... | ... | @@ -5,34 +5,38 @@ MyPiC is a Visual Basic for Application (VBA) macro to be used with Zeiss confoc |
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The macro allows
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* Autofocus based on reflection and fluorescence multi-location time series
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* Fluorescence based tracking using center of mass of fluorescence signal
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* Fluorescence based tracking using the center of mass of the fluorescence signal
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* Multi-location time-lapse experiments
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* Flexible combination of several independent Z-stack and channel settings
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* Flexible combination of imaging with fluorescence correlation spectroscopy (FCS) experiments
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* Usage of several different FCS settings
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* [Adaptive Feedback microscopy support of two triggable imaging and FCS workflows](#adaptiveFeedback)
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Adaptive feedback microscopy allows to combine microscopy acquisition with online image analysis and so perform complex experiments without human supervision.
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> **Disclaimer:**
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> MyPiC for ZEN has been tested on Zeiss LSM 780 microscopes with ZEN 2010, 2011, and 2012, and LSM880 microscopes with ZEN2.1 and ZEN2.3. We don’t guarantee that it will work on other configurations and we don’t take any responsibility for damage occuring during or after use of MyPiC.
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> MyPiC has been tested on Zeiss LSM 780 microscopes with ZEN 2010, 2011, and 2012, and LSM880 microscopes with ZEN2.1 and ZEN2.3. We don’t guarantee that it will work on other configurations and we don’t take any responsibility for damage occuring during or after use of MyPiC.
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## Definitions
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Throughout this manual we will use following naming definitions
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Throughout this manual we will use following definitions
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* **Imaging job**<a name = "imagingjob"></a>: Stores specific settings for imaging. The settings include laser power, Z-stack, pixel-dwell time etc. In principle anything you can define in the ZEN software.
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* **Imaging job**<a name = "imagingjob"></a>: Stores specific settings for imaging. The settings include laser power, Z-stack, pixel-dwell time etc. This are all the settings you can define in the ZEN software.
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* **FCS job**<a name = "fcsjob"></a>: Stores specific settings to be used for FCS measurements.
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* **Task**<a name = "task"></a>: An imaging or FCS job associated 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](#adaptivefeedback).
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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](#default) that is executed at every position and 2 pipelines ([Trigger1 and Trigger2](#trigger))that can be triggered with external commands .
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* **current settings**: These are the settings that are loaded into ZEN.
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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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* Copy the file *MyPiC.lvb* (for ZEN2010 use the file *MyPiC\_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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Load the macro into ZEN| Explanations of the buttons
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--------------|----------------------------
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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/loadMacro.PNG" width = "400px"> | 1. Load the macro. Browse to the file *MyPiC.lvb* <br /> or *MyPiC\_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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... | ... | @@ -43,101 +47,143 @@ Throughout this manual we will use following naming definitions |
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After MyPiC has started the user needs to proceed through several steps before starting an experiment.
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1. [Load/create jobs using JobSetter] (#jobsetter)
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* [Load/create imaging jobs] (#jobsetterimaging)
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1. [Load/create jobs using 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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3. [Execution, saving, and processing options for each 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 saving directory](#saving)
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* [Naming conventions](#naming)
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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=jobsetter></a> Load 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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<div align = "center"><img src="./images/PipCon_upperpart.PNG"> </div>
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In the JobSetter window the user can create several [imaging](#imagingjob) and [FCS](#fcsjob) jobs to be used in one or several [pipelines](#pipeline). The user specify acquisition settings within ZEN and then upload the settings into the 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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In the JobSetter window the user can create several [Imaging](#imagingjob) and [FCS](#fcsjob) jobs to be used in one or several [pipelines](#pipeline). The user specifies acquisition settings within ZEN and then upload the settings into the VBA macro as a job. Imaging jobs can also be created by loading a set of microscopy images (czi or lsm format) previously acquired on the same microscope.
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> **Caution** Do not load settings from images acquired on a different microscope. This may not work and impair the functionality of the system.
