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# Microscopy Pipeline Constructor (MyPiC)
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<table style="width:100px">
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MyPiC is a Visual Basic for Application (VBA) macro to be used with Zeiss confocal microscopes running with the ZEN software (version black).
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The macro allows
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... | ... | @@ -32,8 +61,8 @@ Throughout this manual we will use following naming conventions: |
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<style="width:99%"> Load the macro into ZEN| Explanations of the buttons
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--------------|----------------------------
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<img src="./images/loadMacro.PNG" width="400"> | 1. Load the macro. Browse to the file *PipelineConstructor.lvb* <br /> or *PipelineConstructor\_ZEN2010.lvb* for older ZEN versions. <br /> 2. Run the macro. <br /> 3. To insert the macro in your list of macros click on the tab.
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<img src="./images/assignMacro.PNG" width="400"> | provide a *Menu Entry*, a text, and select *PipelineConstructor.lvb* as project.
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<img src="./images/loadMacro.PNG" width = "400px"> | 1. Load the macro. Browse to the file *PipelineConstructor.lvb* <br /> or *PipelineConstructor\_ZEN2010.lvb* for older ZEN versions. <br /> 2. Run the macro. <br /> 3. To insert the macro in your list of macros click on the tab.
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<img src="./images/assignMacro.PNG" width = "400px"> | provide a *Menu Entry*, a text, and select *PipelineConstructor.lvb* as project.
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### Trouble shooting for the installation:
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... | ... | @@ -80,7 +109,7 @@ After the MyPiC has started the user needs to proceed through several steps befo |
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The **JobSetter** is started by clicking on the corresponding button (1) *JobSetter*
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<div align = "center"><img src="./images/PipCon_upperpart.PNG" width="500"> </div>
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<div align = "center"><img src="./images/PipCon_upperpart.PNG"> </div>
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In the JobSetter window the user can create several [imaging jobs](#imagingjob) and [FCS jobs](#fcsjob) to be used in one or several [pipelines](#pipeline). The user choose imaging and/or FCS settings in ZEN and then upload the settings into VBA macro as a job. Imaging jobs can also be created by loading a set of microscopy images (.czi or .lsm) previously acquired on the same microscope.
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... | ... | @@ -91,7 +120,7 @@ In the JobSetter window the user can create several [imaging jobs](#imagingjob) |
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JobSetter imaging jobs | Explanations of the buttons
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-------| -------------
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<img src="./images/jobSetter.PNG" width="460">| 1. Create a new job with current imaging settings. <br /> 2. Load imaging jobs from saved images (.czi or .lsm). <br/> 3. Change name of current job (highlighted job). <br/> 4. Acquire all or one jobs and save images to a file. <br/> 5. Remove a job from the list. <br/> 6. Update current job with settings from ZEN. <br/> 7. Load settings of current job into ZEN. <br/> 8. Stop acquisition. <br/> 9. Acquire current job. <br/> 10. List of available jobs. <br/> 11. Short description of current job. <br/> 12. Available tracks for current job.
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<img src="./images/jobSetter.PNG" width = "400px">| 1. Create a new job with current imaging settings. <br /> 2. Load imaging jobs from saved images (.czi or .lsm). <br/> 3. Change name of current job (highlighted job). <br/> 4. Acquire all or one jobs and save images to a file. <br/> 5. Remove a job from the list. <br/> 6. Update current job with settings from ZEN. <br/> 7. Load settings of current job into ZEN. <br/> 8. Stop acquisition. <br/> 9. Acquire current job. <br/> 10. List of available jobs. <br/> 11. Short description of current job. <br/> 12. Available tracks for current job.
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#### Remarks for the imaging settings
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... | ... | @@ -107,14 +136,14 @@ For FCS jobs the loading is similar as for imaging jobs. However, you can't load |
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JobSetter FCS jobs | Explanations of the buttons
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-------| -------------
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<img src="./images/jobSetter_FCS.PNG" width="500"> | 1. Switch tab to load jobs for FCS. <br /> 2. Add an FCS job. ZEN asks to save the light-path configuration. <br/>3. Change name of FCS job. <br/> 4. Remove current FCS job. <br/> 5. Update current job with settings from ZEN. <br/> 6. Load settings of current job into ZEN. <br/> 7. Stop acquisition. <br/> 8. Acquire current job. <br/> 9. List of available jobs. <br/> 10. Short description of current job.
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<img src="./images/jobSetter_FCS.PNG" width = "400px"> | 1. Switch tab to load jobs for FCS. <br /> 2. Add an FCS job. ZEN asks to save the light-path configuration. <br/>3. Change name of FCS job. <br/> 4. Remove current FCS job. <br/> 5. Update current job with settings from ZEN. <br/> 6. Load settings of current job into ZEN. <br/> 7. Stop acquisition. <br/> 8. Acquire current job. <br/> 9. List of available jobs. <br/> 10. Short description of current job.
