Stage Automation with VisiView
Steps in Brief
Legacy Mode:
3. Select Stage*
4. Find center in Live View
5. Mark X x Y
6. Update position
7. Test image
Standard Mode:
3. Select Stage*
4. Open the ScanSlide Detail View
5. Navigate around area of interest with joystick
6. Mark Region of Interest
7. Test Image
1. Selecting Magnification
2. Choose Multiposition Mode
Legacy Mode:
3. Uncheck all Acquire tabs except Stage. Only Stage* at the top should have an asterisk next to it. If anything else has an asterisk next to it, that Acquire tab is still checked on.
If it is not selected next to the checkmark, select Scan Slide as the multiposition mode.
You can also use Stage Positions in that drop down to manually select individual positions.
Legacy Mode:
4. Go to the center of your sample in Show Live mode. Turn off Show Live mode to not bleach your sample.
5. Select the number of X positions and number of Y positions. For instance this setting to the right will create a 2x2 Field Of View square.
6. Click Center Area around Current! You must re-click this button after every change you make to the Stage tab or else none of your changes will be recorded.
Legacy Mode:
7. Uncheck Save Sequence to Disk, and take a test Sequence. Adjust as necessary.
If Save Sequence to Disk does not have a checkmark next to it, that means that another Acquire tab (such as Z-Series*) has an Asterisk next to it. If any two Acquire tabs have Asterisks, sequences are automatically saved and there is no checkbox given.
Standard Mode:
Standard Mode:
3. Uncheck all Acquire tabs except Stage. Only Stage* at the top should have an asterisk next to it. If anything else has an asterisk next to it, that Acquire tab is still checked on.
If it is not selected next to the checkmark, select Scan Slide as the multiposition mode.
You can also use Stage Positions in that drop down to manually select individual positions.
Standard Mode:
Standard Mode:
4. Open the ScanSlide Detail View by hovering over the top right of the scan slide area. A box will appear circled in red here, which you click on.
Standard Mode:
Standard Mode:
5. Use live mode to outline the area you want. The boundaries of the box will grow according to your joystick motions.
If you want to reset the area you have outlined with the joystick, hit "Clear"
(You can always close this mode when you do not want it with the top right x button.)
Standard Mode:
Standard Mode:
6. Draw a box or shape around the area you want to image.
Ideally you would press "Overview" here, to see your image before drawing the shape, but this is broken on the MAICO. So there you have to guess.
If you mess up your shapes, you can remove everything with "Clear" , or just the shapes with "Edit->Clear All Regions"
Standard Mode:
6 (cont). If there are multiple regions selected, VisiView will image the selected region. This is too confusing, so I would recommend getting rid of them.
If you mess up your shapes, you can remove everything with "Clear" , or just the shapes with "Edit->Clear All Regions"
Standard Mode:
Standard Mode:
Optional: You can click the center button, to have a MxN option
Standard Mode:
7. Take a test sequence! Clear or Clear All Regions as necessary to retry.
Post-Acquisition: Montaging
You can do this at anypoint later, or you can do this in ImageJ. You can also do this right after taking your data.
Select your muiltiposition data, make sure the panel to the right looks the same as here. Click Start! A new file will be created of the montaged data.
Advanced: Adding New Magnification
Advanced: Adding New Magnification
-Add a new name
-Correctly select parameters
-Magnification is most important
-Refractive Index/NA used for deconvolution
-Calibration/Correction is set in next step.
-> Needs a good guess though!
-Be careful when choosing device positions! These names are actually Microscope Positions 1-6, and the Ti2 will automatically move the device position when each slot is selected.
Advanced: Setting the Overlap
Advanced: Calibrating Magnification
-Draw a little square around a bright dot (colloids work well)
-Hit the calibrate button
-Wait, and watch to see that it looked liked the calibration was successful.
-You may need to rotate the camera, invert the xy, etc.
-You may need to be on bin-1 and have a fast exposure (<100 ms) and be on a fast or standard camera setting
-You may need to come up with a better guess for the original calibration before running this process (see next slide).
Advanced: Noticing wrong calibrations
Correct
Wrong
If correction is wrong enough, you will not be able to calibrate again! Set it to 1 and start over