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* **FCS job**<a name = "fcsjob"></a>: Contains settings used for FCS
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* **Pipeline**<a name = "pipeline"></a>: A concatenation of imaging and/or FCS jobs that will be executed one of the other. In MyPiC we have a Default pipeline that is executed at every position and 2 pipelines that can be triggered with external commands (Trigger1 and Trigger2).
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## Installation
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Copy the file *PipelineConstructor.lvb* (for ZEN2010 use the file *PipelineConstructor\_ZEN2010.lvb*) and the directory *resources* to a directory that can be accessed from the computer running ZEN. In ZEN Open the *Macro* tab (*Alt-F8*), click on *Edit Macro*.
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Load the macro into ZEN| Explanations of the buttons
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## Installation and start
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* Copy the file *PipelineConstructor.lvb* (for ZEN2010 use the file *PipelineConstructor\_ZEN2010.lvb*) and the directory *resources* to a directory that can be accessed from the computer running ZEN.
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* In ZEN Open the *Macro* tab (*Alt-F8*), click on *Edit Macro*.
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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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### Trouble shooting for the installation:
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For any issues please contact one of the developers (at the moment Antonio Politi,
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politi@embl.de or the Ellenberg lab at EMBL).
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For any issues please contact one of the developers (at the moment Antonio Politi, politi@embl.de or the Ellenberg lab at EMBL).
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For errors that appear in the ErrorLog window please provide the PipelineConstructor.err file
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and the full version of your ZEN software (Help->About). If you have a ZEN version >
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2010 (or > 2.1) and the macro complains that it does not find Zeiss.Micro.AIM.ApplicationInterface.dll, you may need to register it manually.
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and the full version of your ZEN software (Help->About). If you have a ZEN version higher than
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2010 and the macro complains that it does not find ```Zeiss.Micro.AIM.ApplicationInterface.dll```, you may need to register it manually.
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You can try this to fix
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• Open a console window (cmd.exe)
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• Go to the ZEN path: “cd ..\..\ZEN
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• Go to the ZEN path: ``cd ..\..\ZEN``
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• Register the Zeiss.Micro.AIM.ApplicationInterface.dll
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• Register the ```Zeiss.Micro.AIM.ApplicationInterface.dll```
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> C:\Windows\Microsoft.NET\Framework\v4.0.30319\RegAsm.exe /tlb /codebase Zeiss.Micro.AIM.ApplicationInterface.
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dll
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``` C:\Windows\Microsoft.NET\Framework\v4.0.30319\RegAsm.exe /tlb /codebase Zeiss.Micro.AIM.ApplicationInterface.dll ```
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• When you uninstall ZEN, make sure to un-register
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>C:\Windows\Microsoft.NET\Framework\v4.0.30319\RegAsm.exe/u /tlb /codebase Zeiss.Micro.AIM.ApplicationInterfa
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```C:\Windows\Microsoft.NET\Framework\v4.0.30319\RegAsm.exe/u /tlb /codebase Zeiss.Micro.AIM.ApplicationInterface.dll```
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## <a name="back"></a> Setting up an experiment with MyPiC
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# <a name="back"></a> Setting up an experiment with MyPiC
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After the PipelineConstructor has started the user needs to proceed through several steps before starting an experiment
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After the MyPiC has started the user needs to proceed through several steps before starting an experiment.
