... | @@ -4,15 +4,15 @@ MyPiC is a Visual Basic for Application (VBA) macro to be used with Zeiss confoc |
... | @@ -4,15 +4,15 @@ 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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The macro allows
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* Autofocus based on reflection and fluorescence multi-location time series
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* Autofocus based on reflection and fluorescence multi-location time series.
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* Fluorescence based tracking using the center of mass of the 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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* 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 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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* Flexible combination of imaging with fluorescence correlation spectroscopy (FCS) experiments.
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* Usage of several different FCS settings
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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 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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> **Adaptive feedback microscopy** combines microscopy acquisition with online image analysis to perform complex experiments without user supervision.
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... | @@ -37,10 +37,10 @@ Throughout this manual we will use following definitions |
... | @@ -37,10 +37,10 @@ Throughout this manual we will use following definitions |
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Load the macro into ZEN| Explanations of the buttons
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Load the macro into ZEN| Explanations of the buttons
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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/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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<img src="./images/assignMacro.PNG" width = "400px"> | provide a *Menu Entry*, a text, and select *MyPiC.lvb* as project.
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### Trouble shooting the installation
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### Trouble shooting the installation
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For any issues please contact one of the developers in the Ellenberg group at EMBL, Heidelberg and provide the version of the software and the full version of your ZEN software (Help->About). If you have a ZEN version higher than 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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For any issues please contact one of the developers in the Ellenberg group at EMBL, Heidelberg and provide the version of the software, the full version of your ZEN software (Help->About), and all the log and err files. If you have a ZEN version higher than 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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You can try this to fix
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... | @@ -233,18 +233,19 @@ A file *PipelineConstructor.ini* is automatically generated when the start-butto |
... | @@ -233,18 +233,19 @@ A file *PipelineConstructor.ini* is automatically generated when the start-butto |
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Start/stop/saving | Explanations of the buttons
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Start/stop/saving | Explanations of the buttons
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:---|:---
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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 registry keys definitions for adpative feedback/online image analysis
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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 registry keys definitions for adaptive feedback/online image analysis
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# [<img src="./images/up.PNG">](#back) <a name="adaptivefeedback"></a> Adaptive feedback microscopy/online image analysis
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# [<img src="./images/up.PNG">](#back) <a name="adaptivefeedback"></a> Adaptive feedback microscopy/online image analysis
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With the adaptive feedback microscopy capability of MyPiC the user can combine online image analysis and image acquisition to perform:
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With the adaptive feedback microscopy capability of MyPiC the user can combine online image analysis and image acquisition to perform:
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* Detection of rare events and start a different imaging protocol
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* Detection of rare events and start a different imaging protocol
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* Automated FCS and imaging experiments
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* Time-lapse FCS measurements
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* Time-lapse FCS measurements
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* Cell tracking
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* Cell tracking
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* Fluorescence recovery after photobleaching (FRAP) experiments
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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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Images are acquired by the microscope and then processed by an image analysis program that is monitoring the arrival of new images to be analyzed. After the image has been analyzed 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 between the image analysis software is through the windows registry, thus any program that can read and write to the windows registry can be used. An example of an application using ImageJ for online image analysis can be found in
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The communication between the image analysis software is through the windows registry, thus any program that can read and write to the windows registry can be used. An example of an application using ImageJ for online image analysis can be found in
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... | @@ -388,18 +389,8 @@ Define two imaging job using the same or similar light-path. Name them LR and HR |
... | @@ -388,18 +389,8 @@ Define two imaging job using the same or similar light-path. Name them LR and HR |
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## <a name= adaptiveFeedback></a>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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With the adaptive feedback microscopy one can perform complex experiments. We provide here an example for high-throughout F(C)CS and imaging using the ImageJ/FiJi macro [Automated FCS](https://git.embl.de/politi/adaptive_feedback_mic_fiji)
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1. Object based tracking in 4D
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2. Automated detection of rare event detection
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3. Automated FC(C)S of single cells
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4. Object based tracking in 4D and FC(C)S
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A small modification of the analysis pipeline also allows for automated fluorescence recovery after photobleaching experiments.
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### Adaptive feedback automated FC(C)S of single cells
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### Adaptive feedback automated FC(C)S of single cells
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The workflow can be used to obtain F(C)CS data in high-throuhput. The FCS measurements and images are linked so that the data can be used for FCS-Calibrated imaging.
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The workflow can be used to obtain F(C)CS data in high-throuhput. The FCS measurements and images are linked so that the data can be used for FCS-Calibrated imaging.
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... | @@ -428,7 +419,7 @@ A two step workflow is used to find cell of interest and start FCS measurements |
... | @@ -428,7 +419,7 @@ A two step workflow is used to find cell of interest and start FCS measurements |
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**JobSetter Imaging** | **JobSetter FCS**| **Default pipeline task 1** | **Default pipeline task 2**
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**JobSetter Imaging** | **JobSetter FCS**| **Default pipeline task 1** | **Default pipeline task 2**
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:--- | :---
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:--- | :--- | :--- | :---
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<img src = './images/fcscalibrated/jobsetter1.png' width = "200px" > | <img src = './images/fcscalibrated/jobsetter2.png' width = "200px" > | <img src = './images/fcscalibrated/default1.png' width = "200px" > | <img src = './images/fcscalibrated/default2.png' width = "200px" >
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<img src = './images/fcscalibrated/jobsetter1.png' width = "200px" > | <img src = './images/fcscalibrated/jobsetter2.png' width = "200px" > | <img src = './images/fcscalibrated/default1.png' width = "200px" > | <img src = './images/fcscalibrated/default2.png' width = "200px" >
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**Trigger1 pipeline task1** | **Trigger1 pipeline task2** | **Trigger1 pipeline task3**
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**Trigger1 pipeline task1** | **Trigger1 pipeline task2** | **Trigger1 pipeline task3**
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<img src = './images/fcscalibrated/trigger1_1.png' width = "200px" >|<img src = './images/fcscalibrated/trigger1_2.png' width = "200px" > | <img src = './images/fcscalibrated/trigger1_3.png' width = "200px" >
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<img src = './images/fcscalibrated/trigger1_1.png' width = "200px" >|<img src = './images/fcscalibrated/trigger1_2.png' width = "200px" > | <img src = './images/fcscalibrated/trigger1_3.png' width = "200px" >
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