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>**Caution** Do not load settings from images acquired on a different microscope. This may not work and impair the functionality of 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 = "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 disk <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 highlighted job <br/> 12. Available tracks for current job
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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 = "400px">| 1. Create a new job with the current imaging settings <br /> 2. Load imaging jobs from saved images <br/> 3. Change name of current job (highlighted job) <br/> 4. Acquire all or one jobs and save images to disk <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 highlighted 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 mode. 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](./workflows_examples.md)).
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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 [Workflow examples](./workflows_examples.md)).
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* **Definite Focus**: If the microscope has Zeiss *Definite focus* this can be be used within the macro. However note that the position of focus stabilizer will be initiated at the current position stored in the macro (see [Workflow examples](./workflows_examples.md)).
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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 mode. 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.
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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.
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* **Definite Focus**: If the microscope has Zeiss *Definite focus* this can be be used within the macro. However note that the position of the focus stabilizer will be initiated at the current position stored in the macro.
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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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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```. Several FCS settings can be stored. For this the user must use different names of the light-path for each FCS job.
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JobSetter FCS jobs | Explanations of the buttons
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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 the user 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. ZEN asks the user to save the light-path configuration <br/> 6. Load settings of current job into ZEN <br/> 7. Stop acquisition <br/> 8. Acquire current FCS measurement <br/> 9. List of available FCS jobs <br/> 10. Short description of current job
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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 = "400px"> | 1. Switch tab to load jobs for FCS <br /> 2. Add an FCS job. ZEN prompts the user 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. ZEN promts user to save the light-path configuration <br/> 6. Load settings of current job into ZEN <br/> 7. Stop acquisition <br/> 8. Acquire current FCS measurement <br/> 9. List of available FCS 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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## [<img src="./images/up.PNG">](#back)<a name=default></a> Add tasks to the Default pipeline
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In a pipeline the user specifies the sequential order of imaging and FCS tasks from jobs previously loaded into the macro using the JobSetter. For an FCS task, 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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<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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Add task to a Default pipeline | Explanations of the buttons
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:-------| :-------------
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<img src="./images/PipCon_2DefaultPipeline_select.PNG" width = "400px"> | 1. Open select job 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 the 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 = "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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For each imaging and FCS [task](#task) several options can be specified. These determine when to execute and save a task. Furthermore, the user can set a ```Z-offset``` with respect to the reference Z-location (see also [Define stage positions for default pipeline](#positions)).
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For imaging tasks the user can specify further processing steps. For instance, the macro can compute a new XYZ position from the center of mass of the fluorescence image. The current reference position can then be updated with the computed new position (options **TrackXY** and/or **TrackZ** are on). This feature is useful to track bright objects in 3D. More complicated processing steps can be achieved when an external image analysis program is used (see also [Adaptive feedback microscopy/Online image analysis](#adaptivefeedback)).
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**Task options**
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<p align= center> <img src="./images/pipcon_track.PNG" width = "400px"> <p/>
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** Explanations of the buttons**
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1. Specifies when task will be acquired. This is every *N* repetitions, or at start or end of repetitions
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2. **Save**: Set if task should be saved or not
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3. **Z-Offset** is added/subtracted from the current reference Z-position. This does not change reference Z-position for subsequent tasks
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4. Execute all tasks in a pipeline once (use to test a pipeline)
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5. Process Image/Tracking methods
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* **None**: Do not perform any analysis
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* **Center of mass (thr)**: Center of mass of upper 10% intensity. Optimal to compute position of glass reflection
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* **Peak**: XYZ coordinate of maximal intensity
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* **Center of mass**: Center of mass for the intensity. Optimal to track sparse bright objects
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* **Online img. analysis**: Adaptive feedback option. Wait for feedback from an external image analysis program
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6. **Channel**: channel to be used for precessing (does not apply for Online img. analysis)
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7. **Track Z**: Update reference Z position with computed value
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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. Each repetition is saved in a seperate file with the suffix
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The user needs to define the time interval and number of repetitions 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 *TXXXX*.
