INsPECT user manual
Contents
1. DAPI 2 DIC 3 DAPI 3 DIC 4 DAPI 4 DIC 5 DAPI 5 DIC 6 DAPI p o e j ad s dd PS Pe i A A S A E A A Ed E ron 6 DIC 7 DAPI 7 DIC 8 Dart 8 DIC 9 DAPI 9 DIC 10 apt 10 DIC 11 DAPI 11 DIC E E Th 2 ve Puts re Ph EZ CIA wes e wy N 12 DAPI 12 DIC a DAPI 13 DIC u DAPI 14 DIC 15 DAPI o BACO DIC Er DAPI 16 DIC 17 DAPI q q ts i i h A The 17 DIC 18 ow 18 DIC re DAPI 19 DIC mi DAPI 20 DIC TT ETT 21 DIC E DAPI 22 DIC Figure 1 1 Right order of putting images in Input Folder Chapter 2 Software Setup and Features When you run the software the main panel will be opened where you can access to options with both menu items and toolbar icons see Fig 2 1 S INSPECT leo File Edit Run Help 13 vou Figure 2 1 Main Panel of INSPECT First of all an Image Viewer with basic Image manipulation operators are embedded in the software These functionalities could be accessed through the main frame of the software Options are listed below Open and Close images Zoom In Zoom Out current image e Invert current Image 2 1 Software s General Flow To process input images and generate results and reports two scenarios are embedded in the software see Fig 2 2 1 Automatic running method in which pipeline parameters are assigned to input images automatically In fact according to experimental and analytical results default values for each parameter are set in this method Chapte2. If you choose DAPI and Phase Contrast option then you should put pairs of such images in Input Folder Alternatively with choosing Just DAPI option software will process input DAPI images only Chapter 2 Software Setup and Features 6 Lj Choose Running Options Choose Input Output Options Specify where input files are located and their type where do you want to save results and what kind of files you want to save Directories Input Folder C inputFolder Browse Output Folder C outputFolder Browse Input Images Type Result Files to Save Pipeline Files to Save 2 DAPI and Phase Contrast v Normal Result y Cell Pipeline Just DAPI y Cell Parasite Marked Result y Parasite Pipeline y Parasites Convex Hull Result y Cytoplasm Pipeline Back Next Cancel Figure 2 3 Running Options Panel Users should specify running options for both automatic and custom scenarios 3 Results Files to Save Normal Result is the processed image with markers and regions boundaries Cell Parasite Marked Result is similar to Normal Result except that cells and parasites are labeled using numbers in such images Finally Parasites Convex Hull Result images show convex hull of related parasites for each cell If checked any of those results will be saved under Output Folder and in relevant sub directory for related input image s 4 Pipeline Files to Save each checkbox namely Cell Pipeline Parasite Pipeline and Cyto
3. Mac OS X and Linux The main functionality of this application is to process fluorescent DNA images with or without corresponding phase contrast DIC image sets of Leishmania infected cells acquired in laboratory conditions and to generate visual and text results Furthermore basic and common image processing operators are embedded in the software as an image viewer package INSPECT is being developed on Windows platform and all parts of the software are coded by the author using Java programming language Chapter 1 Introduction and requirements 2 1 2 Requirements Before running the software you make sure that your device has the minimum requirements for running this software Table 1 1 minimum requirements for running the INSPECT software Resource Value Memory RAM About 1GB Hard Disk space having high quality images and running in Full Option mode maximum 30MB for each input image Processor any x86 or x64 Operating System Windows Mac OS Linux User Permissions Any user can run INSPECT Notice Due to high load of processes and to prevent software crash it is highly recommended to run the program in devices with more RAM space e Try to close or idle ongoing processes while running the software to have timely results e In whatever platform if you have any problems with opening the Jar file or if the progress bar is not moving ahead the system could not probably assign enough memory for the p
4. Black Borders Crop in Results NO Cells Smoothing Level Cell Thresholding Level Cells Minimum Size in Pixels Cells Maximum Size in Pixels Parasites Thresholding Level Parasites Minimum Size in Pixels 0 Parasites Maximum Size in Pixels 200 Cytoplasms Merging Structures Level 50 Cytoplasms Smoothing Level 50 Progress Bar 1 1 tif 2 out of 50 images Processing Cells Figure 2 5 Runtime Log Panel During Process time users can trace flow of running software in this panel Chapter 2 Software Setup and Features 8 Before explaining in details parameter settings of these three panels it should be mentioned that INSPECT software is designed to biologists without image processing background Therefore for all needed parameters more meaningful and touchable synonyms are defined and numeric values are substituted with labels such as Low Medium and High 2 2 Cell Pipeline Parameters Setting In Cell Pipeline Running Parameters panel you can adjust parameters to detect cell nuclei in DAPI input images see Fig 2 6 It includes two subpanels called Parameters and Options 4 Choose Cell Pipeline Running Parameters T e ne Som ae es x D vs Choose Cell Pipeline Running Parameters w s q er Specify your desired cell pipeline parameters for running the software amp J gt If you leave the fields unchanged default values will be used automatically s gt E p ae Paramete