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## [<img src="./images/up.PNG">](#back)<a name=default></a> Add jobs to the Default pipeline
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In a pipeline the users specifies the sequential order of imaging and FCS jobs previously loaded into the macro using the JobSetter. For FCS jobs, FCS positions need to be specified via the adaptive feedback method otherwise the FCS measurement will not be acquired. The ***Default*** pipeline is the imaging workflow executed at every position and repetition.
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Add jobs to a pipeline | Explanations of the buttons
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-------| -------------
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<img src="./images/PipCon_2DefaultPipeline_select.PNG" width = "700"> | 1. Pressing **+** opens a window (3) <br/> 2. Remove current job in pipeline. <br/> 3. Double click on a job to upload it in a pipeline. The GoTo jobs force to switch to a different pipeline.
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<img src="./images/PipCon_2DefaultPipeline_select.PNG" width = "400px"> | 1. Pressing **+** opens a window (3) <br/> 2. Remove current job in pipeline. <br/> 3. Double click on a job to upload it in a pipeline. The GoTo jobs force to switch to a different pipeline.
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## [<img src="./images/up.PNG">](#back)<a name=taskopt></a>Execution, saving, and processing options for a task
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... | ... | @@ -123,7 +152,7 @@ To each imaging and FCS job several options can be specified yielding a |
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Task options | Explanations of the buttons
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-------------| ---------------------------
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<img src="./images/pipcon_track.PNG" width = "700"> | 1. Specify when task will be acquired. This is <br/> every *N* repetitions, or at start or end of repetitions <br/> 2. Set if task should be saved or not <br/> 3. ```Z-Offset``` is added to the current reference Z-position. It does not change reference Z-position for subsequent tasks <br/> 4. Execute all task in a pipeline once (use to test a pipeline) <br/> 5. Processing methods <br/>**None**: Do not perform any analysis <br/> **Center of mass (thr)**: Center of mass of intensity for the upper 10% values. Optimal to compute position of glass reflection<br/>**Peak** XYZ coordinate of maximal intensity<br/>**Center of mass** Center of mass for the intensity. Optimal to track sparse bright objects<br/> **Online img. analysis**: Adaptive feedback option. Wait for feedback from an external image analysis program<br/> 6. Channel to be used for precessing (does not apply for adaptive feedback) <br/> 7. **Track Z**: Update reference Z position with computed value <br/> 8. **Track XY**: Update XY position with computed value
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<img src="./images/pipcon_track.PNG" width = "400px"> | 1. Specify when task will be acquired. This is <br/> every *N* repetitions, or at start or end of repetitions <br/> 2. Set if task should be saved or not <br/> 3. ```Z-Offset``` is added to the current reference Z-position. It does not change reference Z-position for subsequent tasks <br/> 4. Execute all task in a pipeline once (use to test a pipeline) <br/> 5. Processing methods <br/>**None**: Do not perform any analysis <br/> **Center of mass (thr)**: Center of mass of intensity for the upper 10% values. Optimal to compute position of glass reflection<br/>**Peak** XYZ coordinate of maximal intensity<br/>**Center of mass** Center of mass for the intensity. Optimal to track sparse bright objects<br/> **Online img. analysis**: Adaptive feedback option. Wait for feedback from an external image analysis program<br/> 6. Channel to be used for precessing (does not apply for adaptive feedback) <br/> 7. **Track Z**: Update reference Z position with computed value <br/> 8. **Track XY**: Update XY position with computed value
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... | ... | @@ -133,7 +162,7 @@ MyPiC allows the user to define the time interval and number of repetitions that |
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Repetions window | Explanations of the buttons
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-------| -------------
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<img src="./images/pipcon_repetitions.PNG" width = "400"><img src="./images/interval_delay.PNG" width = "400"> | 1. Delay/interval between images <br/> 2. Specify if the delay is in seconds or minutes <br/> 3. If checked time interva is between start of an image and the start o the next image. <br/> 4. Number of repetitions
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<img src="./images/pipcon_repetitions.PNG" width = "400px"><img src="./images/interval_delay.PNG" width = "400px"> | 1. Delay/interval between images <br/> 2. Specify if the delay is in seconds or minutes <br/> 3. If checked time interva is between start of an image and the start o the next image. <br/> 4. Number of repetitions
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## [<img src="./images/up.PNG">](#back)<a name=positions></a> Define stage positions for default pipeline
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... | ... | @@ -143,8 +172,10 @@ MyPiC allows the user to acquire images using different stage positions. Positio |
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* Subpositions: Subpositions are imaging positions defined with respect to wells/positions. Each Well/position can have one or several subpositions.
