired puncta can be removed by right click
13. ze must be gt 4 4 ok Cancel Restore all Puncta Makes all puncta visible Show Hide Dendrites Makes all objects Visible Invisible Invert Ignored Puncta Makes Ignored puncta visible and sets visible puncta to A value of 50 will remove any red puncta that lg nored overlaps thresholded green by 50 Auto Ignore Criteria 1 Auto Ignores puncta based on percent overlap area with chosen thresholded color Will apply set criteria to each new region object added automatically for each new image analyzed Auto Ignore Criteria Overlaping 2 Auto Ignores puncta based on overlap with non thresholded color Set Dendrite Puncta Limit 3 Auto Ignores all puncta smaller than set size Must be gt 4 Clear All Regions Check to remove Red puncta that overlap All regions are permanently removed with green if checked or blue if checked 4 Keep if overlaps Red iKeep if overlaps Green 4 Keep if overlaps Blue 4 Confirm Close _ Auto Ignore
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