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1. [Define one or several imaging jobs using the jobSetter](#jobsetterimaging)
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2. [Define one or several imaging jobs using the jobSetter (optional)](#jobsetterfcs)
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1. [Load imaging jobs in the JobSetter](#jobsetterimaging)
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2. [Load FCS jobs in the JobSetter (optional)](#jobsetterfcs)
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3. [Add jobs to the Default pipeline](#default)
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4. [Define the number of repetitions and repetition interval for the default pipeline](#repetitions)
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5. [Define stage positions and/or a grid](#positions)
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4. [Define the repetitions](#repetitions)
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5. [Define stage positions for default pipeline](#positions)
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6. [Define the directory where to save the images](#saving)
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7. [Define additional pipelines to be triggered](#trigger)
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8. [Reloading settings](#reload)
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### [<img src="./images/up.PNG">](#back) <a name=jobsetterimaging></a> JobSetter for imaging jobs
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## [<img src="./images/up.PNG">](#back) <a name=jobsetterimaging></a> Load imaging jobs in the JobSetter
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The **JobSetter** is started by clicking on the corresponding button (1) *JobSetter*
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<img src="./images/PipCon_upperpart.PNG" width="500">
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In the JobSetter window the user can create several [imaging jobs](#imagingjob) to be used in the [pipelines](#pipeline). The user first set imaging settings in ZEN and then upload them to the VBA macro either by creating a new job or updating it. 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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In the JobSetter window the user can create several [imaging jobs](#imagingjob) to be used in the [pipelines](#pipeline). The user first set imaging settings in ZEN and then upload them to the VBA macro either by creating a new job or updating it. Jobs can also be created by loading a set of microscopy images (.czi or .lsm) previously acquired on the same microscope. Some settings that
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> **Caution** Loading setting from images acquired on a different microscope may not work and may impair/harm the system.
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JobSetter window | Explanations of the buttons
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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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### Remarks for the imaging settings
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* Z-stack: Use the option *Center* instead of *First\Last*, this has proven to be more reliable in the macro. This option is accessed when pressing the *Show all* option in the Z-stack menu-bar. Recenter the stack before loading into MyPiC
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* Time-lapse: The macro has its own time-lapse. For each time point an image is acquired and saved. However, the user can still load a job that contains Time-series. This can be useful for specific workflows (see Workflow examples).
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* Multi-position: The macro allows the user to define a multi-position experiment. Multi-position defined within ZEN is allowed, however in this case the position in the macro should be set to single-position (see also Workflow examples).
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* Definite Focus: Definite focus can be specified for a specific job. The position of focus stabilizer will then be initiated at the current position in the macro. For examples see Workflow examples.
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* Combine several imaging settings: To minimize the hardware time-overhead it is best to optimize the different imaging settings so that the least hardware parts (e.g. MBS, pinhole) are modified between imaging jobs.
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## [<img src="./images/up.PNG">](#back)<a name=jobsetterfcs></a> Load FCS jobs in the JobSetter
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For FCS jobs the loading is similar as for imaging jobs. However, you can't load the settings directly from a file into the JobSetter. If using a file, please load the file into ZEN and press "Re-use".
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JobSetter FCS jobs | Explanations of the buttons
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-------| -------------
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<img src="./images/jobSetter.PNG" width="500">| 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 in 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_FCS.PNG" width="500"> | 1. Switch tab for loading settings for FCS. <br /> 2. Add an FCS job. ZEN will 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/up.PNG">](#back)<a name=repetitions></a> Define the repetitions
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MyPiC allows the user to define the time interval and number of repetitions that will be executed at every position. Note that compared to ZEN ```Time Series``` mode the hardware time-overhead is higher (~0.5-2 depending on the settings) limiting the minimal time-interval. The advantage of MyPiC is to perform different type of images and optionally to combine the workflow with image analysis at every time-point and position.
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Repetions window | Explanations of the buttons
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<p align="center"> <img src="./images/pipcon_repetitions.PNG" width = "500"><img src="./images/interval_delay.PNG" width = "400"> </p> | 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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MyPiC allows the user to acquire images using different stage positions. Positions are defined by moving the stage and pressing the ```Mark``` (8) button.
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Stage positions | Explanations of the buttons
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<p align="center"> <img src="./images/stage_positions.PNG" width = "800"></p> | 1. Acquire at the current stage position <br/> 2. Acquire at multiple positions specified by the user using ```Mark``` (8). <br/> 3. Acquire on a regularly spaced grid + subgrid. The first marked position sets the first point of the grid <br/> 4. For each position specified by the user a subgrid is created (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.
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### [<img src="./images/up.PNG">](#back)<a name=jobsetterimaging></a> JobSetter for FCS jobs
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<img src="./images/grid_subgrid.PNG" width = "200"> |
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