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* **Imaging job**<a name = "imagingjob"></a>: Contains settings used for imaging. They are defined by laser used, Z-stack, pixel-dwell time etc. Basically anything you can define in the ZEN software.
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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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* **Task**<a name = "task"></a>: An imaging or FCS job assicated or not with additional processing steps, e.g. tracking in XY
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* **Pipeline**<a name = "pipeline"></a>: A concatenation of tasks 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 and start
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... | ... | @@ -78,48 +79,53 @@ You can try this to fix |
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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. [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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1. [Load jobs in the JobSetter] (#jobsetter)
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* [Load/create imaging jobs] (#jobsetterimaging)
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* [Load/create FCS jobs (optional)](#jobsetterfcs)
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2. [Add jobs to the Default pipeline](#default)
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3. [Execution, saving, and processing options for a task](#taskopt)
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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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6. [Define saving directory](#saving)
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* [Naming conventions](#naming)
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7. [Start and stop acquisition, save and reload settings](#startstop)
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8. [Adaptive feedback microscopy/Online image analysis](#adaptivefeedback)
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## [<img src="./images/up.PNG">](#back) <a name=jobsetterimaging></a> Load imaging jobs in the JobSetter
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## [<img src="./images/up.PNG">](#back) <a name=jobsetter></a> Load 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. Some settings that
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<div align = "center"><img src="./images/PipCon_upperpart.PNG" width="500"> </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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> **Caution** Loading setting from images acquired on a different microscope may not work and may impair/harm the system.
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### <a name=jobsetterimaging></a> Load/create imaging jobs
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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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#### 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**: If the microscope has Zeiss Definite focus this can be 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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* 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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### <a name=jobsetterfcs></a> Load/create FCS 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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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_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/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/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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... | ... | @@ -128,21 +134,76 @@ Add jobs to a pipeline | Explanations of the buttons |
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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=taskopt></a>Execution, saving, and processing options for a task
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To each imaging and FCS job several options can be specified yielding a
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[task](#task). Each stage position has reference XYZ coordinates. The user can specify a ```Z-offset``` with respect to the reference. Furthermore, simple image analysis tasks are integrated into the macro so that XYZ coordinates can be updated accordingly. For instance the macro can be used to detect the intensity center of mass of a fluorescent image and track bright objects in 3D.
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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/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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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. Each repetition is saved in a seperate file with the suffix
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Repetions window | Explanations of the buttons
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-------| -------------
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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/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/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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MyPiC allows the user to acquire images using different stage positions. Positions are specified by moving the stage and pressing the ```Mark``` (8) button. Positions and subpositions can also be loaded from a file.
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* Wells/Positions: Specifiy a number of positions where imaging is performed. This could be for example wells in a multi-well sample.
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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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<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/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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## [<img src="./images/up.PNG">](#back) <a name=saving></a>Define saving directory
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Images and FCS measurements are saved in a main directory specified by the user. A sub-directory per position/subposition is automatically created.
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Saving directory | Explanations of the buttons
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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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### <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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``` DE_t_WXXXX_PYYYY_TZZZZ.* or basefilename_DE_t_WXXXX_PYYYY_TZZZZ.*```
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where
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* *t*: task number
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* *XXXX*: 4 digit well identifier
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* *YYYY*: 4 digit well identifier
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* *ZZZZ*: 4 digit time point identifier
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Each well is saved in a separate directory
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``` DE_WXXXX_PYYYY```
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If number of subpositions is > 1 then there is a subdirectory per subposition
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```
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DE_WXXXX
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DE_WXXXX_PYYYY
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```
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## [<img src="./images/up.PNG">](#startstop) Start and stop acquisition, save and load settings
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After all the previous steps have been performed, acquisition can be started from the main menu of MyPiC. To reload an experiment without performing every single steps the user requires
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* example-files of each [imaging or FCS job](#jobsetter) and the
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* file containing the task specifications for each pipeline, ```PipelineConstructor.ini```. This file is automatically generated when the start-button is pressed.
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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/grid_subgrid.PNG" width = "200"> |
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