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Repetions window | Explanations of the buttons
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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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Pipeline repetitions | Explanations of the buttons
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:-------| -------------
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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 interval is between start of an image and the start of the next image (see image inset) <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 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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MyPiC allows the user to acquire images using different stage positions. Positions are specified by moving the stage and pressing the ```Mark``` (8) button in MyPiC. 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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<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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Default positions | Example on how (sub)positions are imaged and named
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:---| :---
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<img src="./images/stage_positions.PNG" width = "400px"> | <img src="./images/grid_subgrid.PNG" width = "200px" >
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** Explanations of the buttons**
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1. **Single**: performs imaging at current stage position
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2. **Multiple**: Imaging at multiple positions specified by the user using **Mark** (8).
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3. **Grid**: Use a regularly spaced grid + subgrid. The first marked position sets the first point of the grid
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4. **Multiple + Subgrid**: For each position specified by the user a regular spaced subgrid is used (see 13-14)
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5. Load positions saved in a file **.pos*
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6. Save positions to a file **.pos*
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7. List of positions
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8. Add current stage position to list (7)
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9. Move to highlighted position
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10. Remove highlighted position
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11. Update highlighted position with current stage position
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12. Number of rows and columns for grid and distance between grid points
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13. Number of rows and columns for subgrid and distance between grid points
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14. Position of subgrid with respect to main grid position (red circle)
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* Left: Grid position is first subgrid position.
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* Right: Grid position represents the center of the subgrid.
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15. Order of imaging
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* Left: Image all subpositions of a well and then move to next well.
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* Right: Image one subposition of a well and then move to next well.
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16. File name of **.pos* file 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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Images and FCS measurements are saved in a main directory specified by the user. A sub-directory per position/subposition is automatically generated.
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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 = "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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:--- | :---
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<img src="./images/pipcon_saving.PNG" width = "400px"> | 1. Name of main folder <br/> 2. Browse through folders <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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> **Airy detector** For images acquired with the Airy detector the user must use the czi format.
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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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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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* 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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-----------------|----------------------------
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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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:---|:---
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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/>7. Save pipeline settings to a specific location <br/> 8. Show online image analysis registry keys
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# [<img src="./images/up.PNG">](#back) <a name="adaptivefeedback"></a> Adaptive Feeback Microscopy
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With the adaptive feedback microscopy capability the user can combine online image analysis and image acquisition to perform among other things detection rare events, time-lapse FCS measurement, cell tracking, bleaching experiments. Images are acquired by the microscope and then processed by an image analysis program that is monitoring the arrival of new images to be analysed. After the image has been analysed commands are sent to the microscope in order to start for example an FCS measurement at specific points, a high-resolution 4D imaging, or update the XYZ position for tracking an object in space and time. If the user wish to trigger a different pipeline upon an event detection then the pipeline [Trigger1 and/or Trigger2](#trigger) need to be defined.
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The communication is through the windows registry, thus any program that can read and write to the windows registry can be used.
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With the adaptive feedback microscopy capability the user can combine online image analysis and image acquisition and perform:
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* Detection of rare events and start a different imaging protocol
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* Time-lapse FCS measurements
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* Cell tracking
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* Fluorescence recovery after photobleaching (FRAP) experiments
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Images are acquired by the microscope and then processed by an image analysis program that is monitoring the arrival of new images to be analysed. After the image has been analysed commands are sent to the microscope in order to start a different pipeline or update the XYZ position for tracking an object in space and time. To start a different pipeline upon event detection the settings for the [Trigger1 and/or Trigger2](#trigger) must be defined.
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The communication is through the windows registry, thus any program that can read and write to the windows registry can be used.
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## Windows registry
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Define two imaging job using the same or similar light-path. Name them LR and HR, respectively. Set a large field of view with few pixels for LR. Add to the default pipeline LR as first task with processing **Center of Mass** and **Track Z**, **Track XY**. Specify the channel to process. Add the HR job as second task.
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## Using adaptive feedback
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## <a name= adaptiveFeedback></a>Using adaptive feedback
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With the adaptive feedback microscopy one can perform complex experiments. With the ImageJ/FiJi macro [Automated FCS](https://git.embl.de/politi/adaptive_feedback_mic_fiji) the user can perform
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