5. User s Guide for Image Processing Software INSPECT Version 1 0 Author Ehsan Yazdanparast 1 Contents Introduction and Requirements 1 Ta MOTUHI lt a E a ee A a eS ae oe of 1 1 2 Requirements 2 45 sa soa aa s ba e a Pw ee 2 1 3 Input Images Format and Protocols 2 Software Setup and Features 4 2 1 Software s General Flow a 4 2 2 Cell Pipeline Parameters Setting o 8 2 2 1 Parameters SUDpanel o o 8 2 2 2 Options Subpanel lt a s sa noe sosom a soea d a ee 9 2 3 Parasite Pipeline Parameters Setting 10 2 3 1 Parameters sUbpanel o 11 2 3 2 Options subpanel 0 200000 eee ee 11 2 4 Cytoplasm Pipeline Parameters Setting 12 2 4 1 Parameters subpanel 02 00000005 12 2 4 2 Options subpan l foro ee eee 13 29 OUDS AA A 14 Chapter 1 Introduction and Requirements This guide describes how to install and get started using INSPECT software The following topics are covered e Software Introduction e Requirements Input Images Format and Protocols e Software Setup and Features 1 1 Introduction INSPECT is a public domain Java based Image Processing and analysis framework developed by Ehsan Yazdanparast It runs as a Java application on any computer with Java 1 6 or later installed on it Application is available for Windows
6. ber Cell Area Cell Volume Total Number of Parasites Number of Intracellular Parasites and Number Of Extracellular Parasites Notice Cell Parasites Report could be helpful when one wants to investigate individual cells On the other hand report file is useful for generic analysis
7. mended for users not alter minimum size for parasites unless they are confident that such tiny details are not parasites and could be any other by products e Parasites Color you can choose in which color you want to see the parasites markers in the final output 2 4 Cytoplasm Pipeline Parameters Setting In Cytoplasm Pipeline Running Parameters panel you can adjust parameters to extract cytoplasm traces from Phase Contrast or DIC input images see Fig 2 8 It includes two subpanels called Parameters and Options 4 Choose Cytoplasm Pipeline Running Parameters Le Choose Cytoplasm Pipeline Running Parameters Specify your desired cytoplasm pipeline parameters for running the software If you leave the fields unchanged default values will be used automatically Parameters Options Non Cytoplasm Regions Color Very High Very High Black v High High Merging Structures Level Medium Smoothing Level gt Medium Low Low Very Low Very Low Back Next Cancel Figure 2 8 Cytoplasm Pipeline Running Parameters Panel Chapter 2 Software Setup and Features 12 Notice If you have already chosen the option Just DAPI to run the software then this panel will not be shown to you since processing Phase Contrast or DIC input images are no longer the concern 2 4 1 Parameters subpanel e Merging Structures Level in terms of Image Processing operators this parameter is equivalent to Structuring Element Size of Mo
8. parameter is used locally in subimages to make the binarization of cell portions and background area The default value which is labeled as Medium is calculated automatically based on global standard deviation of current image in process and is set to 1 5 times of this value This is logical when the density of cell is not too high and distribution of cells is not very unbiased across the image For DAPI images with high cell density majority of dark points you can increase Thresholding Level to higher degrees On the other hand for DAPI images with lower cell density you should decrease Thresholding Level to overcome unbiasness of foreground and background pixels Very low label is equivalent to 1 numeric value and Very High label is equivalent to 140 Options subpanel e Cell Minimum Maximum Size in Pixels these two parameters help algorithm to filter out those detected cell portions in final result which do not have sizes between Minimum and Maximum The default values for minimum and maximum size are set to 300 and 12000 pixels respectively Notice Sometimes high density of parasites exist around some cells and denoising them would lead to miss some valuable border data of cells in final results However such kind of misdetected portions can be ignored by setting Cell Minimum Size in Pixels properly Experiments with available image sets in hand showed that size 300 is a suitable choice to filter out those regions while pre
9. plasm Pipeline if checked then related output images for that pipeline will be saved under Output Folder and in relevant sub directory for related input image s Notice that if input images type is chosen to be Just DAPI Cytoplasm Pipeline checkbox will be disabled If you use the software in Automatic mode after choosing above running options the program is ready to run for you And you can run the software in the next panel by clicking Run button see Fig 2 4 and Fig 2 5 If you choose Custom mode then you need to specify parameters manually through three upcoming panels called Cell Pipeline Running Parameters Parasite Pipeline Running Parameters and Cytoplasm Pipeline Running Parameters Chapter 2 Software Setup and Features 7 Run Window r _ A mm gt SA Run Software The parameters and options taken successfully Software is ready for RUN Figure 2 4 Ready to Run Panel After taking parameters and options manually or automatically by clicking on Run button software starts to process the input images Running Log Chosen Method to Run AUTOMATIC Input Folder C workspace Edipse Juno Leishmania inputs Output Folder C workspace Edipse Juno Leishmania Outputs Result Files to Save Normal Result Marked Result Convex Hull Result Pipeline Files to Save none Regions Color in Results Cell Centers Color Blue Parasites Color Green Non Cytoplasm Regions Color