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Stage positions | Explanations of the buttons
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-------| -------------
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<img src="./images/stage_positions.PNG" width = "800px"> <br/> <br/> Example on how positions and subpositions are imaged and naming of files <br/> <div align = 'center'> <img src="./images/grid_subgrid.PNG" width = "200"> </div>| 1. ```Single``` performs imaging at current stage position <br/> 2. ```Multiple``` Imaging at multiple positions specified by the user using ```Mark``` (8). <br/> 3. ```Grid``` Use a regularly spaced grid + subgrid. The first marked position sets the first point of the grid <br/> 4. ```Multiple + Subgrid``` For each position specified by the user a regular spaced subgrid is used (see 13-14) <br/> 5. Load positions saved in a file ```*.pos``` <br/> 6. Save positions to a file ```*.pos``` <br/> 7. List of positions <br/> 8. Add current stage position to list (7) <br/> 9. Move to highlighted position <br/> 10. Remove highlighted position <br/> 11. Update highlighted position with current stage position <br/> 12. Number of rows and columns for grid and distance between grid points. <br/> 13. Number of rows and columns for subgrid and distance between grid points. <br/> 14. Position of subgrid with respect to main grid position (red circle). <br/> Left: Grid position is first subgrid position. Right: Grid position represents the center of the subgrid. </br> 15. Left: Image all subpositions of a well and then move to next well. Right: Image one subposition of a well and then move to next well. <br/> 16. File name of positions to be loaded.
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:---:| :---
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<img src="./images/stage_positions.PNG" width = "400px"> <br/> <br/> Example on how positions and subpositions are imaged and naming of files <br/> <img src="./images/grid_subgrid.PNG" width = "200px" >| 1. ```Single``` performs imaging at current stage position <br/> 2. ```Multiple``` Imaging at multiple positions specified by the user using ```Mark``` (8). <br/> 3. ```Grid``` Use a regularly spaced grid + subgrid. The first marked position sets the first point of the grid <br/> 4. ```Multiple + Subgrid``` For each position specified by the user a regular spaced subgrid is used (see 13-14) <br/> 5. Load positions saved in a file ```*.pos``` <br/> 6. Save positions to a file ```*.pos``` <br/> 7. List of positions <br/> 8. Add current stage position to list (7) <br/> 9. Move to highlighted position <br/> 10. Remove highlighted position <br/> 11. Update highlighted position with current stage position <br/> 12. Number of rows and columns for grid and distance between grid points. <br/> 13. Number of rows and columns for subgrid and distance between grid points. <br/> 14. Position of subgrid with respect to main grid position (red circle). <br/> Left: Grid position is first subgrid position. Right: Grid position represents the center of the subgrid. </br> 15. Left: Image all subpositions of a well and then move to next well. Right: Image one subposition of a well and then move to next well. <br/> 16. File name of positions to be loaded.
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## [<img src="./images/up.PNG">](#back) <a name=saving></a>Define saving directory
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Saving directory | Explanations of the buttons
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-----------------|----------------------------
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<img src="./images/pipcon_saving.PNG" width = "500px"> | 1. Name of main folder <br/> 2. Browse through folder <br/> 3. Prefix before name ID of file <br/> 4. Format of image file. For ***AIRY images*** save as czi only
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<img src="./images/pipcon_saving.PNG" width = "400px"> | 1. Name of main folder <br/> 2. Browse through folder <br/> 3. Prefix before name ID of file <br/> 4. Format of image file. For ***AIRY images*** save as czi only
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### <a name=naming></a>File naming convention
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Each time point and each position is saved in a separate file. The file name contains the ID for well, position, and time point. For the default pipeline this reads
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Start stop | Explanations of the buttons
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<img src="./images/pipcon_startstop.PNG" width = "500px"> | 1. Start acquisition <br/> 2. Start acquisition and send commands to LabView water pump controller (optional) <br/> 3. Stop acquisition <br/> 4. Stop acquistion at the end of current repetition <br/> 5. Pause acquisiotion <br/> 6. Load pipelines settings from ```PipelineConstructor.ini``` file <br/> Save pipeline settings to a specific location <br/> 8. Show online image analysis registry keys
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<img src="./images/pipcon_startstop.PNG" width = "400px"> | 1. Start acquisition <br/> 2. Start acquisition and send commands to LabView water pump controller (optional) <br/> 3. Stop acquisition <br/> 4. Stop acquistion at the end of current repetition <br/> 5. Pause acquisition <br/> 6. Load pipelines settings from ```PipelineConstructor.ini``` file <br/> Save pipeline settings to a specific location <br/> 8. Show online image analysis registry keys
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