Commit 90b9a5f8 authored by Aliaksandr Halavatyi's avatar Aliaksandr Halavatyi
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# ddd
# Overview
The purpose of this software is to automatically locate and carry-out high-throughput imaging of *Drosophila* embryos. Using Zen Black and Fiji / ImageJ, embryos can either be selected manually or automatically and imaged on a Zeiss 780 or 880.
# Quickstart
1) Launch the Zen Black Software.
## ds
2) At the top of the screen, select the "Macro" window and click on "Macro...".
### sfds
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![alt text](img/img01.png "Title text")
3) In the "Edit Macro" panel, click the "load" button and select the file: MyPiC.lvb
![alt text](img/img02.png "Title text")
4) The MyPic window will appear on the screen. Click on "JobSetter" located in the top left corner.
![alt text](img/img03.png "Title text")
5) In the JobSetter window click on the folder icon and select the four Zeiss .lsm files titled: "DE1","DE2","TR1", and "TR2". These are the four jobs that the microscope will carry-out.
![alt text](img/img04.png "Title text")
6) Each job will be loaded into the microscope. To view a job in Zen, simply click on the button "Macro-->Zen" in the JobSetter window. Changes can be made to the job and will be saved by clicking the "ZEN-->Macro" button in the JobSetter window.
7) Return to the MyPiC window and select the folder icon in the bottom right corner.
![alt text](img/img05.png "Title text")
8) Open the file "PipelineConstructor.ini" - these are the settings for the pipeline.
9) Navigate the microscope to the well in the top left corner of the microscope slide and make sure that it is relatively in focus at 5x magnification.
10) In the MyPiC window, select the "Default Positions" button and click the "Mark" Button. The user may have to change these settings depending on how the sampels are mounted on the slide.
![alt text](img/img06.png "Title text")
11) In the MyPiC window, select the "Saving" button
![alt text](img/img07.png "Title text")
12) Select the "..." button and create a new root directory to store the acquired images, subdirectories, and imaging settings.
![alt text](img/img08.png "Title text")
13) In the "Base File Name" enter a unique ID name for the run. It is very important that you change the name of this each time you run the automation.
![alt text](img/img09.png "Title text")
14) Launch Fiji / ImageJ.
15) Select the Macro window and (UPDATE THE PATH TO GET TO THE PLUGIN).
![alt text](img/img10.png "Title text")
16) Embryos can be either automatically located by the macro, or the user can select which specific embryos to image at the beginning of the protocol. To allow the macro to identify embryos, select "Automated Embryo Selection". To select the embryos yourself, select "Manual Embryo Selection".
17) Select the root directory created earlier.
18) Multiple dialog boxes will appear. The user may have to change these settings depending on how the samples are mounted on the slide. The given numbers are the pre-settings used during the screen in the publication: (insert link).
19) Note: Do not press "Stop Monitor" until complete with imaging.
![alt text](img/img11.png "Title text")
20) Return to the MyPiC macro and click the start arrow.
![alt text](img/img12.png "Title text")
21) If you are doing "Automatic Embryo Selection", then you are finished. If you are running "Manual Embryo Selection", continue to the "Manual Embryo Selection" section of this manual.
# Manual Embryo Selection
1) Follow the directions from the Quickstart section of this manual or run the protocol with your own settings.
2) After the second job, TR2 is run, a dialog box will appear. DO NOT CLICK on this box until you have selected the desired embryos.
![alt text](img/img13.png "Title text")
An image will open in a Fiji / ImageJ window. Select individual embryos with the left-click on the mouse, followed by pressing “t” on your keyboard to add the positions to the ROI Manager.
3) When all the embryos are selected, press the “OK” button on in the Select Embryo Positions dialog box.
4) Repeat these steps 1-3 for every well. After embryos positions are manually selected for every well, the microscope will begin the automated feedback imaging.
# Changing / Modifying Job Settings
1) To view a job, select the “JobSetter” panel in the MyPiC window.
2) Select the job of interest by left-clicking on the name.
3) Select "Macro -> Zen" to move the job and its current settings to Zen Black.
4) To modify the job, make any desired changes to the settings in Zen Black.
5) Select "Zen -> Macro" to modify the job settings.
# Viewing data using the Automation Viewer
#### My final images are not correctly rotated. What's going on? ####
The final job may be doing an extra rotation. To fix this:
1) Go to the MyPiC graphical user interface
2) Select the "Jobs" panel
3) Select the final job so that it is highlighted. In the loaded example, this job is called "TR2".
4) Below, click the button "Macro to Zen". The job will load onto the Zen Black interface.
5) Go to "rotation" on Zen Black and make sure the value = "0".
6) Sometimes it is not possible to change the rotation value without first resetting the "zoom" value (located above "rotation" to 0.0). After fixing the rotation, the zoom value should switch back.
#### Why does the plugin not continue to the next step? ####
The macro could be trying to load a file from a previous automation run. To fix this:
1) Close any open images that may be open in Fiji / ImageJ
2) Make sure that the Fiji / ImageJ plugin is still monitoring
2) Go to the MyPiC graphical user interface
3) Select the "Saving" panel
4) Change the name of the run
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