10. r 2 Software Setup and Features 5 j Choose Running Method Lal aa Choose Running Method Choose tomati vith default p 4 Running Type Automatic Use Default Parameter Set and Options for running the software Custom Use Customized Parameter Set and Options for running the software id Next Cancel an ll Figure 2 2 Running method selection panel users can choose Custom and Automatic methods to run the software 2 Custom running method that allows the user to specify his her intended parameters step by step If the user chooses this option cell parasite and cytoplasm pipeline parameters are taken in three steps from the user and then program will go to Ready to Run state Users are recommended to first use the software in Automatic mode to test it and if the results are not satisfactory enough for them they can then switch to Custom mode and feed the software with their parameters Automatic mode s results could also be a very promising way for users to gain ideas regarding parameter ranges Using both scenarios users also should specify some Running Parameters through a panel see Fig 2 3 as follows 1 Directories Select Input Folder that contains input image set and Output Folder to save the results and reports Notice that all results containing images and reports will be saved under Output Folder 2 Input Images Type the software comes with two options for processing input image set
11. rogram heap space In this case you can try to run the program from the command line terminal and adjust memory allocation When you open the terminal you should first go to the directory in which Jar file exists and then type this command Java Xmx lt maxHeapSpace gt m Xms lt minHeapSpace gt m jar INSPECT jar For instance the following command Java Xmx2000m Xms128m jar INSPECT jar will start the INSPECT jar program with minimum and maximum dedicated heap space of 128 megabytes and 2 giga bytes respectively 1 3 Input Images Format and Protocols To feed the software with input you need to put DNA fluorescent images DAPI and light microscopic images DIC or Phase Contrast or alternatively just DAPI images in nput Folder To run the software properly you should follow these instructions before putting images in Input Folder Chapter 1 Introduction and requirements 3 All images should be in 8 bits depth You can simply transform input images using ImageJ software under the menu image gt type gt 8 bit e DAPI images should be in bright background If not try edit gt invert on ImageJ Transformed DAPI and corresponding light microscopic images pairs should be placed exactly after each other Furthermore for each pair DAPI image should be placed before its corresponding DIC or Phase Contrast image see Fig 1 1 gt ra ay y A 4 fee t De wa h Ae E Ka E 1 DAPI 1 DIC 2
12. rphological Closing operator This operator is used to merge and fill the structures of detected cytoplasmic areas after smoothing and thresholding The default value is set to 7 which is labeled as Medium and is acceptable when Phase Contrast or DIC input image is not suffering from very high illumination variance so the thresholded cytoplasm traces need to be merged more If cytoplasmic areas cannot be well detected in a straightforward manner then increasing Merging Structures Level value could help to at least find merged portions for cytoplasms On the other hand if quality of DIC or Phase Contrast images are good and traces are visible and easily detectable then decreasing Merging Structures Level value helps the algorithm to find more exact traces around cells Very Low label is equivalent to 1 numeric value and Very High label is equivalent with a numeric value of 14 Notice DIC or Phase Contrast input images suffer from high illumination variance To compensate such effect we use Merging Structures Level parameter to have at least more smooth and connected regions Accordingly altering this value could sacrifice exact detection of some cytoplasmic regions 2 4 2 e Smoothing Level in terms of Image Processing operators this parameter is equivalent to Median Filter Kernel Radius and it is used to smooth the Morpho logical Closing result fill small holes and discard small detected portions The default value is set to 50
