... | ... | @@ -47,21 +47,21 @@ Throughout this manual we will use following definitions |
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After MyPiC has started the user proceeds through several steps before starting an experiment:
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1. [Create jobs using JobSetter](#jobsetter)
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* [Create jobs using JobSetter](#jobsetter)
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* [Create imaging jobs with the JobSetter](#jobsetterimaging)
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* [Create FCS jobs with the JobSetter](#jobsetterfcs)
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2. [Add tasks to the Default Pipeline](#default)
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3. [Set execution, saving, and processing options of a task](#taskopt)
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4. [Define repetitions](#repetitions)
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5. [Define stage positions for the Default Pipeline](#positions)
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6. [Define the saving options](#saving)
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* [Add tasks to the Default Pipeline](#default)
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* [Set execution, saving, and processing options of a task](#taskopt)
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* [Define repetitions](#repetitions)
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* [Define stage positions for the Default Pipeline](#positions)
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* [Define the saving options](#saving)
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* [Naming conventions](#naming)
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7. [Start acquisition, save and reload settings](#startstop)
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8. [Adaptive feedback microscopy/Online image analysis](#adaptivefeedback)
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* [Start acquisition, save and reload settings](#startstop)
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* [Adaptive feedback microscopy/Online image analysis](#adaptivefeedback)
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## [<img src="./images/up.PNG">](#back)<a name=jobsetter></a> Create jobs in the JobSetter
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## [<img src="./images/up.PNG">](#back)<a name=jobsetter></a> Create jobs in the JobSetter
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The **JobSetter** is started by clicking on the corresponding button (1) *JobSetter*
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... | ... | @@ -231,7 +231,7 @@ With the adaptive feedback microscopy capability of MyPiC the user can combine o |
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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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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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## Windows registry
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... | ... | @@ -349,20 +349,20 @@ Workflows without adaptive feedback do not require any further program. |
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In this workflow at each repetition the microscope acquires a XZ scan and Z-position of the reflection line. Then imaging is performed at a certain offset from this estimated position.
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In MyPiC set in the Default pipeline as first task AF with processing **Center of Mass (thr)** and **Track Z**. The second task is LR. Specify a Z-offset value to achieve the required distance from the cover glass.
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> For the XZ glass-reflection scane do not use `Fast Z line`. This mode is not precise enough.
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> For the XZ reflection of glass scan do not use `Fast Z line`. This mode is not precise enough.
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Define an autofocus and an imaging and name them AF and LR for example. Try to use the same light-path for both jobs. For example use 433 and 561 laser to image GFP and RFP and 633 to measure the glass surface reflection. For the autofocus choose a XZ line scan with several stacks. In absence of a piezo a larger DZ and smaller Z-range needs to be used in order to attain a fast acquisition.
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**LR imaging ** | **AF glass reflection**
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:--- | :---
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<img src="./images/acquisition_imagingSettings.PNG" width = "300px"> | <img src="./images/autofocus_imagingSettings.PNG" width = "300px">
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**AF Scan mode** | **AF Z stack**
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<img src="./images/autofocus_scanMode_line.PNG" width = "300px"> | <img src="./images/autofocus_zStack.PNG" width = "300px">
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Define an autofocus and an imaging and name them AF and LR for example. Try to use the same light-path for both jobs. For example use 433 and 561 laser to image GFP and RFP and 633 to measure the glass surface reflection.
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| Protein job | Reflection job|
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:---: | :---:
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<img src="./images/acquisition_imagingSettings.PNG" width = "400px"> | <img src="./images/autofocus_imagingSettings.PNG" width = "400px">
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For the autofocus choose a XZ line scan with several stacks. In absence of pizo a larger DZ and smaller Z-range needs to be used in order to reach a fast acquisition.
