... | ... | @@ -80,7 +80,7 @@ An example workflow using glass relection autofocus or fluorescence based autofo |
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The interaction with the adaptive feedback microscopy is exaplaied in
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* [Adaptive feedback microscopy/Online image analysis](#adaptivefeedback)
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* [Adaptive feedback microscopy/Online image analysis](./Adaptivefeedback)
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... | ... | @@ -113,14 +113,14 @@ JobSetter > Imaging Jobs | Explanations of the buttons |
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### <a name=jobsetterfcs></a> Create FCS jobs with 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```. 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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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 different 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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:-------| :------
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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 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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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 a 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 task to a Default pipeline | Explanations of the buttons
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:-------| :-------------
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... | ... | @@ -136,7 +136,7 @@ For imaging tasks the user can specify further processing steps. For instance, t |
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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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**Explanations of the buttons**
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1. Set 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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... | ... | @@ -148,7 +148,7 @@ For imaging tasks the user can specify further processing steps. For instance, t |
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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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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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... | ... | @@ -156,7 +156,7 @@ For imaging tasks the user can specify further processing steps. For instance, t |
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## [<img src="./images/up.PNG">](#back)<a name=repetitions></a> Define repetitions
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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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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 sec 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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Pipeline repetitions | Explanations of the buttons
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:-------| -------------
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... | ... | @@ -164,7 +164,7 @@ Pipeline repetitions | Explanations of the buttons |
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## [<img src="./images/up.PNG">](#back)<a name=positions></a> Define stage positions for the 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 in MyPiC. 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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... | ... | @@ -174,17 +174,18 @@ Default positions | Example on how (sub)positions are imaged and named |
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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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5. **Load positions**: Load file with ending **.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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8. **Mark**: 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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... | ... | @@ -245,18 +246,22 @@ Start/stop/saving | Explanations of the buttons |
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## [<img src="./images/up.PNG">](#back) <a name = simpleexample></a> Simple workflows with MyPiC
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Workflows without adaptive feedback do not require any further program.
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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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In this workflow at each repetition the microscope acquires a XZ scan and estimate the Z-position of the glass reflection line. Imaging is then performed at a certain Z-offset from this estimated position.
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Define an autofocus job (named e.g. AF) and an imaging job (named e.g. LR). 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 imaging settings 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 can be used in order to avoid a very slow acquisition.
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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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Example settings are given here
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**LR imaging ** | **AF glass reflection**
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**LR imaging settings** | **AF glass reflection settings**
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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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... | ... | @@ -264,6 +269,6 @@ Define an autofocus and an imaging and name them AF and LR for example. Try to u |
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### Fluorescence 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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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. The pipeline works best for sparse objects. If several bright objects are in the same field of view than the tracking of a single object may fail.
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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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Define a low-resolution job, named LR, with a large field of view (low zoom) and fast acquisition. Define a high-resolution job. Try to use similar light-path settings for the two jobs. 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. |