13. rs Options Cell Minimum Size in Pixels Very High Very High 300 Cell Maximum Size in Pixels High High 2 a 12 000 i Al Cell Centers Color Smoothing Level Medium Thresholding Level gt Medium Blue Z Low Low Exclude Border Cells in Results None Very Low Back Next Cancel Figure 2 6 Cell Pipeline Running Parameters Panel 2 2 1 Parameters subpanel e Smoothing Level in terms of Image Processing operators it is equivalent to Median Kernel Radius It is the main parameter to tell the algorithm how much it should smooth and denoise DAPI input images The default value is set to numeric value 15 which is labeled as Medium and is logical when the quality of input DAPI image is reasonable and doesn t contain that much noise If cells have very clear boundaries and noise does not exist across the image Smoothing Level can be decreased Alternatively it can be increased for low quality images None label is Chapter 2 Software Setup and Features 9 equivalent to O and Very High to 35 numeric values respectively Notice if you have low quality DAPI images and use high Smoothing Levels algorithm will overestimate the volume of the cells For such images low Smoothing Level will also lead to misdetection of cell portions because of the existence of noise and other by products 2 2 2 e Thresholding Level in terms of Image Processing operators it is equivalent to Mean Adaptive Threshold Kernel Radius This
14. serving all other valid spots e Cell Centers Color you can choose in which color you want to see the cells centers markers in final output e Exclude Border Cells in Results Checking this option will allow the algorithm to ignore incomplete cell s nuclei in edges and borders in final visualized results and calculations Chapter 2 Software Setup and Features 10 2 3 Parasite Pipeline Parameters Setting In Parasite Pipeline Running Parameters panel you can adjust parameters for extracting parasites in DAPI input images see Fig 2 7 It includes two subpanels called Parameters and Options 2 3 1 Choose Parasite Pipeline Running Parameters D y a Lo jo i cae Choose Parasite Pipeline Running Parameters 2 o Ff Specify your desired parasite pipeline parameters for running the software a e If you leave the fields unchanged default values will be used automatically s gt j Parameters Options Parasite Minimum Size in Pixels Very High 0 Parasite Maximum Size in Pixels High 200 Thresholding Level gt Medium Parasites Color Green v Low Very Low Back Next Cancel Figure 2 7 Parasite Pipeline Running Parameters Panel Parameters subpanel e Thresholding Level in Image Processing terms this parameter is binded to Structuring Element Size of Black Top Hat Transform operator The size of the Structuring Element is highly dependent on nature of image structures we wan
15. t to analyze keep or suppress Here the components of the interest for suppressing are parasites nuclei Therefore increasing or decreasing the value of Thresholding Level will enable algorithm to suppress those components more or less The default value is set to 2 labeled as Medium and works quite fine for most of DAPI stained images we had to analyze However for any reason if parasites nuclei are bigger or smaller than normal then you could increase or decrease Thresholding Level In this case Structuring Element will be bigger or smaller and as a result it will suppress bigger or smaller candidate areas for parasites Very Low label is equivalent to 1 numeric value and Very High label is equivalent with numeric value 4 Notice Decreasing Thresholding Level is not recommended since using this option often Chapter 2 Software Setup and Features 11 leads to overdetection of parasites However if users are aware of the fact that in reality such density of parasites exists around cell portions they can use this option too 2 3 2 Options subpanel e Parasite Minimum Maximum Size in Pixels these two parameters help the algorithm to filter out those detected parasites which do not meet requirements of size range The default size values for Minimum and Maximum is set to 0 and 100 pixels respectively Notice in some images there may exist detected parasites with very small sizes namely 1 to 10 pixels It is recom
16. which is labeled as Medium This parameter also tries to compensate the effects of bad quality of cytoplasm traces and its task is more or less similar to Merging Structures Level parameter Very Low label is equivalent to 1 numeric value and Very High label is equivalent with a numeric value 50 Options subpanel e Non Cytoplasm Regions Color you can choose in which color you want to see the non cytoplasmic regions in the final output The regions encapsulated inside Chapter 2 Software Setup and Features 13 cytoplasm regions will be shown in original image in results 2 5 Outputs Upon completion of the running of the software visual and text results will be saved under Output Folder directory Visual outputs are already explained in section 2 1 In each run software also generates two report files called report and cell parasites report The report file contains general outcomes of processing each pair or individual FileName Total Number of Cells Number of Infected Cells Total Number of Parasites Total Number of Intracellular Parasites Percentage of Infected Cells The Mean Number of Parasites per Cell and Parasitic Index Percentage of Infected Cells x Mean Number of Parasites Per Cell are the parameters recorded in this file for each image pair or individual The Cell Parasites Report keeps the record of each cells features in image pairs or individual You can find these fields in this report FileName Cell Num
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