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| Autofocus Scan mode | Autofocus Z stack|
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:---: | :---:
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<img src="./images/autofocus_scanMode_line.PNG" width = "400px"> | <img src="./images/autofocus_zStack.PNG" width = "400px">
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... | ... | @@ -377,11 +377,64 @@ With the adaptive feedback microscopy one can perform complex experiments. With |
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1. Object based tracking in 4D
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2. Adaptive feedback rare event detection
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2. Automated detection of rare event detection
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3. Adaptive feedback automated FC(C)S of single cells
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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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With minor modifications also automated fluorescence recovery after photobleaching experiments
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are possible. |
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\ No newline at end of file |
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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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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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For an illustration of the parameters of the image analysis program refer to [https://git.embl.de/politi/adaptive_feedback_mic_fiji.](https://git.embl.de/politi/adaptive_feedback_mic_fiji)
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For FCS analysis software and FCS calibrated imaging please refer to [https://git.embl.de/politi/FCSanalyze.](https://git.embl.de/politi/FCSAnalyze/)
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Cells express a fluorescent protein (mEGFP) and their DNA has been stained with SiR-DNA (far red dye). Following imaging and FCS jobs are used in MyPiC
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* AF: XZ scan with reflection mode on.
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* LR: Low resolution image to detect cells expressing the protein at the correct level.
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* HR: High-resolution image is used to automatically place FCS points. The image can also be used be used for FCS-calibration analysis.
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* fcsPOI: FCS settings to measure the fluorescent protein.
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A two step workflow is used to find cell of interest and start FCS measurements
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* Default pipeline:
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* task1: A XZ scan to find the reflection of the glass
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* task2: A low resolution imaging of the fluorescent protein (Channel 1) and DNA (Channel 2). At the end of the image MyPiC waits for a command from **Automated FCS** (process **Online image Analysis**).
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* Trigger 1:
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* task1: A XZ scan to find the reflection of the glass
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* task2: A high resolution imaging of the fluorescent protein (Channel 1) and DNA (Channel 3). At the end of the image MyPiC waits for a command from **Automated FCS** (process **Online image Analysis**).
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* task3: FCS measurements
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**JobSetter Imaging** | **JobSetter FCS**| **Default pipeline task 1** | **Default pipeline task 2**
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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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**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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The FiJi plugin [**Automated FCS**](https://git.embl.de/politi/adaptive_feedback_mic_fiji) is used analyze the image Default pipeline task2 and Trigger1 pipeline task 2. Following parameters are used
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<div align = "center" > <img src = './images/fcscalibrated/automatedfcs2.png' width = "400px" ></div>
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The **Automated FCS** plugins performs:
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* Job1: Cell detection using DNA channel. Cells where the fluorescent protein is within a certain range are selected. The coordinates of cells that fulfill all criteria are passed to MyPiC to start the Trigger1 pipeline.
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* Job2: detects cells using the DNA stain. Chooses the cell closest to the center where the fluorescent protein is within a certain range. Determines the coordinates of the FCS measurements and pass their values to MyPiC. In task 3 of Trigger1 MyPiC will start FCS measurements at these positions.
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**JOB1: Output generated for every processed image** | **JOB1: Output generated when objects of interest are found**
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:--- | :---
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<img src = './images/fcscalibrated/example_output.png' width = "400px" > | <img src = './images/fcscalibrated/example_output2.png' width = "400px" >
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Segmentation of the DNA channel (red) returned 17 ROIs. <br/>Only for ROI 10 and 13 is the intensity of the fluorescent protein (green) within the specified range. | Outlines for the two cells picked are shown in green.<br/> The coordinates are passed to MyPiC to execute the Trigger1 pipeline for each position.
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**JOB2: Output generated for every processed image**| **JOB2: Output generated when objects of interest are found**
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<img src = './images/fcscalibrated/example_output3.png' width = "400px" > | <img src = './images/fcscalibrated/example_output4.png' width = "400px" >
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Segmenation of the DNA channel (red) returned 2 ROIs. <br/> For ROI 1 and 2 the intensity of the fluorescent protein (green) is within the specified range. | Cell closest to center of image is picked (green outline). <br/>The red outline shows a cell that has not been picked because too far from the center of the image. <br/> Coordinates for the two FCS points are passed to MyPiC. |
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