... | ... | @@ -312,21 +312,43 @@ Workflows without adaptive feedback do not require any further program. |
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## Without adaptive feedback
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### Reflection based Autofocus and imaging
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Define
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### Glass based reflection autofocus and imaging
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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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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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### Intensity center of mass based tracking
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This workflow can be used to perform a tracking of bright objects in low resolution and imaging at high resolution of the object of interest. If several bright objects are in the same field of view than the workflow does not work as it only uses the fluorescence center of mass.
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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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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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### Object based tracking in 4D
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1. Object based tracking in 4D
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### Adaptive feedback rare event detection
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2. Adaptive feedback rare event detection
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### Adaptive feedback automated FC(C)S of single cells
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3. Adaptive feedback automated FC(C)S of single cells
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### Object based tracking in 4D and FC(C)S
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4. Object based tracking in 4D and FC(C)S
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### Automated fluorescence recovery after photobleaching experiments |
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With minor modifications also automated fluorescence recovery after photobleaching experiments
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are possible. |
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